Temperature-responsive injectable double network hydrogel and preparation method and application thereof

Abstract

This invention relates to a temperature-responsive injectable hydrogel, its preparation method, and its application, belonging to the field of dual-network hydrogel construction and application technology. The hydrogel method includes: (1) reacting chitosan oligosaccharide with an allyl reagent to obtain allyl chitosan oligosaccharide; (2) crosslinking and copolymerizing allyl chitosan oligosaccharide with N-isopropylacrylamide under the action of a polymerization initiator to obtain a temperature-sensitive microgel; (3) loading genipin onto the temperature-sensitive microgel to obtain component A; (4) loading β-glycerophosphate onto the temperature-sensitive microgel to obtain component B; (5) mixing component A, component B, and chitosan solution to obtain a temperature-responsive injectable dual-network hydrogel for drug delivery. The temperature-responsive injectable hydrogel provided by this invention satisfies both injectability and strength stability requirements, and can be applied to, but is not limited to, long-term drug delivery via skin wounds or implantation, possessing high application value.

Description

Technical Field

[0001] This invention relates to the field of chitosan modification and application technology, specifically to temperature-responsive injectable dual-network hydrogels, their preparation methods, and applications. Background Technology

[0002] Compared to traditional solid hydrogels, injectable hydrogels have garnered significant attention in clinical and research fields in recent years due to their ability to adapt to irregular shapes and deep wounds, and their potential for localized, targeted, and minimally invasive application to non-superficial tissues. Thermosensitive hydrogels can undergo a sol-gel transition in response to changes in ambient temperature, especially those with a lower critical solution temperature (LCST). Below the LCST, they exist as a solution; above the LCST, they gel. When the LCST of a thermosensitive hydrogel is below human body temperature, it remains in a solution state at room temperature and is injectable. At physiological human temperatures, it transforms into a gel, making it one of the most promising injectable hydrogels in the biomedical field.

[0003] Poly(N-isopropylacrylamide) (PNIPAM) is a typical thermosensitive polymer of this type, easily delivered in liquid form and rapidly transforming into a solid gel at in vivo temperatures, showing promising application prospects. However, achieving effective control of this transformation process remains challenging. On the one hand, excessively rapid gelation and hydrophobic transition can lead to clogging of the infusion device and water retention, increasing the complexity of surgical procedures. On the other hand, the gel durability, biocompatibility, and biodegradability of PNIPAM for long-term in vivo application or implantation are also key areas of focus in clinical translational research. Therefore, ensuring both rapid and controllable gelation while achieving long-term biocompatibility has become a current research priority.

[0004] Chinese patent documents CN 115838536 A and CN115845122A disclose a high-strength dual-network hydrogel method, which uses natural polymers such as hyaluronic acid and gelatin to provide a second network, improving biocompatibility and gel durability. However, this method significantly impacts the temperature-sensitive properties of PNIPAM, thus affecting the injectable gel performance and limiting its in vivo application. In vivo in-situ gel enhancement strategies, which introduce a second network in situ within the hydrogel to enhance its mechanical properties, not only maintain the injectability of the hydrogel but also impart sufficient mechanical strength to ensure durability for in vivo application. However, this method also faces challenges, such as uneven mixing due to dual-injection operations and the complexity of surgical procedures. Therefore, given the urgent need for safer and more efficient gel enhancement technologies, it is necessary to develop innovative in-situ crosslinking strategies to address these issues. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides a temperature-responsive injectable dual-network hydrogel, its preparation method, and its applications. The temperature-responsive dual-network hydrogel is composed of a microgel constructed from N-isopropylacrylamide (NIPAM) and chitosan, along with macromolecular chitosan. Through the hydrophobic transformation of the temperature-sensitive microgel under human body temperature conditions, the loaded β-glycerophosphate sodium and genipin are released in situ sequentially, first forming a physical cross-linked network, and then a chemical cross-linked network. This avoids the problems of surgical complexity, biocompatibility, and gel persistence associated with existing temperature-sensitive hydrogel systems.

[0006] In one aspect of the present invention, a method for preparing a temperature-responsive injectable dual-network hydrogel is provided, comprising the following steps:

[0007] (1) Chitosan oligosaccharide reacts with an allyl reagent to obtain allyl chitosan oligosaccharide; (2) Allyl chitosan oligosaccharide and N-isopropylacrylamide were crosslinked and copolymerized under the action of a polymerization initiator to obtain a thermosensitive microgel; (3) Genipin was loaded onto the thermosensitive microgel to obtain a genipin-loaded microgel; the genipin-loaded microgel was dissolved in water to obtain component A; (4) Thermosensitive microgel is loaded with sodium β-glycerophosphate to obtain thermosensitive microgel loaded with sodium β-glycerophosphate; the thermosensitive microgel loaded with sodium β-glycerophosphate is dissolved in water to obtain component B; (5) Mix component A and component B with a polymer solution to obtain a temperature-responsive injectable hydrogel; wherein the polymer contains amino or amino and carboxyl groups.

[0008] This invention utilizes chitosan oligosaccharide to crosslink N-isopropylacrylamide, constructing a chitosan oligosaccharide-co-N-isopropylacrylamide thermosensitive microgel. This improves the phase transition rate and biocompatibility of traditional poly-N-isopropylacrylamide, allowing the loaded hydrophilic β-glycerophosphate sodium to be released at a relatively stable rate under human body temperature conditions. This facilitates the formation of a more uniform physical crosslinking network with chitosan, resulting in faster physical gelation and addressing the difficulty of injectable surgical procedures. Meanwhile, the less hydrophilic genipin is released at a slower rate, forming a chemical crosslinking network with chitosan. This slower chemical crosslinking results in better stability, addressing the issue of long-term implantation stability.

[0009] In some embodiments of the present invention, the method for preparing chitosan temperature-responsive injectable dual-network hydrogels satisfies at least one of the following conditions: The molecular weight range of the chitosan oligosaccharide is 800 Da to 10000 Da; the allyl reagent includes, but is not limited to, one or more of acrylic anhydride, methacrylic anhydride, acryloyl chloride, and methacryloyl chloride; the polymerization initiator includes, but is not limited to, one or more of potassium persulfate, ammonium persulfate, azobisisobutyronitrile, cuprous chloride, and dithioester; the chitosan solution includes, but is not limited to, one or more of chitosan, carboxymethyl chitosan, and gelatin, and the molecular weight range of the polymer is 10000 Da to 3000000 Da.

[0010] In the preparation method of allyl chitosan oligosaccharide in step (1), the reaction solvent is one or more of water, methanol, and DMSO (dimethyl sulfoxide); the mass ratio of chitosan oligosaccharide, allyl reagent, and solvent is 1:(0.1~10):(0~500), preferably 1:(1~4):(0~100); the reaction temperature is 0~100 ℃, preferably 40~60 ℃; the reaction time is 0.1~20 h, preferably 4~12 h; and the method also includes a purification step, which includes, but is not limited to, one or more of rotary evaporation, solvent precipitation washing, and drying; the solvent in the solvent precipitation washing is one or more of acetone, ethanol, methanol, and ethyl acetate.

[0011] In the preparation method of the microgel in step (2), the reaction solvent is one or more of water, methanol, and DMSO; the mass ratio of allyl chitosan, N-isopropylacrylamide, and initiator is 1:(0.1~20):(0.01~1), preferably 1:(1~3):(0.04~1); the reaction atmosphere is an inert gas, which is nitrogen or argon; the reaction temperature is 0 ℃~100℃, preferably 60~90 ℃; the reaction time is 0.5 h~10 h, preferably 2 h~6 h; and the method also includes a purification step, which includes, but is not limited to, one or more of water dialysis, solvent washing, and freeze drying; during dialysis, the molecular weight cutoff of the dialysis bag used is 800-15000; the solvent used for solvent washing is one or more of acetone, ethanol, methanol, and ethyl acetate. The method for preparing thermosensitive microgels is as follows: allyl chitosan oligosaccharide and N-isopropylacrylamide are placed in a reaction vessel, and after the gas in the reaction vessel is fully exchanged with an inert gas, a free radical initiator is added, and the mixture is heated to carry out cross-linking polymerization. After dialysis separation and purification, the thermosensitive microgels are obtained by freeze drying.

[0012] The method for loading genipin onto the microgel in step (3) is as follows: dissolve genipin in an organic solvent to obtain a genipin solution; then mix the genipin solution with the thermosensitive microgel thoroughly by shaking, remove the organic solvent by rotary evaporation, and wash to obtain the genipin-loaded microgel; the organic solvent includes, but is not limited to, ethanol and DMSO; the ratio of genipin to organic solvent is (0.001~1) g: 20 mL, preferably (0.01~0.1) g: 20 mL, more preferably 0.1 g: 20 mL; the mass ratio of thermosensitive microgel to genipin is 1: (0.001~1), preferably 1: (0.01~1), more preferably 1: (0.1~0.5); washing is performed with an organic solvent, and the number of washing cycles is 3; the organic solvent is one or more of acetone, ethanol, methanol, and ethyl acetate; the concentration of the thermosensitive microgel in component A is 0.001~1 g / mL, preferably 0.01~0.5 g / mL, more preferably 0.1 g / mL.

[0013] The method for loading sodium β-glycerophosphate onto the microgel in step (4) is as follows: Thermosensitive microgel is dissolved in water to obtain a thermosensitive microgel solution; the thermosensitive microgel solution and sodium β-glycerophosphate aqueous solution are thoroughly mixed by shaking, centrifugation, washing, and freeze-drying to obtain a thermosensitive microgel loaded with sodium β-glycerophosphate; the mass ratio of thermosensitive microgel to sodium β-glycerophosphate is 1:(0.001~10), preferably 1:(0.01~5), more preferably 1:(0.5~2); the precipitate is washed with water three times; the concentration of thermosensitive microgel in the thermosensitive microgel solution is 0.001~1 g / mL, preferably 0.01~0.5 g / mL, more preferably 0.1 g / mL; the concentration of sodium β-glycerophosphate aqueous solution is 0.001~1 g / mL, preferably 0.01~0.5 g / mL, more preferably 0.05 g / mL; the concentration of thermosensitive microgel in component B is 0.01~1 g / mL, preferably 0.05~0.5 g / mL. g / mL, more preferably 0.1 g / mL.

[0014] In the preparation method of injectable hydrogel in step (5), the mass ratio of thermosensitive microgel in component A, thermosensitive microgel in component B, and polymer in polymer solution is 1:(0.1~1):(0.1~1), preferably 1:1:0.5; the chitosan solution is a chitosan aqueous solution with a mass concentration of 0.1~10%, preferably 2~4%; the three solutions, component A, component B, and polymer solution, are thoroughly shaken and mixed. When chitosan is used, the pH of the chitosan solution is 6-6.5. The chitosan solution is prepared by dissolving chitosan in hydrochloric acid, then adding sodium hydroxide to adjust the pH to 6-6.5. The hydrochloric acid is a hydrochloric acid solution with a concentration of 0.001-1 M, preferably 0.01 M. The sodium hydroxide is a sodium hydroxide solution with a concentration of 0.001-1 M, preferably 0.1 M. When carboxymethyl chitosan is used, the carboxymethyl chitosan solution is prepared by dissolving carboxymethyl chitosan in water.

[0015] In another aspect of the present invention, the present invention provides a temperature-responsive injectable dual-network hydrogel prepared by the above-described preparation method.

[0016] In another aspect, the present invention proposes the application of the above-mentioned temperature-responsive injectable dual-network hydrogel in skin and mucous membranes, wound dressings, in vivo implantation and drug delivery.

[0017] The temperature-responsive injectable hydrogel provided by this invention releases sodium β-glycerophosphate to form a physical cross-linked network through the sol-gel phase transition of the temperature-sensitive microgel under body temperature conditions, and then gradually releases genipin to form a chemical cross-linked network. This dual-network hydrogel meets both the requirements for injectability and the requirements for strength and stability, and can be applied to, but is not limited to, long-term drug delivery in skin wounds and implantation in the body, and has high application value.

[0018] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention uses chitosan oligosaccharide to crosslink N-isopropylacrylamide to construct chitosan oligosaccharide copolymer N-isopropylacrylamide thermosensitive microgel, which improves the phase transition rate and biocompatibility of traditional poly N-isopropylacrylamide.

[0019] 2. By using the microgel loaded with sodium β-glycerophosphate, under human body temperature conditions, the improved phase transition rate allows the highly hydrophilic sodium β-glycerophosphate to be released at a relatively stable rate, which is beneficial for forming a more uniform physical cross-linking network with chitosan and other substances; the physical cross-linking speed is relatively fast, meeting the requirements for injectability.

[0020] 3. Genipin is loaded onto the microgel described above. Genipin, which has low hydrophilicity, is released at a slower rate and forms a chemical cross-linking network with chitosan. The chemical cross-linking has good stability and meets the requirements for long-term implantation. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a transmission electron microscope image of the microgel provided in Example 16 of the present invention.

[0023] Figure 2 The results are the phase transition rate test results of the microgel provided in Example 16 of this invention.

[0024] Figure 3 These are photographs of the temperature-responsive injectable hydrogel provided in Embodiment 20 of the present invention in its sol state at room temperature and in its gel state after incubation at 37°C for 5 min.

[0025] Figure 4 This is a scanning electron microscope image of the temperature-responsive injectable hydrogel provided in Embodiment 20 of the present invention after incubation at 37°C for 5 min.

[0026] Figure 5 The results are obtained from the rheometer measurement of the temperature-responsive injectable hydrogel provided in Embodiment 20 of the present invention. Detailed Implementation

[0027] The present invention will be described below through specific embodiments. Those skilled in the art will understand that the specific embodiments described below are merely for illustrative purposes and do not limit the scope of the invention in any way. Any product identical or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0028] Furthermore, in the following embodiments, unless otherwise specified, all materials and equipment used are commercially available. If specific processing conditions and methods are not explicitly described in the later embodiments, conditions and methods known in the art can be used for processing.

[0029] In the examples below, the molecular weight of chitosan oligosaccharide is 5000 kDa, and the molecular weight of chitosan and carboxymethyl chitosan is 300 kDa.

[0030] Example 1 0.1 g of chitosan oligosaccharide was dissolved in 10 mL of deionized water, and 0.4 g of acrylic anhydride was added dropwise while stirring. The reaction was carried out at 60 °C for 12 h. After the reaction, acetone was added to precipitate the product. The supernatant was removed, and the precipitate was washed three times with acetone and air-dried to obtain allyl-modified chitosan oligosaccharide (CSAA) solid powder. 0.1 g of CSAA was dissolved in 10 mL of deionized water, and 0.3 g of N-isopropylacrylamide (NIPAM) was added. After stirring and dissolving, the mixture was heated to 50 °C under N2 protection, and 0.1 g of potassium persulfate was added. The temperature was further increased to 80 °C and reacted for 4 h to obtain a temperature-sensitive microgel. The microgel was dialyzed with deionized water (the molecular weight cutoff of the dialysis bag was 3500 Da) for 3 days and then freeze-dried to obtain a temperature-sensitive microgel lyophilized powder.

[0031] Example 2 Weigh 0.01 g of genipin and dissolve it in 20 mL of anhydrous ethanol to obtain a genipin ethanol solution. Weigh 0.1 g of the lyophilized thermosensitive microgel powder prepared in Example 1 and add it to the genipin ethanol solution. Place the solution in a shaker and mix thoroughly at room temperature for 24 h. Remove the ethanol by rotary evaporation. After washing three times with ethanol, dissolve the solution in 1 mL of deionized water to obtain a genipin-loaded thermosensitive microgel solution (component A).

[0032] Example 3 Take 0.1 g of the thermosensitive microgel lyophilized powder prepared in Example 1, dissolve it in 1 mL of deionized water, slowly add 0.05 g / mL of β-glycerophosphate sodium aqueous solution (obtained by dissolving 0.05 g of β-glycerophosphate sodium in 1 mL of deionized water), place it in a shaker, shake and mix thoroughly at room temperature for 24 h, centrifuge at 12000 r / min for 15 min, collect the precipitate, wash the precipitate 3 times with deionized water, and then dissolve it in 1 mL of deionized water to obtain the thermosensitive microgel solution loaded with β-glycerophosphate sodium (component B).

[0033] Example 4 Weigh 0.05 g of chitosan and dissolve it in 2 mL of 0.01 M hydrochloric acid solution. Add 0.1 M sodium hydroxide solution dropwise and adjust the pH to 6-6.5. Then add component A prepared in Example 2 and component B prepared in Example 3. Place the mixture in a shaker and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0034] Example 5 Weigh 0.05 g of carboxymethyl chitosan, dissolve it in 2 mL of deionized water, add component A prepared in Example 2 and component B prepared in Example 3, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0035] Example 6 0.1 g of chitosan oligosaccharide was dissolved in 10 mL of deionized water, and 0.4 g of acrylic anhydride was added dropwise while stirring. The reaction was carried out at 60 °C for 12 h. After the reaction, acetone was added to precipitate the product. The supernatant was removed, and the precipitate was washed three times with acetone and air-dried to obtain allyl-modified chitosan oligosaccharide (CSAA) solid powder. 0.1 g of CSAA was dissolved in 10 mL of deionized water, and 0.3 g of N-isopropylacrylamide (NIPAM) was added. After stirring and dissolving, the mixture was heated to 50 °C under N2 protection, and 0.1 g of potassium persulfate was added. The temperature was further increased to 60 °C, and the reaction was carried out for 4 h to obtain a temperature-sensitive microgel. The microgel was dialyzed with deionized water (the molecular weight cutoff of the dialysis bag was 3500 Da) for 3 days, and then freeze-dried to obtain a temperature-sensitive microgel lyophilized powder.

[0036] Example 7 Weigh 0.01 g of genipin and dissolve it in 20 mL of anhydrous ethanol to obtain a genipin ethanol solution. Weigh 0.1 g of the lyophilized thermosensitive microgel powder prepared in Example 6 and add it to the genipin ethanol solution. Place the solution in a shaker and mix thoroughly at room temperature for 24 h. Remove the ethanol by rotary evaporation. After washing three times with ethanol, dissolve the solution in 1 mL of deionized water to obtain a genipin-loaded thermosensitive microgel solution (component A).

[0037] Example 8 Take 0.1 g of the thermosensitive microgel lyophilized powder prepared in Example 6, dissolve it in 1 mL of deionized water, slowly add 0.05 g / mL of β-glycerophosphate sodium aqueous solution (obtained by dissolving 0.05 g of β-glycerophosphate sodium in 1 mL of deionized water), place it in a shaker, shake and mix thoroughly at room temperature for 24 h, centrifuge at 12000 r / min for 15 min, collect the precipitate, wash the precipitate 3 times with deionized water, and then dissolve it in 1 mL of deionized water to obtain the thermosensitive microgel solution loaded with β-glycerophosphate sodium (component B).

[0038] Example 9 Weigh 0.05 g of chitosan, dissolve it in 2 mL of 0.01 M hydrochloric acid solution, add 0.1 M sodium hydroxide solution dropwise, adjust the pH to 6-6.5, add component A prepared in Example 7 and component B prepared in Example 8, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0039] Example 10 Weigh 0.05 g of carboxymethyl chitosan, dissolve it in 2 mL of deionized water, add component A prepared in Example 7 and component B prepared in Example 8, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0040] Example 11 0.1 g of chitosan oligosaccharide was dissolved in 10 mL of deionized water, and 0.4 g of acrylic anhydride was added dropwise while stirring. The reaction was carried out at 60 °C for 12 h. After the reaction, acetone was added to precipitate the product. The supernatant was removed, and the precipitate was washed three times with acetone and air-dried to obtain allyl-modified chitosan oligosaccharide (CSAA) solid powder. 0.1 g of CSAA was dissolved in 10 mL of deionized water, and 0.1 g of N-isopropylacrylamide (NIPAM) was added. After stirring and dissolving, the mixture was heated to 50 °C under N2 protection, and 0.1 g of potassium persulfate was added. The temperature was further increased to 80 °C and reacted for 4 h to obtain a temperature-sensitive microgel. The microgel was dialyzed with deionized water (the molecular weight cutoff of the dialysis bag was 3500 Da) for 3 days and then freeze-dried to obtain a temperature-sensitive microgel lyophilized powder.

[0041] Example 12 Weigh 0.01 g of genipin and dissolve it in 20 mL of anhydrous ethanol to obtain a genipin ethanol solution. Weigh 0.1 g of the lyophilized thermosensitive microgel powder prepared in Example 11 and add it to the genipin ethanol solution. Place the solution in a shaker and mix thoroughly at room temperature for 24 h. Remove the ethanol by rotary evaporation. After washing three times with ethanol, dissolve the solution in 1 mL of deionized water to obtain a genipin-loaded thermosensitive microgel solution (component A).

[0042] Example 13 Take 0.1 g of the thermosensitive microgel lyophilized powder prepared in Example 11, dissolve it in 1 mL of deionized water, slowly add 0.05 g / mL of β-glycerophosphate sodium aqueous solution (obtained by dissolving 0.05 g of β-glycerophosphate sodium in 1 mL of deionized water), place it in a shaker, shake and mix thoroughly at room temperature for 24 h, centrifuge at 12000 r / min for 15 min, collect the precipitate, wash the precipitate 3 times with deionized water, and then dissolve it in 1 mL of deionized water to obtain the thermosensitive microgel solution loaded with β-glycerophosphate sodium (component B).

[0043] Example 14 Weigh 0.05 g of chitosan, dissolve it in 2 mL of 0.01 M hydrochloric acid solution, add 0.1 M sodium hydroxide solution dropwise, adjust the pH to 6-6.5, add component A prepared in Example 12 and component B prepared in Example 13, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0044] Example 15 Weigh 0.05 g of carboxymethyl chitosan, dissolve it in 2 mL of deionized water, add component A prepared in Example 12 and component B prepared in Example 13, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0045] Example 16 0.1 g of chitosan oligosaccharide was dissolved in 10 mL of deionized water, and 0.4 g of acrylic anhydride was added dropwise while stirring. The reaction was carried out at 60 °C for 12 h. After the reaction, acetone was added to precipitate the product. The supernatant was removed, and the precipitate was washed three times with acetone and air-dried to obtain allyl-modified chitosan oligosaccharide (CSAA) solid powder. 0.1 g of CSAA was dissolved in 10 mL of deionized water, and 0.1 g of N-isopropylacrylamide (NIPAM) was added. After stirring and dissolving, the mixture was heated to 50 °C under N2 protection, and 0.1 g of potassium persulfate was added. The temperature was further increased to 60 °C and reacted for 4 h to obtain a temperature-sensitive microgel. The microgel was dialyzed against deionized water (dialysis bag molecular weight cutoff of 3500 Da) for 3 days, and then freeze-dried to obtain a temperature-sensitive microgel lyophilized powder. Its morphology and particle size were determined by transmission electron microscopy, and the results are shown in the figure. Figure 1 As can be seen from the figure, the synthesized thermosensitive microgel mainly consists of nanoparticles with a size of about 20 nm, with a small amount of aggregation.

[0046] Example 17 Weigh 0.01 g of genipin and dissolve it in 20 mL of anhydrous ethanol to obtain a genipin ethanol solution. Weigh 0.1 g of the thermosensitive microgel lyophilized powder prepared in Example 16, add it to the genipin ethanol solution, place it in a shaker, and shake it thoroughly at room temperature for 24 h. Remove the ethanol by rotary evaporation, dissolve it in 1 mL of deionized water, and obtain a thermosensitive microgel solution loaded with genipin (component A).

[0047] Example 18 Take 0.1 g of the thermosensitive microgel lyophilized powder prepared in Example 16, dissolve it in 1 mL of deionized water, slowly add 0.05 g / mL of sodium β-glycerophosphate aqueous solution (obtained by dissolving 0.05 g of sodium β-glycerophosphate in 1 mL of deionized water), place it in a shaker, and shake and mix thoroughly at room temperature for 24 h to obtain a thermosensitive microgel solution loaded with sodium β-glycerophosphate (component B).

[0048] Example 19 Weigh 0.05 g of chitosan, dissolve it in 2 mL of 0.01 M hydrochloric acid solution, add 0.1 M sodium hydroxide solution dropwise, adjust the pH to 6-6.5, add component A prepared in Example 17 and component B prepared in Example 18, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0049] Example 20 Weigh 0.05 g of carboxymethyl chitosan, dissolve it in 2 mL of deionized water, add component A prepared in Example 17 and component B prepared in Example 18, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0050] Example 21 0.1 g of chitosan oligosaccharide was dissolved in 10 mL of deionized water, and 0.4 g of acrylic anhydride was added dropwise while stirring. The reaction was carried out at 60 °C for 12 h. After the reaction, acetone was added to precipitate the product. The supernatant was removed, and the precipitate was washed three times with acetone and air-dried to obtain allyl-modified chitosan oligosaccharide (CSAA) solid powder. 0.1 g of CSAA was dissolved in 10 mL of deionized water, and 0.2 g of N-isopropylacrylamide (NIPAM) was added. After stirring and dissolving, the mixture was heated to 50 °C under N2 protection, and 0.1 g of potassium persulfate was added. The temperature was further increased to 80 °C and reacted for 4 h to obtain a temperature-sensitive microgel. The microgel was dialyzed with deionized water (the molecular weight cutoff of the dialysis bag was 3500 Da) for 3 days and then freeze-dried to obtain a temperature-sensitive microgel lyophilized powder.

[0051] Example 22 Weigh 0.01 g of genipin and dissolve it in 20 mL of anhydrous ethanol to obtain a genipin ethanol solution. Weigh 0.1 g of the lyophilized thermosensitive microgel powder prepared in Example 21 and add it to the genipin ethanol solution. Place the solution in a shaker and mix thoroughly at room temperature for 24 h. Remove the ethanol by rotary evaporation. After washing three times with ethanol, dissolve the solution in 1 mL of deionized water to obtain a genipin-loaded thermosensitive microgel solution (component A).

[0052] Example 23 Take 0.1 g of the thermosensitive microgel lyophilized powder prepared in Example 21, dissolve it in 1 mL of deionized water, slowly add 0.05 g / mL of β-glycerophosphate sodium aqueous solution (obtained by dissolving 0.05 g of β-glycerophosphate sodium in 1 mL of deionized water), place it in a shaker, shake and mix thoroughly at room temperature for 24 h, centrifuge at 12000 r / min for 15 min, collect the precipitate, wash the precipitate 3 times with deionized water, and then dissolve it in 1 mL of deionized water to obtain the thermosensitive microgel solution loaded with β-glycerophosphate sodium (component B).

[0053] Example 24 Weigh 0.05 g of chitosan, dissolve it in 2 mL of 0.01 M hydrochloric acid solution, add 0.1 M sodium hydroxide solution dropwise, adjust the pH to 6-6.5, add component A prepared in Example 22 and component B prepared in Example 3, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0054] Example 25 Weigh 0.05 g of carboxymethyl chitosan, dissolve it in 2 mL of deionized water, add component A prepared in Example 2 and component B prepared in Example 23, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0055] Example 26 0.1 g of chitosan oligosaccharide was dissolved in 10 mL of deionized water, and 0.4 g of acrylic anhydride was added dropwise while stirring. The reaction was carried out at 60 °C for 12 h. After the reaction, acetone was added to precipitate the product. The supernatant was removed, and the precipitate was washed three times with acetone and air-dried to obtain allyl-modified chitosan oligosaccharide (CSAA) solid powder. 0.1 g of CSAA was dissolved in 10 mL of deionized water, and 0.2 g of N-isopropylacrylamide (NIPAM) was added. After stirring and dissolving, the mixture was heated to 50 °C under N2 protection, and 0.1 g of potassium persulfate was added. The temperature was further increased to 60 °C and reacted for 4 h to obtain a temperature-sensitive microgel. The microgel was dialyzed with deionized water (the molecular weight cutoff of the dialysis bag was 3500 Da) for 3 days and then freeze-dried to obtain a temperature-sensitive microgel lyophilized powder.

[0056] Example 27 Weigh 0.01 g of genipin and dissolve it in 20 mL of anhydrous ethanol to obtain a genipin ethanol solution. Weigh 0.1 g of the lyophilized thermosensitive microgel powder prepared in Example 26 and add it to the genipin ethanol solution. Place the solution in a shaker and mix thoroughly at room temperature for 24 h. Remove the ethanol by rotary evaporation. After washing three times with ethanol, dissolve the solution in 1 mL of deionized water to obtain a genipin-loaded thermosensitive microgel solution (component A).

[0057] Example 28 Take 0.1 g of the thermosensitive microgel lyophilized powder prepared in Example 26, dissolve it in 1 mL of deionized water, slowly add 0.05 g / mL of β-glycerophosphate sodium aqueous solution (obtained by dissolving 0.05 g of β-glycerophosphate sodium in 1 mL of deionized water), place it in a shaker, shake and mix thoroughly at room temperature for 24 h, centrifuge at 12000 r / min for 15 min, collect the precipitate, wash the precipitate 3 times with deionized water, and then dissolve it in 1 mL of deionized water to obtain the thermosensitive microgel solution loaded with β-glycerophosphate sodium (component B).

[0058] Example 29 Weigh 0.05 g of chitosan, dissolve it in 2 mL of 0.01 M hydrochloric acid solution, add 0.1 M sodium hydroxide solution dropwise, adjust the pH to 6-6.5, add component A prepared in Example 27 and component B prepared in Example 28, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0059] Example 30 Weigh 0.05 g of carboxymethyl chitosan, dissolve it in 2 mL of deionized water, add component A prepared in Example 27 and component B prepared in Example 8, place in a shaker, and shake thoroughly at room temperature for 24 h to obtain a temperature-responsive injectable hydrogel.

[0060] Comparative Example 1 The difference from Example 1 is that conventional poly-N-isopropylacrylamide was prepared without the addition of CSAA.

[0061] Example 31: Determination of the phase transition rate of thermosensitive microgels The temperature-sensitive microgel lyophilized powder sample prepared in Example 16 and the conventional poly(N-isopropylacrylamide) sample prepared in Comparative Example 1 were diluted to 0.01 mg / mL, and 1 mL was injected into the sample cell. After degassing, the particle size change with temperature was measured using a nanoparticle size analyzer to evaluate the phase transition rate. The initial temperature was room temperature (25°C), and the particle size was measured every 1°C increase until 40°C was reached. Each temperature point was tested 5 times. Figure 2 The phase transition measurement results of the thermosensitive microgel prepared in Example 16 show that its transition rate is slower than that of conventional poly(N-isopropylacrylamide).

[0062] Example 32 Gel performance determination The hydrogel prepared in Example 20 was placed in a 2 cm × 2 cm × 20 cm mold, placed in a shaker at 37 ℃, and incubated for 0.5 h. Then it was allowed to cool naturally to room temperature for later use.

[0063] When testing is required, the hydrogel is incubated at 37 °C for 5 min. Images of the sol-gel phase transition before and after incubation are captured using a digital camera.

[0064] Rheological tests were performed using a rotational rheometer (Kinexus Pro+, Germany). The hydrogel was transferred to a measuring plate and heated under controlled conditions, with the temperature gradually increased from 25 °C to 45 °C at a rate of 1 °C / min. The storage modulus (G') and loss modulus (G'') were measured. The gelation temperature was determined by the critical point where the storage modulus (G') exceeded the loss modulus (G''), indicating the formation of a stable gel network.

[0065] Figure 3 , 4 Figures 5 and 6 show the sol-gel photograph, transmission electron microscope photograph, and rheometer measurement results of the sample obtained in Example 20, respectively.

[0066] Example 33 Drug Release Performance Determination Doxorubicin hydrochloride (DOX) was used as a model drug to analyze its drug loading performance. 0.005 g of DOX was weighed and dissolved in 2 mL of the temperature-responsive injectable hydrogel solution prepared in Example 20. The solution was placed in a shaker and incubated at room temperature in the dark for 24 hours. The shaker temperature was then adjusted to 37 °C and incubated for 0.5 h. 10 mL of a pH 6.5 (sodium phosphate) solution containing lysozyme (10 mM) was added, and the solution was incubated at 37 °C. The supernatant was analyzed using a UV-Vis spectrophotometer (Shimadzu UV-2600) and a fluorescence spectrometer (FLS1000, Edinburgh Instruments) at days 1, 3, 7, 14, 21, and 30.

[0067] Example 34 Implant Stability Determination The hydrogel prepared in Example 20 was placed in a 2 cm × 2 cm × 20 cm mold and incubated in a shaker at 37 °C for 0.5 h. Then, 10 mL of a pH 6.5 (sodium phosphate) solution containing lysozyme (10 mM) was added, and the mixture was incubated at 37 °C. The supernatant was analyzed by gel permeation chromatography (GPC, Agilent 1260 Infinity II, PL aquagel-OH 30 8µm 300×7.5 mm & PL aquagel-OH MIXED-H 8µm 300×7.5 mM) and transmission electron microscopy (TEM) at days 1, 3, 7, 14, 21, and 30, respectively.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any changes, modifications, substitutions, variations and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a temperature-responsive injectable hydrogel, characterized in that, Includes the following steps: (1) Chitosan oligosaccharide reacts with an allyl reagent to obtain allyl chitosan oligosaccharide; (2) Allyl chitosan oligosaccharide and N-isopropylacrylamide were crosslinked and copolymerized under the action of a polymerization initiator to obtain a thermosensitive microgel; (3) Genipin was loaded onto the thermosensitive microgel to obtain a genipin-loaded microgel; the genipin-loaded microgel was dissolved in water to obtain component A; (4) Thermosensitive microgel is loaded with sodium β-glycerophosphate to obtain thermosensitive microgel loaded with sodium β-glycerophosphate; the thermosensitive microgel loaded with sodium β-glycerophosphate is dissolved in water to obtain component B; (5) Mix component A and component B with a polymer solution to obtain a temperature-responsive injectable hydrogel; wherein the polymer contains amino or amino and carboxyl groups.

2. The method according to claim 1, characterized in that, At least one of the following conditions must be met: The molecular weight of the chitosan oligosaccharide is 800 Da ~ 10000 Da; The allyl reagent includes one or more of acrylic anhydride, methacrylic anhydride, acryloyl chloride, and methacryloyl chloride; The polymerization initiator includes one or more of potassium persulfate, ammonium persulfate, azobisisobutyronitrile, cuprous chloride, and dithioester; The polymer includes one or more of chitosan, carboxymethyl chitosan, and gelatin, and the molecular weight of the polymer is 10,000 Da to 3,000,000 Da.

3. The method according to claim 1, characterized in that, In step (1), the reaction solvent is one or more of water, methanol, and DMSO, and the mass ratio of chitosan oligosaccharide, allyl reagent, and solvent is 1:(0.1~10):(0~500), the reaction temperature is 0~60℃, and the reaction time is 0.1~20 h; It also includes a purification step, which includes one or more of the following: rotary evaporation, solvent precipitation and washing, and drying. The solvent used in the solvent precipitation and washing step is one or more of the following: acetone, ethanol, methanol, and ethyl acetate.

4. The method according to claim 1, characterized in that, In step (2), the reaction solvent is one or more of water, methanol, and DMSO, the mass ratio of allyl chitosan, N-isopropylacrylamide and initiator is 1:(1~20):(0.01~1), the reaction atmosphere is an inert gas, the inert gas is nitrogen or argon, the reaction temperature is 0 ℃~100 ℃, and the reaction time is 0.5 h~10 h; It also includes a purification step, which includes one or more of the following: water dialysis, solvent washing, and freeze drying; during dialysis, the molecular weight cutoff of the dialysis bag used is 800-15000; the solvent used for solvent washing is one or more of the following: acetone, ethanol, methanol, and ethyl acetate.

5. The method according to claim 1, characterized in that, In step (3), the method for loading genipin onto the microgel is as follows: dissolve genipin in an organic solvent to obtain a genipin solution; then shake the genipin solution and the thermosensitive microgel to mix thoroughly, remove the organic solvent by rotary evaporation, and wash to obtain the genipin-loaded microgel; wherein, the mass ratio of the thermosensitive microgel to genipin is 1:(0.001~1); the organic solvent includes one or two of ethanol and DMSO; the ratio of genipin to organic solvent is (0.001~1) g:20 mL; washing is performed with an organic solvent, which is one or more of acetone, ethanol, methanol, and ethyl acetate; the concentration of the thermosensitive microgel in component A is 0.001~1 g / mL.

6. The method according to claim 1, characterized in that, In step (4), the method for loading β-glycerophosphate onto the microgel is as follows: dissolve the thermosensitive microgel in water to obtain a thermosensitive microgel solution; mix the thermosensitive microgel solution and the β-glycerophosphate aqueous solution thoroughly by shaking, centrifugation, washing, and freeze-drying to obtain a thermosensitive microgel loaded with β-glycerophosphate; wherein, the mass ratio of thermosensitive microgel to β-glycerophosphate is 1:(0.001~1); washing is done with water; the concentration of thermosensitive microgel in the thermosensitive microgel solution is 0.001~1 g / mL; the concentration of β-glycerophosphate aqueous solution is 0.001~1 g / mL; and the concentration of thermosensitive microgel in component B is 0.01~1 g / mL.

7. The method according to claim 1, characterized in that, In step (5), the mass ratio of the thermosensitive microgel in component A and the thermosensitive microgel in component B to the polymer solution is 1:(1~10):(1~5), and the chitosan solution is a chitosan aqueous solution with a mass concentration of 0.1~10%; components A and B are thoroughly shaken and mixed with the polymer solution.

8. The temperature-responsive injectable hydrogel prepared by the preparation method according to any one of claims 1-7.

9. The application of the temperature-responsive injectable hydrogel of claim 8 in skin and mucous membranes, wound dressings, and implantation.

10. The use of the temperature-responsive injectable hydrogel of claim 8 in drug delivery.

Images