Thermosensitive hydrogel and preparation method thereof
By hydroxybutylating chitosan and introducing catechol groups, the adhesion properties of thermosensitive hydrogels are enhanced, solving the problem that existing hydrogels are not resistant to blood flow impact in blood vessels, thus achieving effective vascular occlusion and reducing the possibility of residual debris.
Patent Information
- Application Number
- CN202511831501.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-05
- Publication Date
- 2026-02-03
AI Technical Summary
Existing thermosensitive hydrogel embolization materials are not resistant to blood flow impact within blood vessels, which can easily lead to vascular recanalization and the formation of residual fragments, posing a health threat.
A thermosensitive hydrogel with excellent adhesive properties was prepared by hydroxylating and protecting chitosan with hydroxyl groups, introducing catechol groups, and utilizing π-π interactions and π-cation interactions to enhance adhesion.
The prepared thermosensitive hydrogel can be injected in liquid form at low temperatures and solidifies after entering the body. It has excellent adhesion properties, effectively blocking abnormal blood vessels and reducing residual debris and the possibility of vascular recanalization caused by blood flow impact.
Smart Images
Figure CN121445936A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of liquid embolizing agents, and in particular to a thermosensitive hydrogel and its preparation method. Background Technology
[0002] Transcatheter arterial embolization (TAE) is an important technique in interventional therapy. It involves injecting a biocompatible embolic material into a blood vessel through a catheter to reduce blood flow and block blood supply, thus achieving therapeutic goals. It can be used to treat clinical conditions including arteriovenous malformations, aneurysms, tumors, and endovascular hemostasis. With the development of endovascular interventional techniques, the application of endovascular embolization therapy is becoming increasingly widespread.
[0003] Among embolic materials, thermosensitive hydrogels respond to changes in ambient temperature. They are liquid at low temperatures for easy injection, and upon entering the body, undergo a sol-gel phase transition as body temperature rises, effectively sealing abnormal blood vessels. Thermosensitive hydrogels based on natural polysaccharide polymers, such as chitosan (CS), have attracted widespread attention due to their simple preparation process, low cost, good biocompatibility, biodegradability, and antibacterial properties.
[0004] However, existing thermosensitive hydrogels, after embolizing blood vessels, are not resistant to the impact of blood flow. The embolized blood vessels may recanalize, leading to the regeneration of tumor cells. Furthermore, residual fragments formed by the impact of blood flow can easily cause ectopic embolism, thus posing a threat to human health again. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this application provides a thermosensitive hydrogel and its preparation method to improve the problem that existing thermosensitive hydrogel embolization materials are not resistant to blood flow impact after embolization of blood vessels. In a first aspect, embodiments of this application provide a method for preparing a thermosensitive hydrogel, comprising the following steps: S1: Hydroxybutylation of chitosan is performed to obtain hydroxybutyl chitosan; S2: The first intermediate is obtained by protecting the hydroxyl groups on hydroxybutyl chitosan with a hydroxyl protecting agent; S3: The amino sites on the first intermediate are modified by hydroxylation of the hydroxylation modifier to obtain the second intermediate; S4: Obtain the third intermediate by grafting a chain transfer agent onto the hydroxyl group of the second intermediate; S5: A fourth intermediate is obtained by grafting a wet-adhesive functional monomer with a phenolic hydroxyl protecting agent onto the third intermediate using a chain transfer agent. S6: Remove the hydrophobic end group of the chain transfer agent on the fourth intermediate to obtain the fifth intermediate; S7: Remove the phenolic hydroxyl protectant and hydroxyl protectant from the fifth intermediate to obtain a thermosensitive hydrogel. In the above technical solution, this application uses chitosan as a matrix. First, chitosan is hydroxybutylated to form thermosensitive hydroxybutyl chitosan. Then, by protecting the hydroxyl groups on the hydroxybutyl chitosan and introducing new hydroxyl groups at the amino sites, a new hydroxyl group grafting chain transfer agent is used. Finally, a wet adhesive functional monomer with a phenolic hydroxyl group protectant is grafted onto the chain transfer agent, i.e., catechol groups are introduced. Through π-π interactions, π-cation interactions, and oxidative crosslinking between the catechol group segments and the vascular wall tissue, the prepared thermosensitive hydrogel exhibits excellent adhesive properties. In the preparation method of this application, by introducing a chain transfer agent, multiple catechol groups can be introduced through addition polymerization with the wet adhesive functional monomer, thereby greatly enhancing the adhesive properties of the thermosensitive hydrogel.
[0006] The thermosensitive hydrogel prepared in this application is liquid at low temperatures, making it easy to inject. After entering the human body, it transforms into a solidified gel as body temperature rises. The solidified gel exhibits excellent adhesion properties, effectively sealing abnormal blood vessels. Due to its good wet adhesion and embolization properties, it can effectively reduce the possibility of residual fragments formed by blood flow impact during vascular embolization, as well as the possibility of vascular recanalization caused by blood flow impact after embolization.
[0007] Secondly, embodiments of this application provide a thermosensitive hydrogel prepared by the preparation method of the first aspect of this application. The thermosensitive hydrogel includes a chitosan matrix and hydroxybutyl side chains and catechol group side chains grafted onto the chitosan matrix.
[0008] In the above technical solution, the thermosensitive hydrogel uses chitosan as a matrix, with hydroxyl sites modified by hydroxybutylation, chain transfer agents grafted onto amino sites, and further grafted with catechol groups. It remains liquid at low temperatures for easy injection, and upon entering the human body, it transforms into a solidified gel as body temperature rises. The solidified gel exhibits excellent adhesion properties, effectively sealing abnormal blood vessels. Due to its good wet adhesion and embolic properties, it reduces the possibility of residual fragments formed by blood flow impact during vascular embolization, and the possibility of vascular recanalization after embolization. Attached Figure Description
[0009] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0010] Figure 1A process flow diagram of a method for preparing a thermosensitive hydrogel provided in this application embodiment.
[0011] Figure 2 Optical images of the thermosensitive hydrogel prepared in Example 2 of this application in solution state (A) and gel state (B). Detailed Implementation
[0012] The following detailed description, with appropriate reference to the accompanying drawings, discloses embodiments of the thermosensitive hydrogel and its preparation method thereof, but some unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of actually identical structures may be omitted. This is to avoid unnecessarily lengthy descriptions and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided to enable those skilled in the art to fully understand this application and are not intended to limit the subject matter of the claims.
[0013] It should be noted that the terms "and / or" in this application, such as "feature 1 and / or feature 2", refer to the three cases where it can be "feature 1" alone, "feature 2" alone, or "feature 1" plus "feature 2".
[0014] In addition, in the description of this application, unless otherwise stated, "one or more" means two or more; the range of "numerical value a to numerical value b" includes the two endpoints "a" and "b"; and "unit of measurement" in "numerical value a to numerical value b + unit of measurement" represents the "unit of measurement" of both "numerical value a" and "numerical value b".
[0015] Figure 1 For a process flow diagram of a method for preparing a thermosensitive hydrogel provided in this application embodiment, please refer to [link / reference]. Figure 1 The preparation method of this thermosensitive hydrogel includes the following steps: S1: Hydroxybutylation of chitosan to obtain hydroxybutyl chitosan.
[0016] In this process, the alkyl hydroxyl sites of chitosan are chemically modified to form hydroxybutyl chitosan. The introduction of hydroxybutyl groups can endow the hydrogel with preliminary temperature-sensitive properties.
[0017] In some embodiments, step S1 includes: alkalizing chitosan and then reacting it with 1,2-epoxybutane for a substitution reaction; after the reaction is completed, separating, purifying, and drying the chitosan to obtain the hydroxybutyl chitosan.
[0018] Furthermore, the temperature of the substitution reaction can be 50℃~60℃, and the time can be 24h~72h.
[0019] Further, step S1 includes: alkalizing chitosan in sodium hydroxide solution, filtering, adding isopropanol and 1,2-epoxybutane, and carrying out a substitution reaction under heating and stirring conditions. After the reaction is completed, the chitosan is neutralized, filtered, and dried to obtain hydroxybutyl chitosan.
[0020] The chemical structural formula of the hydroxybutyl chitosan is shown in formula (1):
[0021] Equation (1).
[0022] S2: The first intermediate is obtained by protecting the hydroxyl groups on hydroxybutyl chitosan with a hydroxyl protecting agent.
[0023] In particular, since specific amino sites need to be chemically modified subsequently, using hydroxyl protectants to protect the hydroxyl groups to prevent them from being accidentally activated is beneficial to improving reaction specificity.
[0024] In some embodiments, step S2 includes: mixing hydroxybutyl chitosan and a hydroxyl protectant to carry out a substitution reaction to obtain a first intermediate.
[0025] In some embodiments, the hydroxyl protectant includes at least one of hydrocarbon ether protectants, silicone ether protectants, ester protectants, or acetal protectants.
[0026] Furthermore, the hydrocarbon ether protecting agent includes at least one of methyl ether protecting agents, tert-butyl ether protecting agents, benzyl ether protecting agents, p-methoxybenzyl ether protecting agents, or triphenylmethyl ether protecting agents.
[0027] Furthermore, the silyl ether protective agent includes at least one of the following: trimethyl silyl ether protective agent, triethyl silyl ether protective agent, dimethyl tert-butyl silyl ether protective agent, triisopropyl silyl ether protective agent, or tert-butyldiphenyl silyl ether protective agent.
[0028] Furthermore, the ester protecting agent includes at least one of the following: acetate protecting agent, neopentyl ester protecting agent, or benzoic acid ester protecting agent.
[0029] Furthermore, acetal protectants include at least one of methoxymethyl ether protectants, methoxyethoxymethyl ether protectants, benzyloxymethyl ether protectants, trimethylsilylethoxymethyl ether protectants, or tetrahydropyran ether protectants.
[0030] In some embodiments, the temperature of the substitution reaction is -10°C to 10°C, for example -10°C, -5°C, 0°C, 5°C, or 10°C. Preferably, the temperature of the substitution reaction is 0°C (ice-water bath).
[0031] In some embodiments, step S2 includes: dissolving hydroxybutyl chitosan in a solvent, then sequentially adding imidazole and trimethylchlorosilane (TMSCl), carrying out a substitution reaction under stirring conditions, and after the reaction is completed, diluting, washing, filtering, and drying to obtain trimethylsilyl ether-protected hydroxybutyl chitosan, i.e., the first intermediate.
[0032] Among them, imidazole is used as a basic nucleophilic catalyst; trimethylchlorosilane is used as a trimethylsilyl ether protecting agent. Its silicon atom undergoes a nucleophilic substitution reaction with the hydroxyl oxygen atom on hydroxybutyl chitosan to generate a trimethylsilyl ether protecting group. This group is a reversible hydroxyl protecting group and can be subsequently removed by acid removal.
[0033] When trimethylchlorosilane is used as a hydroxyl protecting agent, the chemical structure of the first intermediate is shown in formula (2):
[0034] Equation (2).
[0035] S3: The amino site on the first intermediate is hydroxylated by a hydroxylation modifier to obtain the second intermediate.
[0036] In some embodiments, step S3 includes: mixing the first intermediate, the hydroxylated modifier and the first condensing agent, and performing a dehydration condensation reaction to obtain the second intermediate.
[0037] In some embodiments, the hydroxylated modifier includes at least one of carboxylic acid compounds containing hydroxyl groups and their derivatives, epoxide alkane compounds, haloalcohol compounds, and aldehydes or ketones containing hydroxyl groups.
[0038] Furthermore, carboxylic acid compounds containing hydroxyl groups and their derivatives include at least one of glycolic acid, 3-hydroxypropionyl chloride, 4-hydroxybutyryl-4-hydroxybutyrate, or ethyl 3-hydroxypropionate.
[0039] Furthermore, the alkylene oxide compounds include at least one of ethylene oxide or propylene oxide.
[0040] Furthermore, the haloalcohols include at least one of 3-chloro-1-propanol or 5-chloro-2-pentanol.
[0041] Furthermore, the aldehyde or ketone compound containing a hydroxyl group includes at least one of 4-hydroxybutanal or 4-hydroxy-2-butanone.
[0042] Optionally, the first condensing agent comprises a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS).
[0043] In some embodiments, step S3 includes: dissolving the first intermediate in water, adjusting the pH value to less than 5, adding glycolic acid, water-soluble 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS), carrying out a dehydration condensation reaction under room temperature and light-protected conditions, and filtering, washing and drying after the reaction to obtain an ethanol-acylated second intermediate.
[0044] When glycolic acid is used as the hydroxylation modifier, the chemical structure of the second intermediate is shown in formula (3):
[0045] Equation (3).
[0046] S4: Obtain the third intermediate by grafting a chain transfer agent onto the hydroxyl group of the second intermediate.
[0047] In some embodiments, step S4 includes: mixing the second intermediate, the chain transfer agent, and the second condensing agent, and performing an esterification reaction to obtain the third intermediate.
[0048] Understandably, the chain transfer agent used in this application is a reversible addition-fragmentation chain transfer (RAFT) agent.
[0049] In some embodiments, the chain transfer agent includes at least one of trithiocarbonates, aromatic dithioesters, thiocarbamates, or thiocarbonates.
[0050] Further, the chain transfer agent includes at least one of S-1-dodecyl-S′(α,α′-dimethyl-α″-acetic acid) trithiocarbonate, S-dodecyl-S-(2-cyano-4-carboxyl)but-2-yl trithiocarbonate, 2-cyano-2-propylbenzodisulfide, 4-cyano-4-(dodecylthioalkylthiocarbonyl)thioalkylpentanoic acid, or 2,2'-(thiocarbonylbis(thioalkyldiyl))bis(2-methylpropionic acid).
[0051] Optionally, the second condensing agent includes dicyclohexylcarbodiimide (DCC).
[0052] In some embodiments, the molar ratio of the second intermediate, the chain transfer agent, and the second condensing agent is 1:(0.5~1.5):(0.5~2), such as 1:1:1.5, 1:0.5:0.5, 1:1.5:2, etc.
[0053] In some embodiments, step S4 includes: dissolving the second intermediate in a solvent, adding S-1-dodecyl-S′(α,α′-dimethyl-α″-acetic acid) trithiocarbonate (DDACT) and dicyclohexylcarbodiimide (DCC), and carrying out an esterification reaction at room temperature. After the reaction is completed, precipitation, washing, and drying are performed to obtain the third intermediate. At this time, the chemical structure of the third intermediate is shown in formula (4):
[0054] Equation (4).
[0055] S5: A wet-adhesive functional monomer with a phenolic hydroxyl protecting agent is grafted onto the third intermediate using a chain transfer agent to obtain the fourth intermediate.
[0056] In some embodiments, step S5 includes: mixing the third intermediate, the wet adhesion functional monomer with a phenolic hydroxyl protectant, and the initiator, and carrying out an addition polymerization reaction to obtain a wet adhesion functional precursor, namely the fourth intermediate.
[0057] It should be noted that the wet adhesive functional monomer with phenolic hydroxyl protecting agent in this application refers to the derivative after the phenolic hydroxyl group in the functional monomer containing catechol group (catechol structure, containing 2 phenolic hydroxyl groups) is protected and modified.
[0058] In some embodiments, the wet adhesion functional monomer includes acrylate compounds containing catechol groups.
[0059] Furthermore, the wet adhesion functional monomer includes at least one of 2-(3,4-dihydroxyphenyl)ethyl acrylate or 4-allyl catechol.
[0060] In some embodiments, the molar ratio of the third intermediate, the initiator, and the wet adhesion functional monomer is 1:0.1:(1~100), such as 1:0.1:30, 1:0.1:50, 1:0.1:100, etc.
[0061] In some embodiments, the temperature of the addition polymerization reaction is 30°C to 150°C, for example, 30°C, 50°C, 100°C, 150°C, etc.
[0062] In some embodiments, step S5 includes: dissolving the third intermediate in a solvent, adding the wet-adhesive functional monomer 2-(3,4-dihydroxyphenyl)ethyl acrylate and the initiator azobisisobutyronitrile (AIBN), and carrying out an addition polymerization reaction under heating conditions. After the reaction is complete, precipitation, washing, and drying are performed to obtain the fourth intermediate. At this time, the chemical structural formula of the fourth intermediate is shown in formula (5-1):
[0063] Equation (5-1).
[0064] In some embodiments, step S5 further includes: grafting a wet adhesion enhancing monomer with an amino protecting agent onto the third intermediate using a chain transfer agent; wherein the wet adhesion enhancing monomer comprises a flexible segment compound containing an amino group.
[0065] It should be noted that the wet adhesion-enhancing monomer with amino protecting agent in this application refers to the derivative after the amino group is protected and modified in a flexible segment monomer containing an amino group.
[0066] In this embodiment, by further grafting a wet adhesion-enhancing monomer with an amino protectant to introduce hydrophilic amino flexible segments, the wetting ability of the catechol groups can be effectively improved, thereby enhancing the adhesion performance of the catechol groups. Amino ionization can also form electrostatic interactions with the cell wall, further enhancing adhesion. Furthermore, the introduction of hydrophilic amino flexible segments improves the hydrophilicity of the thermosensitive chitosan system, increasing the low critical solution temperature (LCST) of the thermosensitive embolic agent, preventing premature solidification at room temperature, and facilitating product storage, transportation, and preparation before clinical use. Simultaneously, the strong hydrophilicity of the amino functional groups can effectively regulate the solidification time of the thermosensitive gel system, thereby extending the in vitro operation time before the liquid embolic agent solidifies.
[0067] In some embodiments, the wet adhesion-enhancing monomer comprises an acrylate compound containing an amino functional group.
[0068] Furthermore, the wet adhesion-enhancing monomer includes at least one of 3-aminopropyl-2-enoate or 4-pentenyl-1-amine.
[0069] Furthermore, the molar ratio of the wet adhesion functional monomer to the wet adhesion enhancing monomer is 1:(0~10), for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, etc. Even further, the molar ratio of the wet adhesion functional monomer to the wet adhesion enhancing monomer is 1:(1~4).
[0070] Further, step S5 includes: dissolving the third intermediate in a solvent, adding 2-(3,4-dihydroxyphenyl)ethyl acrylate, 3-aminopropyl-2-enoate, and azobisisobutyronitrile (AIBN), and carrying out an addition polymerization reaction under heating conditions. After the reaction is complete, precipitation, washing, and drying are performed to obtain the fourth intermediate. At this point, the chemical structural formula of the fourth intermediate is shown in formula (5-2):
[0071] Equation (5-2).
[0072] S6: Remove the hydrophobic end group of the chain transfer agent on the fourth intermediate to obtain the fifth intermediate.
[0073] In some embodiments, step S6 includes: removing the hydrophobic end group of the chain transfer agent from the fourth intermediate using an ammonolysis reagent to obtain a mercapto-terminated polymer; and then obtaining the hydroxyl-terminated fifth intermediate through a click chemistry reagent addition reaction.
[0074] In some embodiments, the chemical reaction reagents include olefin compounds and their derivatives.
[0075] Furthermore, the chemical reaction reagent includes at least one of allyl alcohol or hydroxyethyl acrylate.
[0076] In some embodiments, the ammonolysis reagent includes liquid ammonia, ammonia water, urea, ammonium salts (such as ammonium carbonate), and organic amines (such as n-hexylamine). Preferably, the ammonolysis reagent is an organic amine.
[0077] Further, step S6 includes: dissolving the fourth intermediate in a solvent, adding an ammonolysis reagent, a click chemistry reagent and a catalyst, mixing them and carrying out an ammonolysis reaction and an addition reaction, and after the reaction is completed, precipitating, purifying and drying to obtain the fifth intermediate.
[0078] In step S6, corresponding to the fourth intermediate in formula (5-1), the chemical structural formula of the fifth intermediate obtained is shown in formula (6-1):
[0079] Equation (6-1).
[0080] In step S6, corresponding to the fourth intermediate in formula (5-2), the chemical structural formula of the fifth intermediate obtained is shown in formula (6-2):
[0081] Equation (6-2).
[0082] S7: Remove the phenolic hydroxyl protectant and hydroxyl protectant from the fifth intermediate to obtain a thermosensitive hydrogel. In some embodiments, step S7 further includes removing the amino protecting agent from the fifth intermediate.
[0083] In some embodiments, step S7 includes: mixing the fifth intermediate with an acid hydrolysis reagent to carry out a hydrolysis reaction to obtain a thermosensitive hydrogel.
[0084] In some embodiments, the acid hydrolysis reagent includes a 95% trifluoroacetic acid solution.
[0085] In step S7, the chemical structure of the thermosensitive hydrogel obtained corresponding to the fifth intermediate of formula (6-1) is shown in formula (7-1):
[0086] Equation (7-1).
[0087] In step S7, the chemical structure of the thermosensitive hydrogel obtained corresponding to the fifth intermediate in formula (6-2) is shown in formula (7-2):
[0088] Equation (7-2).
[0089] This application also provides a thermosensitive hydrogel prepared by the above-described method. The thermosensitive hydrogel comprises a chitosan matrix, and hydroxybutyl side chains and catechol side chains grafted onto the chitosan matrix.
[0090] In some embodiments, the thermosensitive hydrogel further includes flexible side chains containing amino groups grafted onto a chitosan matrix.
[0091] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0092] Example 1 This embodiment provides a thermosensitive hydrogel, the preparation method of which includes the following steps: S1: Take 10g of chitosan (degree of deacetylation ≥ 95%), add sodium hydroxide solution, and alkalize under nitrogen protection at room temperature for 24h. Discard the excess alkali solution, add 100mL of isopropanol, and add 200mL of 1,2-epoxybutane with magnetic stirring. Heat to 50℃ and stir for 72h. After the reaction, filter to remove the supernatant and collect the precipitate. Dissolve the precipitate in deionized water and adjust the pH to 7.0 with 10% acetic acid. Finally, use acetone to precipitate hydroxybutyl chitosan, and dry in a 40℃ oven to obtain hydroxybutyl chitosan.
[0093] S2: The hydroxybutyl chitosan obtained in step S1 was dissolved in dry DMF. Imidazole and trimethylchlorosilane (TMSCl) were added in an ice-water bath (0°C) at a molar ratio of hydroxybutyl chitosan:imidazolium:TMSCl = 1:3:3. After stirring at 0°C for 1.5 h, the mixture was diluted with ethyl acetate (EtOAc), washed with water and then with brine. The resulting product was then vacuum dried at 40°C to obtain trimethylsilyl ether-protected hydroxybutyl chitosan (HBCS-TMS), which is the first intermediate.
[0094] S3: The first intermediate obtained in step S2 was dissolved in deionized water, and the pH was adjusted to less than 5 using hydrochloric acid and sodium hydroxide solution. Glycolic acid (GA), water-soluble 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (HS) were added in a molar ratio of first intermediate:GA:EDC:HS = 1:1.5:0.1:0.2. The reaction was carried out at room temperature in the dark for 12 hours. After the reaction, the pH was adjusted to 7.0 using sodium hydroxide, and the product was washed four times alternately with acetone and deionized water. The resulting product was dried under vacuum at 60°C to obtain the second intermediate.
[0095] S4: The second intermediate obtained in step S3 was dissolved in dry DMF, and S-1-dodecyl-S′(α,α′-dimethyl-α″-acetic acid) trithiocarbonate (DDACT) and dicyclohexylcarbodiimide (DCC) were added. The molar ratio of the three substances was second intermediate:DDACT:DCC = 1:1:1.5. The mixture was reacted at room temperature for 24 h. The resulting mixture was poured into ice water, the precipitate was collected, and the product was washed thoroughly with acetone. The product was then dried under vacuum at 60 °C to obtain the third intermediate.
[0096] S5: The third intermediate obtained in step S4 was dissolved in anhydrous DMF and magnetically stirred under argon protection. Azobisisobutyronitrile (AIBN) and a wet adhesive functional monomer (2-(3,4-dihydroxyphenyl) ethyl acrylate) with a phenolic hydroxyl protecting agent were added. The molar ratio of the three components was third intermediate: AIBN: wet adhesive functional monomer = 1:0.1:30. The reaction was carried out at 70°C for 9 hours. After the reaction was completed, the product was precipitated with diethyl ether and then extracted with acetone. The resulting product was dried under vacuum at 60°C to obtain the fourth intermediate.
[0097] S6: The fourth intermediate prepared in step S5 was dissolved in anhydrous tetrahydrofuran (THF) and magnetically stirred under argon protection. Hexylamine, azobisisobutyronitrile (AIBN), and allyl alcohol were added in a molar ratio of fourth intermediate: hexylamine: AIBN: allyl alcohol = 1:1.5:0.1:1.5. The reaction was carried out at room temperature for 6 hours. After the reaction was complete, the product was precipitated with diethyl ether and then extracted with acetone. The resulting product was dried under vacuum at 60°C to obtain the fifth intermediate.
[0098] S7: The fifth intermediate obtained in step S6 was dissolved in dichloromethane (DCM), and 95% trifluoroacetic acid (TFA) solution was added. The volume ratio of dichloromethane (DCM) to trifluoroacetic acid (TFA) was 80:20. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the product was washed with water and collected. The product was then washed with ethanol and extracted. The product was dried under vacuum at 60°C to obtain the thermosensitive hydrogel.
[0099] Example 2 This embodiment provides a thermosensitive hydrogel, the preparation method of which differs from that of Example 1 in that: In step S5, a wet adhesion-enhancing monomer (3-aminopropyl-2-enoate) with an amino protecting agent is also added. The molar ratio of the wet adhesion functional monomer to the wet adhesion-enhancing monomer is 1:1.
[0100] Example 3 This embodiment provides a thermosensitive hydrogel, the preparation method of which differs from that of Example 2 in that: In step S5, the molar ratio of the wet adhesion functional monomer and the wet adhesion enhancing monomer is 1:2.
[0101] Example 4 This embodiment provides a thermosensitive hydrogel, the preparation method of which differs from that of Example 2 in that: In step S5, the molar ratio of the wet adhesion functional monomer and the wet adhesion enhancing monomer is 1:4.
[0102] Comparative Example 1 This comparative example provides a thermosensitive hydrogel, the preparation method of which includes the following steps: Take 10g of chitosan (degree of deacetylation ≥ 95%), add sodium hydroxide solution, and alkalize under nitrogen protection at room temperature for 24h. Discard the excess alkali solution, add 100mL isopropanol, and add 200mL 1,2-epoxybutane with magnetic stirring. Heat to 50℃ and stir the reaction. After 72h, filter to remove the supernatant and collect the precipitate. Dissolve the precipitate in deionized water and adjust the pH to 7.0 with 10% acetic acid. Finally, use acetone to precipitate hydroxybutyl chitosan, and dry in an oven at 40℃ to obtain hydroxybutyl chitosan.
[0103] Comparative Example 2 This comparative example provides a thermosensitive hydrogel, the preparation method of which includes the following steps: The hydroxybutyl chitosan prepared in Comparative Example 1 was added to hydrochloric acid solution and stirred to dissolve, thus obtaining a hydroxybutyl chitosan solution.
[0104] Separately, 3,4-dihydroxyphenylalanine was dissolved in hydrochloric acid solution, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added to obtain an activated solution of 3,4-dihydroxyphenylalanine. The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and 3,4-dihydroxyphenylalanine was 1:2:4.
[0105] The above-mentioned 3,4-dihydroxyphenylalanine activation solution was added dropwise to the above-mentioned hydroxybutyl chitosan solution at a volume ratio of 1:4. The pH was adjusted to 5. Under nitrogen protection, the reaction was stirred for 20 hours. Then, glycine solution was added to terminate the reaction. After dialysis and freeze-drying, modified hydroxybutyl chitosan was obtained.
[0106] Performance testing and results analysis 1. Temperature sensitivity test The thermosensitive hydrogels prepared in Examples 1-4, Comparative Examples 1 and 2 were dissolved in deionized water at 5°C to prepare 3% gel solutions. A suitable amount of each gel solution was placed in a 10mL vial and placed in a constant-temperature circulating water system for thermosensitivity testing. The experiment employed a gradient heating process, where the temperature was increased from 5°C to 40°C by raising it by 1°C, holding the temperature for 1 minute, and recording the phase transition temperature and time during the experiment. The test results are shown in Table 1 and... Figure 2 As shown: Table 1. Results of thermosensitive hydrogel test
[0107] Figure 2 Optical images of the thermosensitive hydrogel prepared in Example 2 of this application in solution state (A) and gel state (B). From Figure 2 As can be seen from Table 1, the hydroxybutyl chitosan in Comparative Example 1, as well as the thermosensitive hydrogels obtained after further modification in Examples 1-4 and Comparative Example 2, all have good thermosensitive properties.
[0108] Compared to Comparative Examples 1 and 2, the gel phase transition temperature of Example 1 was lower, which may be due to the addition of the modified wet adhesion functional monomer, which reduced the phase transition temperature of the gel solution. In Examples 2 to 4, as the proportion of the modified wet adhesion functional monomer increased, both the phase transition temperature and the phase transition time of the gel solution increased.
[0109] 2. Adhesion and injectability tests The thermosensitive hydrogels prepared in Examples 1-4 and Comparative Examples 1-2 were dissolved in deionized water at 5°C to prepare 3% gel solutions. 5 mL of each gel solution was placed in a 10 mL vial, and the vials were sealed with rubber stoppers and aluminum caps. After moist heat sterilization, the vials were placed at 5°C until they reverted to a gel solution before use.
[0110] Take aluminum plates, pig skin and pig arteries respectively, wash them with PBS solution, cut them into strips of 3cm×1cm, and place them in beakers containing PBS solution (pH=7.4, 0.8mmol / L) at a constant temperature of 37℃.
[0111] Using a syringe, the sterilized gel solutions from Examples 1-4, Comparative Examples 1 and 2 were drawn up and connected to a 2.7F microcatheter. Each gel solution was injected onto the surface of the elongated aluminum plate, pig skin, and pig artery, respectively, while simultaneously observing the injectability (catheter permeability). After standing for 5 minutes to allow the gel to completely solidify, the elongated aluminum plate, pig skin, and pig artery were removed, and their adhesion properties were quickly tested using a universal testing machine.
[0112] The test results are shown in Table 2: Table 2. Results of injectability and adhesion tests for thermosensitive hydrogels.
[0113] As can be seen from Table 2, the gel solutions in Examples 1 to 4, as well as Comparative Examples 1 and 2, can all pass smoothly through the microcatheter, indicating that they all have good injectability.
[0114] All sample gel solutions were able to solidify on aluminum plates, pig skin, and pig arteries. However, according to the mechanical test data of adhesion properties, the samples of Comparative Example 1 and Comparative Example 2 had almost no adhesion properties to the three experimental substrates, while the samples of each embodiment had good adhesion properties on aluminum plates, pig skin, and pig arteries. This indicates that the modification scheme in this application can effectively increase the wet adhesion of the thermosensitive hydrogel material.
[0115] The adhesion performance test results of Examples 1 and Examples 1-3 show that the adhesion performance of Examples 2-4 is better than that of Example 1, and Example 3 has better wet adhesion performance, indicating that the introduction of flexible side chains containing amino groups can further increase the wet adhesion of thermosensitive hydrogel materials.
[0116] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.
Claims
1. A method for preparing a thermosensitive hydrogel, characterized in that, Includes the following steps: S1: Hydroxybutylation of chitosan is performed to obtain hydroxybutyl chitosan; S2: Hydroxyl groups on the hydroxybutyl chitosan are protected by a hydroxyl protectant to obtain the first intermediate; S3: The amino sites on the first intermediate are modified by hydroxylation of the hydroxylation modifier to obtain the second intermediate; S4: Obtain the third intermediate by grafting a chain transfer agent onto the hydroxyl group of the second intermediate; S5: A fourth intermediate is obtained by grafting a wet-adhesive functional monomer with a phenolic hydroxyl protectant onto the third intermediate using the chain transfer agent; S6: Remove the hydrophobic end group of the chain transfer agent on the fourth intermediate to obtain the fifth intermediate; S7: Remove the phenolic hydroxyl protectant and the hydroxyl protectant from the fifth intermediate to obtain the thermosensitive hydrogel.
2. The preparation method according to claim 1, characterized in that, Step S2 includes: mixing the hydroxybutyl chitosan and the hydroxyl protecting agent to carry out a substitution reaction to obtain the first intermediate; and / or, In step S2, the hydroxyl protecting agent includes at least one of hydrocarbon ether protecting agents, silicone ether protecting agents, ester protecting agents, or acetal protecting agents; Optionally, the hydrocarbon ether protective agent includes at least one of methyl ether protective agents, tert-butyl ether protective agents, benzyl ether protective agents, p-methoxybenzyl ether protective agents, or triphenylmethyl ether protective agents; Optionally, the silyl ether protective agent includes at least one of trimethyl silyl ether protective agent, triethyl silyl ether protective agent, dimethyl tert-butyl silyl ether protective agent, triisopropyl silyl ether protective agent, or tert-butyldiphenyl silyl ether protective agent; Optionally, the ester protective agent includes at least one of acetate protective agents, neopentyl ester protective agents, or benzoic acid ester protective agents; Optionally, the acetal protective agent includes at least one of methoxymethyl ether protective agents, methoxyethoxymethyl ether protective agents, benzyloxymethyl ether protective agents, trimethylsilylethoxymethyl ether protective agents, or tetrahydropyran ether protective agents; Optionally, in step S2, the temperature of the substitution reaction is -10℃ to 10℃.
3. The preparation method according to claim 1, characterized in that, Step S3 includes: mixing the first intermediate, the hydroxylated modifier, and the first condensing agent, and performing a dehydration condensation reaction to obtain the second intermediate; and / or, In step S3, the hydroxylated modifier includes at least one of the following: carboxylic acid compounds containing hydroxyl groups and their derivatives, epoxide alkane compounds, haloalcohol compounds, aldehyde compounds containing hydroxyl groups, or ketone compounds containing hydroxyl groups. Optionally, the carboxylic acid compounds containing hydroxyl groups and their derivatives include at least one of glycolic acid, 3-hydroxypropionyl chloride, 4-hydroxybutyryl-4-hydroxybutyrate, or ethyl 3-hydroxypropionate; Optionally, the alkylene oxide compound includes at least one of ethylene oxide or propylene oxide; Optionally, the haloalcohol compound includes at least one of 3-chloro-1-propanol or 5-chloro-2-pentanol; Optionally, the aldehyde compound containing a hydroxyl group includes 4-hydroxybutyraldehyde; Optionally, the ketone compound containing a hydroxyl group includes 4-hydroxy-2-butanone.
4. The preparation method according to claim 1, characterized in that, Step S4 includes: mixing the second intermediate, the chain transfer agent, and the second condensing agent, and performing an esterification reaction to obtain the third intermediate; and / or, In step S4, the chain transfer agent includes at least one of trithiocarbonates, aromatic dithioesters, thiocarbamates, or thiocarbonates. Optionally, the molar ratio of the second intermediate, the chain transfer agent, and the second condensing agent is 1:(0.5~1.5):(0.5~2). Optionally, the chain transfer agent comprises at least one of S-1-dodecyl-S′(α,α′-dimethyl-α″-acetic acid) trithiocarbonate, S-dodecyl-S-(2-cyano-4-carboxyl)but-2-yl trithiocarbonate, 2-cyano-2-propylbenzodisulfide, 4-cyano-4-(dodecylthioalkylthiocarbonyl)thioalkylpentanoic acid, or 2,2'-(thiocarbonylbis(thionediyl))bis(2-methylpropionic acid).
5. The preparation method according to claim 1, characterized in that, Step S5 includes: mixing the third intermediate, the wet adhesive functional monomer with a phenolic hydroxyl protecting agent, and an initiator, and carrying out an addition polymerization reaction to obtain the fourth intermediate; and / or, In step S5, the wet adhesion functional monomer includes acrylate compounds containing catechol groups; Optionally, the wet adhesion functional monomer includes at least one of 2-(3,4-dihydroxyphenyl)ethyl acrylate or 4-allyl catechol; Optionally, the molar ratio of the third intermediate, the initiator, and the wet adhesion functional monomer is 1:0.1:(1~100). Optionally, in step S5, the temperature of the addition polymerization reaction is 30°C to 150°C.
6. The preparation method according to claim 1, characterized in that, Step S5 further includes: grafting a wet adhesion enhancing monomer with an amino protecting agent onto the third intermediate via the chain transfer agent; wherein the wet adhesion enhancing monomer comprises a flexible segment compound containing an amino group; Optionally, the wet adhesion-enhancing monomer includes acrylate compounds containing amino groups; Optionally, the wet adhesion-enhancing monomer includes at least one of 3-aminopropyl-2-enoate or 4-pentenyl-1-amine; Optionally, the molar ratio of the wet adhesion functional monomer to the wet adhesion enhancing monomer is 1:(0~10).
7. The preparation method according to claim 1 or 6, characterized in that, Step S6 includes: removing the hydrophobic end groups of the chain transfer agent from the fourth intermediate using an ammonolysis reagent to obtain a mercapto-terminated polymer; and then performing an addition reaction with a click chemistry reagent to obtain the hydroxyl-terminated fifth intermediate. Optionally, the click chemical reaction reagent includes olefin compounds and their derivatives; Optionally, the click chemical reaction reagent includes at least one of allyl alcohol or hydroxyethyl acrylate.
8. The preparation method according to claim 6, characterized in that, Step S7 further includes: removing the amino protecting agent from the fifth intermediate; and / or, Step S7 includes: mixing the fifth intermediate with an acid hydrolysis reagent and carrying out a hydrolysis reaction to obtain the thermosensitive hydrogel; Optionally, the acid hydrolysis reagent includes a 95% trifluoroacetic acid solution.
9. A thermosensitive hydrogel, prepared by the method according to any one of claims 1 to 8, characterized in that, It includes a chitosan matrix, and hydroxybutyl side chains and catechol side chains grafted onto the chitosan matrix.
10. The thermosensitive hydrogel according to claim 9, characterized in that, Also includes: Flexible side chains containing amino groups grafted onto the chitosan matrix.