Modified chitosan and homogeneous hydroxyalkylation / quaternization modification method and application thereof

Abstract

The homogeneous hydroxyalkylation / quaternization modification method comprises the following steps: dissolving chitosan in a water phase by using a weakly acidic dissolution promoter, carrying out a ring-opening reaction with a 1, 2-epoxide or a quaternization reagent containing the 1, 2-epoxide under a weak base condition, introducing a hydroxyalkyl / quaternary ammonium group on a chitosan molecule, and carrying out homogeneous hydroxyalkylation / quaternization modification on the chitosan molecule to obtain the modified chitosan. The hydroxyalkylated chitosan or the quaternized chitosan is obtained. Compared with the prior art, the method provided by the invention has the advantages of simple conditions, safety, environmental protection, no need of strong base and toxic solvent, low energy consumption and controllable substitution degree. The obtained product has good solubility and transparency in a neutral aqueous solution, and is suitable for the fields of biomedical hydrogel, drug delivery, antibacterial coatings, lubricating materials and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of modified chitosan materials and biomaterials, and particularly relates to a modified chitosan and a homogeneous hydroxyalkylation / quaternization modification method and application thereof. BACKGROUND

[0002] As a natural cationic polysaccharide, chitosan has good biocompatibility, biodegradability and intrinsic antibacterial activity, and thus has attracted extensive attention in the fields of biological medicine, tissue engineering and functional materials. In particular, chitosan shows important potential in biomedical applications such as lubricating materials, medical dressings and injectable hydrogels. However, the presence of a large number of intramolecular and intermolecular hydrogen bonds in the molecular chain of chitosan leads to its strong hydrophobicity and poor water solubility. In particular, it is difficult to dissolve in a neutral to weak alkaline environment related to physiology, and usually relies on acidic solution or strong alkaline conditions to achieve dissolution. This characteristic seriously restricts the homogeneous functional modification of chitosan materials under neutral or near physiological pH conditions and their subsequent biological applications.

[0003] To improve the hydrophilicity, aqueous solution stability and biological function of chitosan, researchers often use chemical modification methods, among which hydroxyalkylation and quaternization are two important modification strategies. By introducing hydroxyalkyl groups (such as hydroxybutyl) or quaternary ammonium groups on the chitosan backbone, the hydration capacity can be effectively enhanced, the biocompatibility can be adjusted, and specific functions such as temperature sensitivity and enhanced antibacterial properties can be further imparted, thereby expanding its applications in high-end biological materials such as tissue lubrication and antibacterial films.

[0004] Currently, the processes for realizing chitosan hydroxyalkylation mainly include heterogeneous and homogeneous methods. For example, patent publication CN116655942A discloses a preparation method of a temperature-responsive chitosan solution, which modifies chitosan with 1,2-epoxybutane as a hydroxyalkylation reagent in an alkaline organic solvent system. However, since chitosan does not dissolve in this reaction medium, the reaction is actually a heterogeneous process, which often needs to be assisted by organic dispersants such as isopropyl alcohol for dispersion, resulting in poor uniformity of substitution, inconsistent product structure, and complex process flow with environmental burden.

[0005] Another patent publication CN110386995A proposes a homogeneous synthesis path for hydroxybutyl chitosan, which pre-disperses chitosan in a potassium hydroxide / urea aqueous solution, forms a transparent solution through low-temperature freeze-thaw treatment, and then performs ring-opening reaction with 1,2-epoxide (such as epoxybutane). Although this method realizes homogeneous reaction in an alkaline medium to some extent, it relies on strong base, low temperature and even freeze-thaw pretreatment, which puts high requirements on equipment corrosion resistance and operation safety, and the process conditions are harsh, which is difficult to adapt to large-scale or conventional production needs.

[0006] In addition, although there are reports of improving the reactivity of chitosan in aqueous phase with the help of reaction aids, the reaction types are mostly focused on condensation paths such as carboxylation and amidation, and are not suitable for hydroxyalkylation modification based on the ring-opening of 1,2-epoxide.

[0007] In summary, the poor solubility of chitosan material in neutral and weak alkaline environments due to strong hydrogen bonding is the core bottleneck restricting its homogeneous chemical modification. In the non-homogeneous or particle dispersion state, the ring-opening reaction of epoxy is easy to form a substitution layer on the surface of chitosan particles, hindering the diffusion of reagents to the interior of the particles, making it difficult to fully utilize the core reaction sites, and ultimately leading to uneven distribution of product substitution degree, large difference in side chain length, and poor structure repeatability. These problems will directly cause the salting-out phenomenon, microphase separation and decrease in light transmittance of the modified product in the physiological salt environment, seriously affecting the consistency and application reliability of the material performance, and bringing significant batch difference and risk of scale-up production.

[0008] Therefore, there is an urgent need in the art to develop a homogeneous hydroxyalkylation / quaternization modification method of chitosan that can be realized under the conditions of normal pressure, mild alkaline and even near neutral aqueous phase, to overcome the dependence on strong alkali, organic solvents, low temperature and other conditions in the prior art, improve the reaction uniformity, process safety and product structure controllability, and further promote the wider application of chitosan-based biomaterials in the field of high-end medical and tissue engineering. SUMMARY

[0009] The purpose of the present application is to overcome the defects of the prior art and provide a modified chitosan and a homogeneous hydroxyalkylation / quaternization modification method and application thereof, which can realize homogeneous hydroxyalkylation / quaternization modification of chitosan under the conditions of normal pressure, mild alkaline and even near neutral aqueous phase, improve the reaction uniformity, process safety and product structure controllability, and further promote the wider application of chitosan-based biomaterials in the field of high-end medical and tissue engineering.

[0010] The purpose of the present application can be achieved by the following technical solutions: One of the technical solutions of the present application is to provide a homogeneous hydroxyalkylation / quaternization modification method of chitosan solution, which utilizes a weakly acidic solubilizing agent to dissolve chitosan in aqueous phase, and then undergoes ring-opening reaction with 1,2-epoxide or 1,2-epoxide-containing quaternization reagent under weakly alkaline conditions to introduce hydroxyalkyl / quaternary ammonium groups on the chitosan molecule, thereby obtaining hydroxyalkylated chitosan or quaternized chitosan.

[0011] Further, the weak acid promoter is benzotriazole or its nitrogen analogues, selected from one or more of 1-hydroxybenzotriazole (HOBt), 1-oxylbenzotriazole (NOBt), 5-methyl-1-hydroxybenzotriazole, 5-chloro-1-hydroxybenzotriazole, 5-bromo-1-hydroxybenzotriazole, 5-nitro-1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole sodium salt, 1-hydroxybenzotriazole lithium salt, O-benzotriazole-N-tetramethyluronium tetrafluoroborate, benzotriazole-1-acyl-1-methylpyridinium hexafluorophosphate, benzotriazole-1-acyloxytris(dimethylamino) phosphonium hexafluorophosphate, 3-[(trisdimethylamino)sulfonyl]-2-oxo-4-benzotriazole, 3-[(diphenylphosphoryl)oxy]-4-benzotriazole, or substituted derivatives thereof, preferably HOBt.

[0012] Further, the aqueous phase is a urea aqueous solution, having a mass concentration of 0.01% to 20%, preferably 0.1% to 10%, and further preferably 0.2%. Further, the molar ratio of the chitosan and the weak acid promoter is 1.0 to 5.0:1, preferably 1.5 to 3:1; and the mass ratio of the chitosan and the aqueous phase is 0.01 to 0.05:1, preferably 0.025 to 0.035:1.

[0013] Further, the weak base condition is obtained by adding a weak base reagent, the molar amount of the weak base reagent being 0.1 to 1.0 times, preferably 0.2 to 0.5 times, and further preferably 0.3 times the molar amount of the chitosan glucosamine unit. The weak base reagent is an organic weak base or an inorganic weak base; the organic weak base is triethanolamine; and the inorganic weak base includes one or more of sodium bicarbonate, sodium carbonate, amino acid salt, or magnesium hydroxide, preferably sodium bicarbonate.

[0014] Further, the weak base reagent is mixed with an aqueous imidazole catalyst to obtain the weak base condition. The aqueous imidazole catalyst is selected from one or more of imidazole, 2-methylimidazole, 4-methylimidazole, 5-methylimidazole, benzimidazole, 2-hydroxymethylimidazole, or 2-phenylimidazole, preferably 2-methylimidazole. The molar amount of the aqueous imidazole catalyst is 0.05 to 0.2 times, and preferably 0.1 to 0.15 times the molar amount of the chitosan glucosamine unit.

[0015] Further, the 1,2-epoxide is selected from one or more of linear or branched 1,2-alkene oxide, or functionalized epoxide species. The linear or branched 1,2-alkylene oxide is selected from one or more of 1,2-ethylene oxide, 1,2-propylene oxide, 1,2-butylene oxide, 1,2-pentylene oxide, 1,2-hexylene oxide or 1,2-octylene oxide; The functionalized epoxide is selected from one or more of glycidol, glycidyl ether or glycidyl ester; The 1,2-epoxy-containing quaternization reagent is glycidyl trimethylammonium chloride (GTMAC); The molar amount of the 1,2-epoxide or 1,2-epoxy-containing quaternization reagent is 0.1-30 times, preferably 5-20 times, the molar amount of the chitosan glucosamine unit.

[0016] Further, the conditions of the ring-opening reaction are: temperature is 15-50℃, preferably 20-35℃; pH is 7.0-9.0, preferably 8.0-8.8; time is 0.5-48h, preferably 24-48h.

[0017] Further, the specific steps of the homogeneous hydroxyalkylation / quaternization modification method are: S1, dispersing a weakly acidic solubilizing agent and chitosan in an aqueous phase to obtain a homogeneous chitosan solution by stirring; S2, adding a weak base reagent to adjust the homogeneous chitosan solution to be weakly alkaline; S3, adding 1,2-alkylene oxide or 1,2-epoxy-containing quaternization reagent dropwise in batches while stirring to carry out ring-opening reaction, introducing hydroxyalkyl / quaternary ammonium groups on the chitosan molecules to obtain hydroxyalkylated chitosan or quaternized chitosan, which is in the form of a solution at this time.

[0018] Further, the hydroxyalkylated chitosan or quaternized chitosan solution is titrated to neutral with dilute hydrochloric acid, then dialyzed with a dialysis bag, and freeze-dried to obtain hydroxyalkylated chitosan or quaternized chitosan.

[0019] Further, the concentration of the dilute hydrochloric acid is 0.01-1 mol / L.

[0020] Further, the temperature of the freeze-drying is -50--100℃, preferably -70--90℃.

[0021] The second technical solution of the present application provides a modified chitosan prepared by the homogeneous hydroxyalkylation / quaternization modification method.

[0022] Further, the structural formula of the modified chitosan is shown in the following formula (1): Formula (1), Wherein, n is 200-1000, m is 0-6, wherein m is 0 Indicated as 1,2-oxirane.

[0023] The third technical scheme of the present application provides an application of modified chitosan, which is used for preparing a drug delivery carrier, a hydrogel, an antibacterial coating or a lubricating material, and has certain temperature sensitivity and antibacterial properties.

[0024] Compared with the prior art, the present application has the following advantages: Green and efficient and easy to operate: The present application realizes the homogeneous hydroxyalkylation / quaternization modification of chitosan under a dispersant system at room temperature for the first time, the reaction process is green and environmentally friendly, avoids the dependence on strong alkali, low temperature and even freeze-thaw pretreatment, reduces energy consumption and equipment requirements, and is convenient for smooth scaling up of production.

[0025] Superior reaction uniformity: The present application uses benzotriazole or its azo analogues as a solvent, so that chitosan can be dissolved in a homogeneous solution, thereby ensuring uniform distribution of the reactants. Compared with the traditional heterogeneous grafting method, the heterogeneous method usually starts from solid chitosan, and local incomplete dissolution and uneven reaction often occur during the dissolution process. Since 1-hydroxybenzotriazole effectively promotes the uniform dissolution of chitosan through hydrogen bonding, the entire reaction system remains uniform, avoiding the situation of uneven grafting after solid dissolution, thereby significantly improving the uniformity of the grafting reaction and the consistency of the product.

[0026] Higher process safety: The benzotriazole or its azo analogue system used in the present application can dissolve chitosan and carry out grafting reaction in a mild weak acid environment, and the pH value during the reaction process is maintained in the range close to neutral (pH 7±1.5). Compared with the traditional method of using strong alkali (such as NaOH, KOH) catalyzed grafting reaction, the strong alkali system usually requires high pH conditions (pH>13), which is easy to cause large pH fluctuation and potential safety threat to equipment and operating personnel. The 1-hydroxybenzotriazole system avoids the side reactions such as overheating, deamination of amino group or degradation of main chain in strong alkali environment by mild control of pH, ensuring the stability and safety of the reaction process.

[0027] More moderate reaction control: When benzotriazole or its azo analogue solution is used for chitosan dissolution, the competition of hydrogen bonding not only improves the solubility of chitosan, but also maintains the reactivity of amino group during the reaction process, without causing excessive protonation or unnecessary side reactions. In contrast, the strong alkaline solution used in the traditional method easily causes side reactions such as deamination or chain scission, thereby affecting the selectivity of the reaction and the structure control of the product. The mildness of 1-hydroxybenzotriazole solution effectively avoids these problems, improving the structure controllability of the product.

[0028] The present application can realize homogeneous hydroxyalkylation / quaternization of chitosan under normal temperature and pressure, mild alkaline or even near neutral aqueous phase conditions, because: benzotriazole or its nitrogen analogs and urea are used to cooperatively compete to destroy hydrogen bonds in chitosan particles, to untangle and promote solubility to form a molecular level homogeneous system; bicarbonate / organic amine and other weak bases are used to construct a buffer zone, so that -NH2 is only partially deprotonated and deacetylation and β-elimination are inhibited, and weak alkaline conditions are provided for epoxide ring opening; imidazole / 2-methylimidazole is used as a proton shuttle and a mild epoxide ring opening catalyst, which reduces the activation energy and improves the selectivity of N-site etherification, thereby realizing the preparation of homogeneous modified chitosan without organic solvents and without relying on low temperature. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 For the preparation process of the homogeneous hydroxyalkylation / quaternization modification method of the chitosan solution of the present application, (a) step S1, (b) steps S2, S3; Figure 2 For the infrared spectra of chitosan, sodium bicarbonate HBCs, triethanolamine HBCs, and HACC; Figure 3 For (a) the structural formula of quaternary ammonium chitosan, (b) the nuclear magnetic hydrogen spectrum of chitosan, quaternary ammonium chitosan; Figure 4 For the Zeta potential test results of chitosan and quaternary ammonium chitosan; Figure 5 For (a) the structural formula of hydroxybutyl chitosan, (b) the nuclear magnetic hydrogen spectrum of chitosan, sodium bicarbonate HBCs, and triethanolamine HBCs; Figure 6 For the test results of the gelation transition temperature point of sodium bicarbonate HBCs; Figure 7 For the experimental process schematic diagram of (a) step (1) and (b) step (2) of Comparative Example 1; Figure 8 For (a) Comparative Example 2 and (b) the reaction system dissolution situation schematic diagram of Example 1. DETAILED DESCRIPTION

[0030] The present application will be described in detail below in conjunction with the drawings and specific examples. The present embodiment is implemented on the premise of the technical solution of the present application, and detailed implementation and specific operation processes are given, but the protection scope of the present application is not limited to the following examples. Based on the given examples, all other examples obtained by those of ordinary skill in the art without making creative efforts are within the scope of the present application.

[0031] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present application are conventional reagents, methods, instruments and equipment in the art.

[0032] The following products were purchased from Macklin: Chitosan (deacetylation degree ≥ 95%, viscosity 100-200 mpa.s), 1-hydroxybenzotriazole (analytical pure, 99%), urea (analytical pure, 99%), triethanolamine (99% biotechnology grade), glacial acetic acid (analytical pure, 99%). The following products were purchased from Adamas: Glycidyltrimethylammonium chloride (analytical pure, 99%), 2-methylimidazole (analytical pure, 98%), hydrochloric acid was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Unless otherwise specified, the reagents used were commercially available analytical pure or above grade and were used without further purification.

[0033] A homogeneous hydroxyalkylation / quaternization modification method of chitosan solution, which utilizes a weakly acidic solubilizing agent to dissolve chitosan in an aqueous phase, and then undergoes ring-opening reaction with 1,2-epoxide or quaternization reagent containing 1,2-epoxide under weakly basic conditions to introduce hydroxyalkyl / quaternary ammonium groups on the chitosan molecule, obtaining hydroxyalkylated chitosan or quaternized chitosan.

[0034] In some specific embodiments, the weakly acidic solubilizing agent is benzotriazole or its azo analogues, selected from one or more of HOBt, NOBt, 5-methyl-1-hydroxybenzotriazole, 5-chloro-1-hydroxybenzotriazole, 5-bromo-1-hydroxybenzotriazole, 5-nitro-1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole sodium salt, 1-hydroxybenzotriazole lithium salt, O-benzotriazole-N-tetramethylurea tetrafluoroborate, benzotriazole-1-acyl-1-methylpyridinium hexafluorophosphate, benzotriazole-1-acyloxytris(dimethylamino) phosphonium hexafluorophosphate, 3-[(tridimethylamino)sulfonyl]-2-oxo-4-benzotriazole, 3-[(diphenyl phosphoryl)oxy]-4-benzotriazole, or substituted derivatives thereof, preferably HOBt.

[0035] In some specific embodiments, the aqueous phase is a urea aqueous solution with a mass concentration of 0.01% to 20%, preferably 0.1% to 10%, and further preferably 0.2%. In some specific embodiments, the molar ratio of chitosan to weakly acidic solubilizing agent is 1.0 to 5.0:1, preferably 1.5 to 3:1. The mass ratio of chitosan to aqueous phase is 0.01 to 0.05:1, preferably 0.025 to 0.035:1.

[0036] In some specific embodiments, the weakly basic condition is obtained by adding a weak base reagent, and the molar amount of the weak base reagent is 0.1 to 1.0 times the molar amount of the chitosan glucosamine unit, preferably 0.2 to 0.5, and further preferably 0.3. The weak base reagent is an organic weak base or an inorganic weak base; the organic weak base is triethanolamine; the inorganic weak base includes one or more of sodium bicarbonate, sodium carbonate, amino acid salt or magnesium hydroxide, preferably sodium bicarbonate.

[0037] In some specific embodiments, the weak base reagent is mixed with the water-soluble imidazole catalyst to obtain the weak base condition; The water-soluble imidazole catalyst is selected from one or more of imidazole, 2-methylimidazole, 4-methylimidazole, 5-methylimidazole, benzimidazole, 2-hydroxymethylimidazole or 2-phenylimidazole, preferably 2-methylimidazole; The molar amount of the water-soluble imidazole catalyst is 0.05-0.2 times, preferably 0.1-0.15 times, the molar amount of the chitosan glucosamine unit.

[0038] In some specific embodiments, the 1,2-epoxide is selected from one or more of a linear or branched 1,2-alkene oxide, or a functionalized epoxide species; The linear or branched 1,2-alkene oxide is selected from one or more of 1,2-ethylene oxide, 1,2-propylene oxide, 1,2-butylene oxide, 1,2-pentylene oxide, 1,2-hexylene oxide or 1,2-octylene oxide; The functionalized epoxide is selected from one or more of glycidol, glycidyl ether or glycidyl ester; The 1,2-epoxy-containing quaternization reagent is GTMAC; The molar amount of the 1,2-epoxide or 1,2-epoxy-containing quaternization reagent is 0.1-30 times, preferably 5-20 times, the molar amount of the chitosan glucosamine unit.

[0039] In some specific embodiments, the conditions of the ring-opening reaction are: a temperature of 15-50°C, preferably 20-35°C, further preferably 25°C; a pH of 7.0-9.0, preferably 8.0-8.8; and a time of 0.5-48h, preferably 24-48h.

[0040] In some specific embodiments, as shown in Figure 1 The specific steps of the homogeneous hydroxyalkylation / quaternization modification method are: S1, dispersing a weakly acidic solubilizing agent and chitosan in an aqueous phase to obtain a homogeneous chitosan solution; S2, adding a weak base reagent to adjust the homogeneous chitosan solution to be weakly basic; S3, while stirring, dropwise add 1,2-alkylene oxide or 1,2-epoxy-containing quaternary ammonium reagent in batches, perform ring-opening reaction, introduce hydroxyalkyl / quaternary ammonium groups on the chitosan molecules, to obtain hydroxyalkylated chitosan or quaternary ammonium chitosan, which is in solution form at this time.

[0041] In some specific embodiments, the hydroxyalkylated chitosan or quaternary ammonium chitosan solution is titrated to neutral using dilute hydrochloric acid, and then dialyzed using a dialysis bag, and freeze-dried to obtain hydroxyalkylated chitosan or quaternary ammonium chitosan.

[0042] In some specific embodiments, the concentration of the dilute hydrochloric acid is 0.01-1 mol / L.

[0043] In some specific embodiments, the temperature of the freeze-drying is -50 to -100℃, preferably -70 to -90℃.

[0044] A modified chitosan prepared by the homogeneous hydroxyalkylation / quaternization modification method.

[0045] In some specific embodiments, the structural formula of the modified chitosan is as shown in the following formula (1): Formula (1), wherein n is 200-1000, and m is 0-6, wherein when m is 0 represents 1,2-ethylene oxide.

[0046] An application of a modified chitosan, the modified chitosan being used for preparing a drug delivery carrier, a hydrogel, an antibacterial coating, or a lubricating material, which has certain temperature-sensitive properties and antibacterial properties.

[0047] Any of the above embodiments can be implemented alone, or in any two or more combinations.

[0048] The following will be described in conjunction with specific embodiments.

[0049] Embodiment 1 A homogeneous hydroxyalkylation modification method of a chitosan solution, specifically comprising the following steps: (1) At room temperature, mix 1 g of chitosan powder with 0.98 g of 1-hydroxybenzotriazole, dissolve in 80 ml of urea aqueous solution (weigh 0.120 g of urea, dissolve in deionized water and dilute to 80.0 mL), and stir until clear to obtain a homogeneous chitosan solution.

[0050] (2) 0.140 g of sodium bicarbonate was dissolved in 20 ml of deionized water and stirred until transparent to prepare a weak alkali solution. The weak alkali solution was slowly dropped into the homogeneous chitosan solution and stirred for 5 min, and then 9.8 ml of 1,2-epoxybutane was added dropwise in batches while stirring, and the mechanical stirring was 400 rpm, and the reaction was carried out at room temperature for 24 hours.

[0051] (3) The reacted solution was titrated to pH = 7.0-7.4 using 0.1 mol / L dilute hydrochloric acid, and dialyzed for 5-7 days (molecular weight cutoff 8000-12000 Da), after dialysis was completed, -80℃ drying in a freeze dryer for 48-72 hours to obtain hydroxybutyl chitosan (sodium bicarbonate HBCs).

[0052] Example 2 A homogeneous hydroxy quaternization modification method of chitosan solution, specifically comprising the following steps: (1) 1 g of chitosan powder was mixed with 0.98 g of 1-hydroxybenzotriazole, dissolved in 80 ml of urea aqueous solution (urea 0.120 g was weighed, dissolved in deionized water and diluted to 80.0 mL), and stirred until clear to obtain a homogeneous chitosan solution. (2) Then, 0.140 g of sodium bicarbonate was mixed with 0.052 g of 2-methylimidazole, dissolved in 20 ml of deionized water, and stirred until transparent to prepare a mixed weak alkali solution. 4.96 g of glycidyltrimethylammonium chloride was dissolved in 5 ml of deionized water, and stirred until the solution was light yellow to prepare a glycidyltrimethylammonium chloride aqueous solution.

[0053] (3) The mixed weak alkali solution was slowly dropped into the homogeneous chitosan solution and stirred for 5 min, and then the glycidyltrimethylammonium chloride aqueous solution was added, and the mechanical stirring was 400 rpm, and the reaction was carried out at room temperature for 24 hours.

[0054] (4) The reacted solution was titrated to pH = 7.0-7.4 using 0.1 mol / L dilute hydrochloric acid, and dialyzed for 6 days (molecular weight cutoff 8000-12000 Da), after dialysis was completed, -80℃ drying in a freeze dryer for 64 hours to obtain quaternary ammonium chitosan.

[0055] Example 3 A homogeneous hydroxyalkylation modification method of chitosan solution, specifically comprising the following steps: (1) 1 g of chitosan powder was mixed with 0.98 g of 1-hydroxybenzotriazole, dissolved in 80 ml of urea aqueous solution (urea 0.120 g was weighed, dissolved in deionized water and diluted to 80.0 mL), and stirred until clear to obtain a homogeneous chitosan solution.

[0056] (2) Then, 1.11 g of triethanolamine and 0.052 g of 2-methylimidazole were mixed and dissolved in 20 ml of deionized water, and stirred to be transparent to prepare a mixed weak base solution. The mixed weak base solution was slowly dropped into the homogeneous chitosan solution and stirred for 5 min, and then 9.8 ml of 1,2-epoxybutane was added, and mechanically stirred at 400 rpm for 24 hours at room temperature.

[0057] (3) The reacted solution was titrated to pH = 7.0-7.4 using 0.1 mol / L dilute hydrochloric acid, and dialyzed for 6 days (molecular weight cut-off 8000-12000 Da), and after dialysis was completed, dried in a freeze dryer at -80°C for 64 hours to obtain hydroxybutyl chitosan (triethanolamine HBCs).

[0058] Example 4 A homogeneous hydroxyalkylation modification method of a chitosan solution, specifically comprising the following steps: (1) At room temperature, 1 g of chitosan powder and 0.98 g of 1-hydroxybenzotriazole were mixed and dissolved in 80 ml of urea aqueous solution (urea 0.120 g was weighed, dissolved in deionized water and diluted to 80.0 mL), and stirred to be clear to obtain a homogeneous chitosan solution.

[0059] (2) Then, 0.140 g of sodium bicarbonate and 0.052 g of 2-methylimidazole were mixed and dissolved in 20 ml of deionized water, and stirred to be transparent to prepare a mixed weak base solution. The mixed weak base solution was slowly dropped into the homogeneous chitosan solution and stirred for 5 min, and then 8.8 ml of 1,2-epoxypropane was added, and mechanically stirred at 400 rpm for 24 hours at room temperature.

[0060] (3) The reacted solution was titrated to pH = 7.0-7.4 using 0.1 mol / L dilute hydrochloric acid, and dialyzed for 6 days (molecular weight cut-off 8000-12000 Da), and after dialysis was completed, dried in a freeze dryer at -80°C for 64 hours to obtain hydroxypropyl chitosan.

[0061] Example 5 A homogeneous hydroxyalkylation modification method of a chitosan solution, specifically comprising the following steps: (1) At room temperature, 2 g of chitosan powder and 1.96 g of 1-hydroxybenzotriazole were mixed and dissolved in 80 ml of urea aqueous solution (urea 0.240 g was weighed, dissolved in deionized water and diluted to 80.0 mL), and stirred to be clear to obtain a homogeneous chitosan solution; (2) Subsequently, 0.280 g of sodium bicarbonate and 0.104 g of 2-methylimidazole were mixed and dissolved in 20 ml of deionized water and stirred until transparent to prepare a mixed weak base solution. The mixed weak base solution was slowly added dropwise to the homogeneous chitosan solution and stirred for 5 min. Then, 19.8 ml of 1,2-epoxybutane was added, and the mixture was mechanically stirred at 500 rpm and reacted at room temperature for 24 hours.

[0062] (3) The solution after the reaction was titrated with 0.1 mol / L dilute hydrochloric acid to pH=7.0-7.4 and dialyzed for 6 days (molecular weight cutoff 8000-12000 Da). After the dialyz was completed, it was dried in a freeze dryer at -80℃ for 64 hours to obtain hydroxybutyl chitosan.

[0063] The hydroxybutyl chitosan sample obtained in Example 1 was pressed into a pellet using potassium bromide, and its structure was tested using Fourier transform infrared spectroscopy. The structure was then verified using proton nuclear magnetic resonance spectroscopy.

[0064] like Figure 2 As shown, the infrared spectroscopy results indicate that the modified hydroxybutyl chitosan sample exhibits a high spectral density at 1463 cm⁻¹. -1 A distinct absorption peak appears at 1153 cm⁻¹, corresponding to the bending vibration of the –CH₂– group; at 1153 cm⁻¹... -1 A new absorption peak appears at the point, which is a characteristic peak of C–O–C stretching vibration, indicating that a hydroxyalkyl side chain was successfully introduced after the ring-opening of 1,2-epoxybutane.

[0065] like Figure 3 As shown, the 1H NMR results revealed that the products modified with butane oxide (HBCs) exhibited a new methyl (–CH3) signal at 0.91 ppm and a methylene (–CH2–) signal at 1.48 ppm, both characteristic peaks belonging to the hydroxybutyl side chain. These newly emerging signals demonstrate that butane oxide was successfully grafted onto the chitosan backbone via a ring-opening reaction. Simultaneously, the modified samples retained the main signals of the chitosan backbone in the 3.0–5.0 ppm range, indicating that the modification process did not disrupt the polysaccharide backbone structure. Therefore, this method can achieve hydroxybutylation modification of chitosan under mild conditions, producing products with clear structures and effective grafting.

[0066] The quaternized chitosan sample obtained in Example 2 was pressed into a pellet using potassium bromide, and the structure of the quaternized chitosan was tested using a Fourier transform infrared spectroscopy (FTIR) instrument.

[0067] like Figure 2 As shown, the infrared spectroscopy results indicate that quaternized chitosan has a concentration at 2970 cm⁻¹. -1 A significantly enhanced absorption peak for the –CH3 / –CH2– stretching vibration appears at 1480 cm⁻¹.-1 Near -N + (CH3)3 characteristic absorption peak, while the C-N stretching vibration absorption peak intensity at 1153 cm -1 significantly enhanced, indicating that the quaternary ammonium group has been successfully introduced into the chitosan molecule, thereby proving that the scheme has obtained a quaternary ammonium chitosan with cationicity.

[0068] As Figure 3 shown, the nuclear magnetic hydrogen spectrum result shows that the quaternary ammonium chitosan has a new peak at 3.22 ppm, corresponding to the characteristic signal of -N + (CH3)3 protons, while no such peak is observed in the original chitosan. The appearance of this characteristic peak indicates that the quaternary ammonium group has been successfully introduced into the chitosan molecule through the glycidyl trimethyl ammonium chloride ring-opening reaction, thereby obtaining a quaternary ammonium chitosan with permanent positive charge.

[0069] As Figure 4 shown, the Zeta potential test result shows that the unmodified chitosan only has a low positive potential under neutral conditions, while the quaternary ammonium chitosan modified by the method of the present application exhibits a significantly increased positive potential under the same conditions, proving that the stable -N + (CH3)3 group has been introduced into the molecule, thereby verifying the success of the quaternary ammonium grafting.

[0070] Take the hydroxybutyl chitosan sample prepared in Example 3, press the sample using potassium bromide, and test the structure of the quaternary ammonium chitosan using a Fourier transform infrared spectrometer.

[0071] As Figure 2 shown, the infrared spectrum result shows that the modified hydroxybutyl chitosan sample has a clear absorption peak at 1463 cm -1 , corresponding to the bending vibration of -CH2- group; a new absorption peak appears at 1153 cm -1 , which is the characteristic peak of C-O-C stretching vibration, indicating that the hydroxyalkyl side chain has been successfully introduced after the ring-opening of 1,2-epoxybutane.

[0072] As Figure 5 shown, the nuclear magnetic hydrogen spectrum result shows that the product (HBCs) modified by epoxybutane has a new methyl (-CH3) signal at 0.91 ppm and a methylene (-CH2-) signal at 1.48 ppm, both of which belong to the characteristic peaks of hydroxybutyl side chain. These newly appearing signals prove that the epoxybutane has been successfully grafted onto the chitosan backbone through ring-opening reaction.

[0073] The gel transition temperature test is performed on the hydroxybutyl chitosan prepared in Example 1.

[0074] The gelation transition temperature point of the hydroxybutyl chitosan aqueous solution prepared in Example 1 was tested using the temperature scanning mode of a rotational rheometer, wherein the cross point of the storage modulus (G') and the loss modulus (G") was the gelation temperature.

[0075] As shown in Figure 6 , G' gradually increased with the increase of temperature, and finally was greater than G", indicating that the hydroxybutyl chitosan prepared by the scheme involved herein was converted from a solution to a gel, and the gelation temperature was about 46℃.

[0076] The hydroxyalkylated chitosan and quaternized chitosan obtained by the present application have excellent water solubility, biocompatibility and surface activity characteristics, and can be widely applied in the fields of biomedical and functional materials. The obtained material can be used to prepare a hydrogel system with high transparency and stable existence under physiological conditions, and is suitable for eye surface lubrication, wound healing, mucosal drug delivery and cell culture scaffold, etc. biological medical scenes; the positively charged quaternary ammonium group endows the material with excellent antibacterial and anti-biofilm adhesion capacity, which can be further used for antibacterial coating, oral care, contact lens lubricant and tissue engineering support material.

[0077] Comparative Example 1 Most of them are the same as Example 1, except that step (1) is different: Under room temperature conditions, 1 g of chitosan powder was dissolved in an acetic acid aqueous solution with pH = 5.5-6.5, and stirred until clear to obtain a homogeneous chitosan weak acid solution.

[0078] (2) 0.140 g of sodium bicarbonate was dissolved in 20 ml of deionized water and stirred until transparent to prepare a weak base solution. The weak base solution was slowly added to the homogeneous chitosan solution and stirred for 5 min, and then 9.8 ml of 1,2-epoxybutane was added batchwise while stirring, and the mechanical stirring was 400 rpm, and the reaction was carried out at room temperature for 24 hours.

[0079] As can be seen from Figure 7 , the acetic acid weak acid solution cannot cooperate with sodium bicarbonate and other weak bases to provide a reaction environment for the ring opening of epoxy, and chitosan precipitates immediately after addition, while the weak acid environment provided by 1-hydroxybenzotriazole can cooperate with sodium bicarbonate and other weak base solutions to slowly improve the reaction environment for the ring opening of epoxy and reduce the phase separation of chitosan.

[0080] Comparative Example 2 Most of them are the same as Example 1, except that step (2) is different: (1) Under room temperature conditions, 1 g of chitosan powder was mixed with 0.98 g of 1-hydroxybenzotriazole and dissolved in 80 ml of urea aqueous solution (0.120 g of urea was weighed, dissolved in deionized water and diluted to 80.0 mL), and stirred until clear to obtain a homogeneous chitosan solution.

[0081] (2) Add 9.8 ml of 1,2-epoxybutane dropwise while stirring, and stir mechanically at 400 rpm for 24 hours at room temperature.

[0082] (3) Titrate the solution after reaction with 0.1 mol / L dilute hydrochloric acid to pH=7.0-7.4, and dialyze for 5-7 days (molecular weight cutoff 8000-12000 Da). After dialysis, dry in a freeze dryer at -80℃ for 48-72 hours to obtain hydroxybutyl chitosan (HBCs without weak base).

[0083] Depend on Figure 8 It can be seen that in Comparative Example 2, without the addition of a weak base and HOBt, the grafting amount was limited and the solubility was extremely poor, while the reaction system with the addition of a weak base had extremely high solubility. In contrast, the solubility of Example 1 was relatively high after the addition of a weak base.

[0084] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for homogeneous hydroxyalkylation / quaternization modification of chitosan solution, characterized in that, Chitosan is dissolved in an aqueous phase using a weakly acidic solvent, and then undergoes a ring-opening reaction with a 1,2-epoxide or a quaternizing agent containing a 1,2-epoxide under weakly alkaline conditions to introduce hydroxyalkyl / quaternary ammonium groups onto the chitosan molecule, thus obtaining hydroxyalkylated chitosan or quaternized chitosan.

2. The method for homogeneous hydroxyalkylation / quaternization modification of chitosan solution according to claim 1, characterized in that, The weakly acidic co-solvent is benzotriazole or its aza analogue, selected from one or more of 1-hydroxybenzotriazole, 1-oxybenzotriazole, 5-methyl-1-hydroxybenzotriazole, 5-chloro-1-hydroxybenzotriazole, 5-bromo-1-hydroxybenzotriazole, 5-nitro-1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole sodium salt, 1-hydroxybenzotriazole lithium salt, O-benzotriazole-N-tetramethylurea tetrafluoroborate, benzotriazole-1-acyl-1-methylpyridinium hexafluorophosphate, benzotriazole-1-acyloxytris(dimethylamino)phosphonium hexafluorophosphate, 3-[(tris(dimethylamino)sulfonyl]-2-oxy-4-benzotriazole, 3-[(diphenylphosphonyl)oxy]-4-benzotriazole or its substituted derivatives.

3. The method for homogeneous hydroxyalkylation / quaternization modification of chitosan solution according to claim 1, characterized in that, The aqueous phase is an aqueous solution of urea with a mass concentration of 0.01% to 20%.

4. The method for homogeneous hydroxyalkylation / quaternization modification of chitosan solution according to claim 1, characterized in that, The molar ratio of chitosan to the weakly acidic co-solvent is 1.0~5.0:1, and the mass ratio of chitosan to the aqueous phase is 0.01~0.05:

1.

5. The method for homogeneous hydroxyalkylation / quaternization modification of chitosan solution according to claim 1, characterized in that, The weakly alkaline conditions are obtained by adding a weakly alkaline reagent, wherein the molar amount of the weakly alkaline reagent is 0.1 to 1.0 times the molar amount of chitosan glucosamine units. The weak base reagent is an organic weak base or an inorganic weak base. The organic weak base is triethanolamine, and the inorganic weak base includes one or more of sodium bicarbonate, sodium carbonate, amino acid salts, or magnesium hydroxide.

6. The method for homogeneous hydroxyalkylation / quaternization modification of chitosan solution according to claim 5, characterized in that, The weak base reagent is mixed with a water-soluble imidazole catalyst and added to obtain the weak base conditions. The water-soluble imidazole catalyst is selected from one or more of imidazole, 2-methylimidazolium, 4-methylimidazolium, 5-methylimidazolium, benzimidazole, 2-hydroxymethylimidazolium, or 2-phenylimidazolium; The molar amount of the water-soluble imidazole catalyst is 0.05 to 0.2 times the molar amount of the chitosan glucosamine unit.

7. The method for homogeneous hydroxyalkylation / quaternization modification of chitosan solution according to claim 1, characterized in that, The 1,2-epoxide is selected from one or more of straight-chain or branched 1,2-epoxyalkanes or functionalized epoxides. Wherein, the straight-chain or branched 1,2-epoxyalkane is selected from one or more of 1,2-epoxyethylene, 1,2-epoxypropane, 1,2-epoxybutane, 1,2-epoxypentane, 1,2-epoxyhexane or 1,2-epoxyoctane. The functionalized epoxide is selected from one or more of glycidyl ether, glycidyl ester, or glycidyl ester; The quaternizing agent containing 1,2-epoxy is glycidyltrimethylammonium chloride; The molar amount of the 1,2-epoxide or the quaternizing agent containing 1,2-epoxide is 0.1 to 30 times the molar amount of the chitosan glucosamine unit.

8. The method for homogeneous hydroxyalkylation / quaternization modification of chitosan solution according to claim 1, characterized in that, The conditions for the ring-opening reaction are: temperature 15~50℃, pH 7.0~9.

0.

9. A modified chitosan, prepared by the homogeneous hydroxyalkylation / quaternization modification method according to any one of claims 1 to 8.

10. The application of the modified chitosan as described in claim 9, characterized in that, The modified chitosan is used to prepare drug delivery carriers, hydrogels, antibacterial coatings, or lubricating materials.

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