CN111234263A - Preparation method of injectable polyethylene glycol active hydrogel - Google Patents

Preparation method of injectable polyethylene glycol active hydrogel Download PDF

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CN111234263A
CN111234263A CN202010038211.XA CN202010038211A CN111234263A CN 111234263 A CN111234263 A CN 111234263A CN 202010038211 A CN202010038211 A CN 202010038211A CN 111234263 A CN111234263 A CN 111234263A
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詹静
罗巧洁
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/002—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from unsaturated compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2387/00—Characterised by the use of unspecified macromolecular compounds, obtained otherwise than by polymerisation reactions only involving unsaturated carbon-to-carbon bonds

Abstract

The invention provides a preparation method of injectable polyethylene glycol active hydrogel, which is characterized in that dihydroxy of oligo-polyethylene glycol, maleic anhydride and thiomalic acid are subjected to polycondensation reaction under mild conditions of scandium trifluoromethanesulfonate catalysis, a synthesized hydrogel precursor contains a plurality of sulfydryl and double bonds, and the double bonds and the sulfydryl in the gel precursor can be completely and efficiently reacted through sulfydryl-alkene click reaction to form the injectable hydrogel. The method of the invention has high yield of water-soluble PEG derivatives and no residue of respective precursors. The observation and analysis of the gelation time show that the gelation time can be changed by the fine adjustment of the pH value so as to adapt to the actual operation requirement of the clinical in-situ forming in the future.

Description

Preparation method of injectable polyethylene glycol active hydrogel
Technical Field
The invention belongs to the technical field of medical material manufacturing, and relates to a preparation method of injectable polyethylene glycol active hydrogel.
Background
With the increasing requirements of people on the repair of bone defects caused by trauma, deformity, tumor surgery and the like and the increasing pursuit of the function and aesthetics of implant repair, both orthopedic repair and oral implant repair often face the embarrassment that bone regeneration and functional repair can be realized only by relying on bone grafting and other methods due to insufficient bone mass, which is also the most common clinical challenge.
PEG is considered to be one of the best artificially synthesized medical macromolecular materials at present because of the advantages of hydrophilicity, no toxicity, no sensitization, capability of quickly removing degradation products from the body and the like. Currently, PEG hydrogels are usually obtained by modifying the terminal hydroxyl group of PEG to a functional group that can be chemically cross-linked, and then gelling by chemical cross-linking. However, the synthesis steps of the thiol group and the double bond of the hydrophilic polymer are complicated and expensive; there are also problems such as a small number of functional groups and complicated synthesis of functional groups, and biocompatibility being affected. Dihydroxy oligo (ethylene glycol), DHPEG, is one of the basic raw materials in the chemical industry, is cheap and can be directly obtained from the market. Therefore, from the perspective of popularization and application, the synthesis of multifunctional PEG hydrogel based on DHPEG is very interesting. However, how to prepare PEG gel precursor with good biocompatibility and multifunctional functional groups by using easily available raw materials and simple and mild synthesis method is the key point for preparing medical PEG gel.
Disclosure of Invention
The invention aims to provide a preparation method of injectable polyethylene glycol active hydrogel, which is realized by the following steps:
(1) synthesis of gel precursor (poly (oligo) maleate), POEGM) containing multiple double bonds
Dihydroxyoligoethylene glycol (ethylene glycol), OEG, was placed in a 250mL eggplant-shaped flask9) (40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf) as a catalyst were added3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours under nitrogen protection, then slowly heating to 100 ℃, condensation polymerization for 12 hours, dissolving the crude product in water, placing in a cellulose acetate dialysis bag, placing in PBS with 2L0.01M concentration, and dialyzing for 1W to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying is carried out again to obtain 41.6g of the product with a yield of 87%. This stepIn the step, the oligoethylene glycol and maleic acid are subjected to condensation polymerization reaction under the catalysis of scandium trifluoromethanesulfonate to form a gel precursor POEGM containing a plurality of double bonds. POEGM polycondensation reaction formula:
Figure BDA0002366780470000021
(2) synthesis of Multi-thiol-group-containing gel precursor (poly (oligo (ethylene glycol) mercaptosuccinate), POEGMS) OEG was placed in a 250mL eggplant-shaped bottle9(40.0g, 1.0mol) and thiomalic acid (15.0g, 1.0mol) and scandium triflate (Sc (OTf))3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours, then slowly heating to 120 ℃, condensation polymerization for 12 hours. The crude product was dissolved in aqueous solution and dialyzed 7d to remove the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying gave 42.1g of product in 81% yield. In the step, the oligoethylene glycol and the thiomalic acid are subjected to condensation polymerization reaction under the catalysis of scandium trifluoromethanesulfonate to form a gel precursor POEGMS with a plurality of sulfydryl groups. POEGMS polycondensation reaction chemical formula:
Figure BDA0002366780470000022
(3) preparation of gel and calculation of gelation time: both precursors synthesized above were dissolved in PBS solution at pH 7.0, 7.4, 8.0 (n ═ 3) according to 5%, 10%, 20% (solid content, wt%) to form equimolar gel precursor solutions of clickable groups. And uniformly mixing two liquid gel precursors POEGM and POEGMS preheated at 37 ℃ in a vial according to an equimolar ratio of clickable functional groups to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel. The gel formation time was calculated by the test-tube inversion method (i.e.the time when the vials were inverted from time to time and when no liquid flow in the vials was observed is the gelation time). In the step, two gel precursors POEGM and POEGMS which are preheated at 37 ℃ are uniformly mixed in a small bottle according to the equal molar ratio of click functional groups to generate thiol-ene click reaction to form a molecular skeleton of the hydrogel. No additional catalyst, initiator or light stimulation is required. Under the conditions of pH7.4 and 37 ℃ (similar to the physiological conditions of human bodies), the gelation time is reduced from 2 minutes to 30 seconds, the requirement of clinical injection operation can be met, and the result also indicates that the gelation time can change the injectable time through the fine adjustment of the pH value, so as to adapt to different clinical requirements in the future.
The invention provides a preparation method of injectable polyethylene glycol active hydrogel, which is characterized in that dihydroxy of oligo-polyethylene glycol, maleic anhydride and thiomalic acid are subjected to polycondensation reaction under mild conditions catalyzed by scandium trifluoromethanesulfonate, a synthesized hydrogel precursor contains a plurality of sulfydryl and double bonds, the yield of water-soluble PEG derivatives is high, and no residue of each precursor exists. And then through mercapto-alkene "click" reaction, the double bond and mercapto in the gel precursor can all finish the reaction high-efficiently, the molecular skeleton that forms hydrogel can inject hydrogel, the observation and analysis of gelation time shows that the gelation time can be changed through the fine adjustment of pH value, in order to meet the actual operation needs of clinical in-situ forming in the future. The present invention does not require additional catalysts, initiators or light stimuli. Under the conditions of pH7.4 and 37 ℃ (similar to the physiological condition of human body), the gelation time is reduced from about 2 minutes to about 30 seconds, which can meet the requirement of clinical injection operation, and the result also indicates that the gelation time can change the injectable time through the fine adjustment of the pH value, so as to adapt to different clinical requirements in the future.
Drawings
FIG. 1 is a schematic representation of the thiol-ene "click" reaction of POEGM and POEGMS at 37 ℃.
FIG. 2 is a rheological behavior test of hydrogels of different solids contents, wherein G': storage modulus, G ": loss modulus.
FIG. 3 is a graph of the degradation curves of hydrogels of different solids contents.
Detailed Description
The invention is further explained by the accompanying drawings and examples. But merely as a better illustration of the features of the invention and not as a complete inclusion of the invention.
Example one
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and then maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf))3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours under the protection of nitrogen, then slowly raising the temperature to 100 ℃, condensation polymerization for 12 hours. The crude product was dissolved in water and placed in a cellulose acetate dialysis bag and dialyzed against 2L of 0.01M PBS for 1W to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain product POEGM 41.6 g. POEGM polycondensation reaction formula:
Figure BDA0002366780470000031
in 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) and thiomalic acid (15.0g, 1.0mol) and scandium triflate (Sc (OTf)3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours, then slowly heating to 120 ℃, condensation polymerization for 12 hours, dissolving the crude product in water solution, and dialyzing for 7d to remove the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain 42.1g of product POEGMS. POEGMS polycondensation reaction chemical formula:
Figure BDA0002366780470000032
both precursors synthesized above were first dissolved in PBS solution at pH 7.0 (n ═ 3) at 5% (solid content, wt%) to form equimolar solutions of clickable groups (gel precursor groups). And uniformly mixing two gel precursors POEGM and POEGMS which are preheated at 37 ℃ according to an equimolar ratio and can click functional groups in a small bottle to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel.
Example two
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and then maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf))3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours under the protection of nitrogen, then slowly raising the temperature to 100 ℃, condensation polymerization for 12 hours. The crude product was dissolved in water and placed in a cellulose acetate dialysis bag and dialyzed against 2L of 0.01M PBS for 1W to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain product POEGM 41.6 g.
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) and thiomalic acid (15.0g, 1.0mol) and scandium triflate (Sc (OTf)3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours, then slowly heating to 120 ℃, condensation polymerization for 12 hours, dissolving the crude product in water solution, and dialyzing for 7d to remove the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain 42.1g of product POEGMS.
Both precursors synthesized above were first dissolved in PBS solution at pH7.4 (n ═ 3) at 5% (solid content, wt%) to form equimolar solutions of clickable groups (gel precursor groups). And uniformly mixing two gel precursors POEGM and POEGMS which are preheated at 37 ℃ according to an equimolar ratio and can click functional groups in a small bottle to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel.
EXAMPLE III
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and then maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf))3) (5.0g,1.0mol), esterifying at 80 deg.C for 12 hr under nitrogen protection, slowly heating to 100 deg.C, performing condensation polymerization for 12 hr, dissolving the crude product in water, placing in cellulose acetate dialysis bag, and placing into 2L0.01M dialysis bagWas dialyzed against PBS for 1W to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain product POEGM 41.6 g.
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) and thiomalic acid (15.0g, 1.0mol) and scandium triflate (Sc (OTf)3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours, then slowly heating to 120 ℃, condensation polymerization for 12 hours. The crude product was dissolved in aqueous solution and dialyzed 7d to remove the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain 42.1g of product POEGMS.
Both precursors synthesized above were first dissolved in PBS solution at pH 8.0 (n ═ 3) at 5% (solid content, wt%) to form equimolar solutions of clickable groups (gel precursor groups). And uniformly mixing two gel precursors POEGM and POEGMS which are preheated at 37 ℃ according to an equimolar ratio and can click functional groups in a small bottle to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel.
Example four
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and then maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf))3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours under nitrogen protection, then slowly heating to 100 ℃, condensation polymerization for 12 hours, dissolving the crude product in water, placing in a cellulose acetate dialysis bag, placing in 2L PBS with 0.01M concentration, and dialyzing for 1W to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain product POEGM 41.6 g.
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) and thiomalic acid (15.0g, 1.0mol) and scandium triflate (Sc (OTf)3) (5.0g,1.0mol), esterifying at 80 deg.C for 12 hr, slowly heating to 120 deg.C, condensation polymerizing for 12 hr, dissolving the crude product in water solution,dialysis 7d removes the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain 42.1g of product POEGMS.
Both precursors synthesized above were dissolved in PBS solution at pH 7.0 (n ═ 3) at 10% (solid content, wt%) to form equimolar solutions of clickable groups (gel precursor groups). And uniformly mixing two gel precursors POEGM and POEGMS which are preheated at 37 ℃ according to an equimolar ratio and can click functional groups in a small bottle to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel.
EXAMPLE five
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and then maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf))3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours under nitrogen protection, then slowly heating to 100 ℃, condensation polymerization for 12 hours, dissolving the crude product in water, placing in a cellulose acetate dialysis bag, placing in 2L PBS with 0.01M concentration, and dialyzing for 1W to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain product POEGM 41.6 g.
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) and thiomalic acid (15.0g, 1.0mol) and scandium triflate (Sc (OTf)3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours, then slowly heating to 120 ℃, condensation polymerization for 12 hours. The crude product was dissolved in aqueous solution and dialyzed 7d to remove the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain 42.1g of product POEGMS.
Both precursors synthesized above were dissolved in PBS solution at pH7.4 (n ═ 3) at 10% (solid content, wt%) to form equimolar solutions of clickable groups (gel precursor groups). And uniformly mixing two gel precursors POEGM and POEGMS which are preheated at 37 ℃ according to an equimolar ratio and can click functional groups in a small bottle to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel.
EXAMPLE six
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and then maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf))3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours under nitrogen protection, then slowly heating to 100 ℃, condensation polymerization for 12 hours, dissolving the crude product in water, placing in a cellulose acetate dialysis bag, placing in 2L PBS with 0.01M concentration, and dialyzing for 1W to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain product POEGM 41.6 g.
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) and thiomalic acid (15.0g, 1.0mol) and scandium triflate (Sc (OTf)3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours, then slowly heating to 120 ℃, condensation polymerization for 12 hours. The crude product was dissolved in aqueous solution and dialyzed 7d to remove the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain 42.1g of product POEGMS.
Both precursors synthesized above were dissolved in PBS solution (n ═ 3) at pH 8.0 (solid content, wt%) to form equimolar solutions of clickable groups (gel precursor). And uniformly mixing two gel precursors POEGM and POEGMS which are preheated at 37 ℃ according to an equimolar ratio and can click functional groups in a small bottle to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel.
EXAMPLE seven
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and then maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf))3)(5.0g,1.0mol), esterifying at 80 deg.C for 12 hr under nitrogen protection, slowly heating to 100 deg.C, condensation polymerizing for 12 hr, dissolving the crude product in water, placing in cellulose acetate dialysis bag, and dialyzing in 2L0.01M PBS for 1W to remove catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain product POEGM 41.6 g.
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) and thiomalic acid (15.0g, 1.0mol) and scandium triflate (Sc (OTf)3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours, then slowly heating to 120 ℃, condensation polymerization for 12 hours. The crude product was dissolved in aqueous solution and dialyzed 7d to remove the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain 42.1g of product POEGMS.
Both precursors synthesized above were dissolved in PBS solution at pH 7.0 (n ═ 3) at 20% (solid content, wt%) to form equimolar solutions of clickable groups (gel precursor groups). And uniformly mixing two gel precursors POEGM and POEGMS which are preheated at 37 ℃ according to an equimolar ratio and can click functional groups in a small bottle to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel.
Example eight
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and then maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf))3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours under nitrogen protection, then slowly heating to 100 ℃, condensation polymerization for 12 hours, dissolving the crude product in water, placing in a cellulose acetate dialysis bag, placing in 2L PBS with 0.01M concentration, and dialyzing for 1W to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain product POEGM 41.6 g.
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) and thiomalic acid (15.0g,1.0mol) and scandium triflate (Sc (OTf)3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours, then slowly heating to 120 ℃, condensation polymerization for 12 hours, dissolving the crude product in water solution, and dialyzing for 7d to remove the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain 42.1g of product POEGMS.
Both precursors synthesized above were dissolved in PBS solution at pH7.4 (n ═ 3) at 20% (solid content, wt%) to form equimolar solutions of clickable groups (gel precursor groups). And uniformly mixing two gel precursors POEGM and POEGMS which are preheated at 37 ℃ according to an equimolar ratio and can click functional groups in a small bottle to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel.
Example nine
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) was azeotropically dewatered with toluene in a 110 ℃ oil bath, and then maleic acid (11.6g, 1.0mol) and scandium trifluoromethanesulfonate (Sc (OTf))3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours under nitrogen protection, then slowly heating to 100 ℃, condensation polymerization for 12 hours, dissolving the crude product in water, placing in a cellulose acetate dialysis bag, placing in 2L PBS with 0.01M concentration, and dialyzing for 1W to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain product POEGM 41.6 g.
In 250mL eggplant-shaped bottles, OEG was added9(40.0g, 1.0mol) and thiomalic acid (15.0g, 1.0mol) and scandium triflate (Sc (OTf)3) (5.0g,1.0mol), esterification at 80 ℃ for 12 hours, then slowly heating to 120 ℃, condensation polymerization for 12 hours, dissolving the crude product in water solution, and dialyzing for 7d to remove the catalyst. The resulting solution was dialyzed against 2L of 0.01M PBS in a cellulose acetate dialysis bag for 1w to remove the catalyst. The water changing interval frequency is 1h (3 times), 2h (3 times), 6h (4 times), 12h (8 times) and 24h (2 times) in sequence. Vacuum freeze-drying to obtain 42.1g of product POEGMS.
Both precursors synthesized above were dissolved in PBS solution (n ═ 3) at pH 8.0 (solid content, wt%) to form equimolar solutions of clickable groups (gel precursor). And uniformly mixing two gel precursors POEGM and POEGMS which are preheated at 37 ℃ according to an equimolar ratio and can click functional groups in a small bottle to generate a thiol-ene click reaction to form a molecular skeleton of the hydrogel.
Example ten
The rheological properties of the samples were characterized using a TA AR-G2 rotational rheometer. The samples from example two, example five, example eight were placed in parallel jaws with a diameter of 20 mm. The test temperature was 37 ℃ before the examination started; the angular frequency scanning test range is 0.05 to 50 rad/s; the fixed strain was set to 2%. The storage modulus and loss modulus are respectively represented by sum.
As shown in fig. 2, the storage modulus of the hydrogel with all solids content is much greater than the loss modulus, indicating that the material being tested is in a solid state and undergoes mainly elastic deformation. Its storage modulus G' increases with increasing precursor solids content. The storage modulus increased from 0.7kPa to 69.2kPa as the bulk solids content increased from 5% to 20%. In the angular frequency range used in the test, almost all of the storage modulus G' did not change with frequency, indicating that the gel was very well crosslinked and gradually increased in elasticity with increasing solid content.
EXAMPLE eleven
The experimental conditions of the hydrolysis of the prepared hydrogel are set to be similar to the environment of the physiological temperature of a human body, so that the in-vivo degradation process is simulated to the maximum extent. Samples of the second, fifth and eighth examples were each prepared by placing 1ml of the gel in 10ml of PBS solution (0.01M, pH7.4, n 3 × 19) in a 37 ℃ water bath, replacing the fresh PBS solution every day, removing hydrogel samples at prescribed time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29(d)), rinsing with deionized water 3 times, freeze-drying and weighing. And finally, presenting the degradation process of the hydrogel through a relation curve of the mass loss fraction of the gel and the degradation time.
As shown in FIG. 1, the hydrogel with 5% gel solids content rapidly degraded within 5 days, but the degradation of the hydrogels with 10% and 20% solids content clearly divided into two stages. In the first stage (2 w) the mass loss of the gel is very small with only minor changes. This further demonstrates the high efficiency of the thiol-ene "click" reaction in the hydrogel process prepared in this study, with few unreacted precursors, and hydrolytic cleavage of the ester bond only at the very few sites where incomplete "click" reactions are likely to occur. In the second stage (2-4w), the gel mass continues to decrease rapidly and the degradation is accelerated exponentially until complete degradation occurs after 25-29 days.
Example twelve
For biosafety evaluation, we adopted ISO standards (ISO 10993-1: 1997). The sample extracts of the second, fifth and eighth examples were evaluated for their cell compatibility with Hela cells (TCTU 187, China academy of sciences type culture Collection cell Bank) and osteogenic precursor cells (MC3T3-E1, China academy of sciences type culture Collection cell Bank), respectively; a hemolytic reaction experiment is used as a rapid toxicity test index for evaluating the blood compatibility of the material. All samples had better cell compatibility.
EXAMPLE thirteen
0.2g of POEGM and POEGMS were dissolved in 2ml (10% solids content) and 1ml (20% solids content) of α MEM medium containing 10% FBS, respectively, and after filtration and sterilization, the solutions were mixed in equal volumes, and then added to a 96-well plate (100. mu.l/well) immediately before ungelled, until they gelled spontaneously.
The experiments of adhesion, proliferation and spreading of the osteogenic precursor cells on the gel surface show that the osteogenic precursor cells can be well adhered, spread and proliferated on the hydrogel surface with the solid content of 10 percent and 20 percent, and can successfully generate osteogenic differentiation under the osteogenic inducing culture condition.
Example fourteen
To further optimize the hydrogel with proper solid content, we observed the direct loading of osteogenic precursor cells and the invasive growth of cell aggregates in the gel, respectively.
Gel precursor solutions were prepared with solids contents of 10% and 20%. And (3) rapidly and uniformly mixing the cell suspension with the prepared ungelled gel solution by using the osteogenic precursor cell suspension in the exponential growth phase, and carrying out a cell load growth observation experiment and a cell invasion growth observation experiment. The results show that the hydrogel with the solid content of 10 percent and 20 percent can be loaded with osteogenic precursor cells and maintain the activity of the osteogenic precursor cells, and the cells can successfully invade and grow in the gel; however, the hydrogel with the solid content of 10% is more excellent in the aspects of cell growth and cell invasion, and is a hydrogel material with a prospect of bone transplantation repair application.

Claims (6)

1. The preparation method of the injectable polyethylene glycol active hydrogel is characterized by comprising the following steps:
(1) synthesis of gel precursor containing multiple double bonds: carrying out azeotropic dehydration on chemically pure oligo-polyethylene glycol and toluene in an oil bath at 110 ℃ in a 250mL eggplant-shaped bottle, adding maleic acid and a catalyst, esterifying for 12 hours at 80 ℃ under the protection of nitrogen, slowly heating to 100 ℃, carrying out condensation polymerization for 12 hours, dissolving a crude product in water, putting the crude product into a cellulose acetate dialysis bag, putting the bag into PBS with the concentration of 2L0.01M, dialyzing for 1W to remove the catalyst, and carrying out vacuum freeze drying to obtain a gel precursor POEGM;
(2) synthesis of gel precursor containing multiple thiol groups: the same as in step (1), the OEG is added9Reacting with thiomalic acid and a catalyst at 80 ℃ for 12 hours, slowly heating to 120 ℃, performing condensation polymerization reaction for 12 hours, dissolving the crude product in an aqueous solution, dialyzing to remove the catalyst, putting the solution in a cellulose acetate dialysis bag, putting the solution in 2L of 0.01M PBS, dialyzing to remove the catalyst, and performing vacuum freeze drying to obtain a gel precursor POEGMS;
(3) preparation of gel and calculation of gelation time: firstly, respectively dissolving the two precursors synthesized in the step (1) and the step (2) in a PBS solution to form an equimolar gel precursor solution with clickable groups, then uniformly mixing the two gel precursors POEGM and POEGMS preheated at 37 ℃ in a vial according to an equimolar ratio with clickable functional groups to generate mercaptoene click reaction to form a molecular skeleton of hydrogel, and calculating the gel formation time by a vial inversion method.
2. The production method according to claim 1, wherein the frequency of water change intervals in the dialysis of step (2) is sequentially 1 hx 3 times, 2 hx 3 times, 6 hx 4 times, 12 hx 8 times, and 24 hx 2 times.
3. The method according to claim 1, wherein the two precursors are dissolved in 5%, 10%, 20% PBS solution at pH 7.0, 7.4, 8.0 in step (3).
4. The method according to claim 1, wherein the thiol-ene click reaction occurs in step (3) under conditions of pH7.4 and 37 ℃.
5. The method according to claim 1, wherein the gel forming time calculated by the bottle inversion method in the step (3) is: the vials were occasionally inverted and the time when no liquid flow in the vial was observed was the gel formation time.
6. The method according to claim 1, wherein the catalyst used in steps (1) and (2) is scandium trifluoromethanesulfonate.
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