Process for the preparation of a very low degree of deacetylation carboxylated chitin

Abstract

This invention provides a method for preparing carboxylated chitin with extremely low deacetylation degree, relating to the field of biomaterials technology. By employing a more reactive bromoacid as the carboxylating agent and optimizing the pretreatment method and reaction parameters (molar ratio, temperature, and time), the nucleophilic substitution reaction rate is significantly accelerated, enabling the initial reaction phase to be completed rapidly within a very short reaction time. The increased reaction rate effectively shortens the time the chitin molecular chain is exposed to an alkaline environment, thereby greatly suppressing the accompanying deacetylation side reaction, ultimately achieving an extremely low degree of deacetylation while maintaining a high degree of substitution. The extremely low DD value indicates that the product retains a large number of natural acetamide groups, which helps reduce the cytotoxicity of the material and improves its hydrophobic interactions, laying the foundation for subsequent regulation of material properties (such as viscoelasticity).

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more particularly to a method for chemically modifying chitin, especially a method for preparing 1-carboxyethyl chitin with extremely low degree of deacetylation. Background Technology

[0002] Hyaluronic acid (HA) is widely used in the biomedical field due to its excellent moisturizing properties, viscoelasticity, and biocompatibility. However, its production faces challenges such as limited sources, complex processes, and a wide molecular weight distribution. Carboxymethyl chitosan (CMCS) is a potential alternative to HA, but its high degree of deacetylation (DD) leads to higher cytotoxicity and insufficient viscoelasticity, limiting its application in the medical field.

[0003] To address these issues, researchers have attempted carboxymethylation modification of chitin to prepare carboxymethyl chitin (CMCT) with a lower degree of deacetylation. For example, existing techniques using β-chitin as a raw material and chloroacetic acid for carboxymethylation yield products with low cytotoxicity, but their aqueous solutions have low viscosity and unsatisfactory viscoelasticity. The fundamental reason is that CMCT with a high degree of substitution (DS) introduces excessive hydrophobic acetyl groups and hydrophilic carboxymethyl groups at the C2 and C6 positions of the sugar ring, lacking sufficient hydroxyl groups to form effective physical crosslinks (hydrogen bonds); while CMCT with low DS suffers from insufficient water solubility. Liu et al. prepared a CMCT thermosensitive hydrogel with a DS of only 18% at low temperature in an alkali-urea system using α-chitin. Its thermosensitivity stemmed from the weakening of hydroxyl hydrogen bond crosslinking and the enhancement of hydrophobic group effects as the temperature increased, thus achieving gelation. This suggests that by simultaneously introducing hydrophilic carboxyl groups and alkyl groups that facilitate hydrophobic crosslinking onto chitin, it is possible to obtain materials with properties similar to HA.

[0004] However, in existing technologies, whether using chloroacetic acid or 2-chloropropionic acid for modification, it is difficult to achieve a high degree of substitution (DS) while controlling the degree of deacetylation at an extremely low level (DD≤10%). High DD values ​​(>10%) are considered one of the key factors leading to cytotoxicity. Therefore, developing a method for the efficient and rapid preparation of carboxylated chitin with extremely low DD and high DS is of great significance for obtaining excellent alternatives to HA. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is: how to provide a method for preparing carboxylated chitin that can simultaneously obtain high degree of substitution and extremely low degree of deacetylation.

[0006] On one hand, the present invention provides a method for preparing carboxylated chitin with extremely low degree of deacetylation, comprising the following steps: S1, raw material pretreatment: dispersing chitin raw material in isopropanol and performing swelling pretreatment to obtain swollen chitin; S2, carboxylation reaction: mixing the swollen chitin with bromoic acid and alkali, and performing a carboxylation reaction at a reaction temperature of 45℃~55℃ for 30min~50min to obtain a carboxylation reaction product; wherein, the molar ratio of chitin, bromoic acid and alkali is 1.0:7.0~8.2:11.5~13.0; S3, product purification: post-processing the carboxylation reaction product to obtain carboxylated chitin with a degree of deacetylation DD≤10%.

[0007] Optionally, the chitin raw material is β-chitin; the bromoacid is 2-bromopropionic acid.

[0008] Optionally, in step S1, the swelling pretreatment time is 20h~28h.

[0009] Optionally, in step S1, the swelling pretreatment is performed at room temperature.

[0010] Optionally, in step S2, the molar ratio of β-chitosan, 2-bromopropionic acid and alkali is 1:7.6:12.16.

[0011] Optionally, in step S2, the carboxylation reaction is carried out at a temperature of 50°C.

[0012] Optionally, in step S2, the carboxylation reaction takes 40 minutes.

[0013] Optionally, in step S3, the post-processing includes: terminating the reaction, washing with alcohol, dissolving, adjusting pH, filtering, ultrafiltration, and lyophilizing.

[0014] Optionally, the alcohol washing solution is a mixture of ethanol and 5M acetic acid aqueous solution for the first alcohol washing, and a mixture of ethanol and purified water for subsequent alcohol washing.

[0015] On the other hand, the present invention also provides a 1-carboxyethyl chitosan prepared by the above-described method for preparing carboxylated chitosan with extremely low degree of deacetylation, wherein the degree of deacetylation of the 1-carboxyethyl chitosan is DD≤10%.

[0016] The implementation of this invention offers the following advantages: By employing bromoacids under specific reaction conditions, the carboxylation reaction products rapidly form a viscous precipitate. This precipitate effectively hinders further transport of reactants, thereby physically suppressing the continued deacetylation side reaction and achieving an extremely low degree of deacetylation. Specifically, this invention uses bromoacids with higher reactivity as carboxylating agents and optimizes the pretreatment method and reaction parameters (molar ratio, temperature, time), significantly accelerating the nucleophilic substitution reaction rate. This allows the initial reaction phase to be completed rapidly within a very short reaction time, completing the main carboxylation process. The increased reaction rate effectively shortens the time the chitin molecular chains are exposed to an alkaline environment, thus greatly suppressing the accompanying deacetylation side reaction. Ultimately, this successfully achieves an extremely low degree of deacetylation in the product while maintaining a high degree of substitution. The extremely low DD value indicates that the product retains a large number of natural acetamide groups, which helps reduce the cytotoxicity of the material and improves its hydrophobic interactions, laying the foundation for subsequent regulation of material properties (such as viscoelasticity). Attached Figure Description

[0017] Figure 1 This is a schematic flowchart of the preparation method of carboxylated chitin with extremely low degree of deacetylation; Figure 2-1 This is a schematic diagram comparing the elastic modulus G′-frequency curves of 1-CECT and CMCT. Figure 2-2 This is a schematic diagram comparing the loss modulus G″-frequency curves of 1-CECT and CMCT. Figure 2-3 This is a schematic diagram comparing the shear viscosity η-shear rate curves of 1-CECT and CMCT. Figure 3-1 This is a schematic diagram comparing the TG thermogravimetric curves of β-chitosan and 1-CECT. Figure 3-2 This is a schematic diagram comparing the DSC heat flow curves of β-chitosan and 1-CECT; Figure 4 This is a schematic diagram illustrating the effect of carboxylic acid type on the DD / DS content of the product; Figure 4-1 This is a schematic diagram comparing the moisture absorption rate (Ra) of different samples at a relative humidity (RH) of 43%. Figure 4-2 This is a schematic diagram comparing the moisture absorption rate (Ra) of different samples at a relative humidity (RH) of 81%. Figure 4-3 This is a schematic diagram comparing the moisturizing rate (Rr) of different samples; Figure 5 This is a state diagram of the crude product modified by carboxylation of 2-bromopropionic acid; Figure 5-1This is a diagram showing the inverted (without alkaline solution) state of the crude product modified by carboxylation of 2-bromopropionic acid; Figure 5-2 The comparison is based on the UV full-spectrum scan of 1-CECT at different degradation times; Figure 5-3 This is the OD value-lysozyme degradation time curve at 218 nm from the UV full spectrum scan of 1-CECT. Figure 6 These are the FT-IR spectra of β-chitin and its carboxylation products; Figure 6-1 This is a schematic diagram comparing the cell viability of the DD5 group and the DD22 group at different culture times; Figures 6-2a to 6-2d This is an in vitro morphological image of L929 mouse fibroblasts (comparison between DD5 and DD22 groups). Figure 6-3 This is a comparison of cell viability between the DD5 and DD18 groups at different culture times; Figures 6-4a to 6-4d This is a morphological image of L929 mouse fibroblasts cultured for 48 h in vitro (comparison between DD5 and DD18 groups). Figures 6-5a to 6-5d This is a morphological image of L929 mouse fibroblasts cultured for 72 h in vitro (comparison between DD5 and DD18 groups). Figure 7 It is a carboxylation product of 2-chloropropionic acid. 1 HNMR spectrum; Figure 7-1 This is an elemental mapping scan of the 1-carboxyethyl chitosan sample; Figure 8 It is a carboxylated modified product of 2-bromopropionic acid. 1 HNMR spectrum; Figure 9 This is a graph showing the differences in DD and DS of the orthogonal groups; Figure 10 This is a graph showing the effect of alkaline solution swelling time on DD and DS at room temperature; Figure 11 This is a graph showing the effect of alkaline freezing time on the product DD and DS. Figure 12 This is a graph showing the differences in carboxylation modification effects under different pretreatment methods; Figure 13 This is a graph showing the difference in carboxylation modification effects between the pure freezing group and the untreated group; Figure 14 This is a graph showing the effect of acid dosage on DD and DS. Figure 15 This is a graph showing the effect of reaction temperature on the products DD and DS; Figure 16This is a graph showing the effect of alkali dosage on the product DD and DS. Figure 17 This is a graph showing the effect of reaction time on the products DD and DS. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The step numbers in the following embodiments are only for ease of explanation and do not limit the order of the steps. The execution order of each step in the embodiments can be adapted according to the understanding of those skilled in the art.

[0019] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0020] In the following description, when referring to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims. In the description of this application, it should be understood that the terms "first," "second," "third," etc., are used only to distinguish similar objects and are not necessarily used to describe a specific order or sequence, nor should they be construed as indicating or implying relative importance. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0021] Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0022] Example 1 In this embodiment, as Figure 1 The method for preparing a carboxylated chitin with extremely low degree of deacetylation shown includes the following steps: S1. Raw material pretreatment: β-chitosan raw material is dispersed in isopropanol and subjected to swelling pretreatment to obtain swollen β-chitosan; S2, Carboxylation reaction: The swollen β-chitosan is mixed with 2-bromopropionic acid and a base, and the carboxylation reaction is carried out at a reaction temperature of 45℃~55℃ for 30min~50min to obtain the carboxylation reaction product; wherein, the molar ratio of β-chitosan raw material, 2-bromopropionic acid and base is 1.0:7.0~8.2:11.5~13.0; S3. Product purification: The carboxylation reaction product is post-processed to obtain carboxylated chitin with extremely low degree of deacetylation.

[0023] The core of this embodiment lies in the discovery and use of bromoacids to carboxylate β-chitin. β-Chitin has weak intermolecular hydrogen bonds, a loose aggregate structure, and high reactivity; while the bromine atom in bromoacids has a stronger leaving ability than the chlorine atom in chloroacids such as chloroacetic acid and 2-chloropropionic acid.

[0024] In this embodiment, 2-bromopropionic acid was used as the bromoacid. β-Chitosan underwent swelling pretreatment with isopropanol to reduce the deacetylation initiation point, and reaction parameters (molar ratio, temperature, and time) were precisely controlled. Due to the extremely high reactivity of 2-bromopropionic acid, the main carboxylation reaction rate was much higher than the deacetylation side reaction rate, allowing the reaction system to pass through the initial reaction stage and complete the main carboxylation process in a very short time (e.g., within 40 minutes). This significant increase in reaction rate fundamentally shortened the total time that the chitosan molecular chains were exposed to a strongly alkaline environment, thereby kinetically inhibiting the deacetylation side reaction. Furthermore, in the later stages of the reaction, some carboxylation products formed a viscous system or localized precipitation, further restricting the movement of molecular chains and the diffusion of reagents, playing a certain auxiliary role in inhibiting side reactions. Thus, while obtaining a high degree of substitution (DS), the time window of the side reaction (i.e., the deacetylation reaction) was greatly shortened, ultimately achieving the goal of preparing carboxylated chitosan with extremely low degree of deacetylation (DD≤10%).

[0025] In this embodiment, in step S1, isopropanol is used to pre-treat β-chitosan by swelling at room temperature, and the swelling time is 20h~28h.

[0026] In this embodiment, in step S2, the molar ratio of β-chitosan raw material, 2-bromopropionic acid and alkali is controlled to be 1.0:7.6:12.16; and the temperature of the carboxylation reaction is controlled to be 50°C for 40 minutes.

[0027] Weigh 2g of β-chitosan and disperse it in 20 mL of isopropanol. Stir at room temperature for 24 h. Then add a certain amount of 40% (w / w) sodium hydroxide aqueous solution for preliminary alkalization. After the chitosan has been alkalized for 1 h, add dropwise a mixed solution of isopropanol containing the modifier. Heat to the preset temperature and carry out the reaction for the preset time to obtain the carboxylation reaction product. Then, the carboxylation reaction product is post-processed.

[0028] In this embodiment, room temperature refers to 20℃~25℃.

[0029] In this embodiment, step S3 includes the following post-processing steps: terminating the reaction, alcohol washing, dissolving, adjusting pH, filtration, ultrafiltration, and lyophilization. The alcohol washing uses a mixture of ethanol and 5M acetic acid aqueous solution for the first alcohol wash, and a mixture of ethanol and purified water for subsequent alcohol washes.

[0030] Specifically, the solid-liquid mixture of the carboxylation reaction products after the reaction is completed is filtered to remove the liquid phase mixture and quickly terminate the reaction, yielding a solid recovery. The solid recovery is first washed with a mixture of ethanol and 5M acetic acid aqueous solution (80:20 volume ratio) to remove some of the small molecule modifier (isopropanol) and adjust the pH of the product to acidic. The reaction is then terminated to prevent further deacetylation of the carboxylated chitin. A second and third wash are then performed with a mixture of ethanol and purified water (80:20 volume ratio) to ensure that the small molecule modifier is largely removed. After washing, the carboxylation reaction products are placed in a polytetrafluoroethylene mold, and the ethanol is evaporated in a fume hood for 1 hour. They are then fully dissolved in 300 mL of purified water, and the pH is adjusted to 8-8.3 with sodium hydroxide aqueous solution. The mixture was filtered through a 450-mesh plastic filter membrane to remove the sticky filter residue. The resulting solution was then transferred to a cup ultrafilter for further ultrafiltration at a pressure of 2 atmospheres. Each ultrafiltration cycle was controlled to reduce the cup ultrafilter volume from 95% to 30%. The ultrafiltration operation was repeated five times to theoretically ensure that the content of small molecule impurities was below 0.05%. After ultrafiltration, the resulting carboxylated chitin sample was frozen at -20°C and then freeze-dried to prepare a white, spongy product.

[0031] In this embodiment, the theoretical molecular structure of the 2-bromopropionic acid carboxylated chitin product is shown in the figure below. The effect of each successful carboxylation or deacetylation reaction on the C / N ratio is different. Carboxylation increases the C content, which increases the C / N ratio. Therefore, the C / N ratio can reflect the progress of the reaction.

[0032] In this embodiment, the theoretical reaction formula for the carboxylation of 2-bromopropionic acid is as follows: This embodiment also provides a comparative experiment on carboxylated chitin generated from bromoacid and chitin generated from chloroacid.

[0033] In the comparative experiment, the modifiers used for the carboxylation reaction were 2-bromopropionic acid and chloroacid, with chloroacetic acid and chloropropionic acid being selected from the chloroacids.

[0034] In the comparative experiment, the ratio of β-chitoxin, modifier, and alkali was set to 1:4:9, the reaction temperature was 60℃, and the reaction time was 4 h. The experimental results obtained based on the above parameters are shown in Table 1. Table 1. Differences in carboxylation modification effects of chloroacetic acid, 2-chloropropionic acid, and 2-bromopropionic acid under the same reaction conditions. In the comparative experiment, the effect of the type of carboxylic acid on the DD and DS values ​​of the modified product was as follows: Figure 4 As shown, the DS value of carboxylated chitosan obtained by modification with 2-bromopropionic acid is greater than that of carboxylated chitosan obtained by modification with 2-chloropropionic acid, and is similar to that of carboxylated chitosan obtained by modification with chloroacetic acid. The DD value of carboxylated chitosan obtained by modification with 2-bromopropionic acid is less than that of carboxylated chitosan obtained by modification with 2-chloropropionic acid, but greater than that of carboxylated chitosan obtained by modification with chloroacetic acid.

[0035] In this embodiment, the crude product modified with 2-bromopropionic acid (the carboxylation reaction product in step S2, i.e., carboxylated chitin that has not undergone product purification) is in the following state: Figure 5 and Figure 5-1 As shown, the 2-bromopropionic acid modified product forms a relatively viscous precipitate after a period of reaction, thus interfering with subsequent reactions and reducing the reaction rate of β-chitosan with the modifier. From Figure 5 It is known that the carboxylated chitin obtained by modification with 2-bromopropionic acid forms a relatively viscous precipitate in isopropanol, thereby blocking or reducing the further deacetylation reaction of carboxylated chitin and preventing further increase in DD value. Figure 5-1 The experiment showed that even when the container was inverted, the relatively viscous precipitate still adhered to the bottom of the container, demonstrating that the relatively viscous precipitate has good mutual adhesion.

[0036] In this embodiment, in order to measure the composition and properties of the carboxylated chitin obtained according to the above steps and parameters, the different carboxylated chitins obtained by different modifiers in the comparative experiment were subjected to elemental characterization analysis, potentiometric titration analysis, FT-IR (Fourier Transform Infrared Spectroscopy) characterization, and other methods. 1Characterization using 1H NMR (Proton Nuclear Magnetic Resonance).

[0037] In this embodiment, the elemental characterization analysis is performed as follows: Weigh 1.9–2.2 mg of modified chitin sample, wrap the sample in a tin boat, and then place it in an elemental analyzer for analysis. Calculate the C / N ratio based on the mass percentages of C and N elements in the analysis results. Repeat the analysis three times and take the average C / N ratio.

[0038] In this embodiment, the potentiometric titration characterization is performed as follows: Weigh 0.25–0.30 g of carboxylated chitin product and dissolve it in 30 mL of 0.1 M sodium chloride solution. Then, add 0.1 M hydrochloric acid solution dropwise until the pH of the solution decreases to 4.0. Finally, perform potentiometric titration with 0.05 M sodium hydroxide solution. The quantitative relationship between DD and DS is shown in formulas (1) and (2): (1); (2); Wherein, 203 is the molecular weight of undeacetylated chitin, 72 is the molecular weight of carboxylated chitin (carboxyethyl in this example), and 42 is the molecular weight of deacetylated chitin.

[0039] Based on formulas (1) and (2), formulas (3) and (4) are derived. Then, the experimental values ​​obtained from potentiometric titration and elemental analysis are combined. C / N Enter the numerical values ​​into formulas (3) and (4) to calculate the sample's... DD Numerical values ​​and DS Numerical value.

[0040] (3); (4).

[0041] In this embodiment, the FT-IR characterization operation is as follows: The carboxylated product appears as a loose, porous, white sponge. After thorough drying, the sponge-like product is clamped onto the detection port of an infrared spectrometer for testing, with a scanning range of 4000–500 cm⁻¹. -1 .

[0042] Specifically, such as Figure 6 As shown, FT-IR spectra of unmodified β-chitin and carboxylated chitin obtained by modification with three different modifiers in the comparative experiment are presented. Compared with β-chitin, the carboxylated chitin obtained by modification with 2-bromopropionic acid, 2-chloropropionic acid, and chloroacetic acid shows a higher FT-IR spectrum at 1585 cm⁻¹.-1 And 1413 cm -1 There is a clear difference in peak shape at 1585 cm⁻¹. -1 The difference in peak shape reflects the absorption peak of the symmetric contraction vibration of carboxylates; at 1413 cm⁻¹ -1 The difference in peak shape reflects the asymmetric contraction vibration absorption peak of carboxylates. At 2983 cm⁻¹... -1 2930 cm -1 And 1460 cm -1 At this point, the carboxylation product of 2-bromopropionic acid shows a more pronounced absorption peak compared to 2-chloropropionic acid, indicating that 2-bromopropionic acid has better reactivity than 2-chloropropionic acid. This is consistent with the difference in DS derived by potentiometric titration in section 1.3.1, where the peak at 2983 cm⁻¹ is significant. -1 and 2930 cm -1 The stretching vibration peak corresponding to -CH3 is 1460 cm⁻¹. -1 This corresponds to the asymmetric angle-shifting vibration peak of -CH3. And at 2983 cm⁻¹... -1 2930 cm -1 And 1460 cm -1 At this point, the carboxylation product of 2-bromopropionic acid has a more obvious absorption peak than that of 2-chloropropionic acid, which means that 2-bromopropionic acid has better reactivity than 2-chloropropionic acid. This is consistent with the difference in DS derived by potentiometric titration in formulas (3) and (4), where 2983 cm⁻¹ -1 And 2930 cm -1 The stretching vibration peak corresponding to -CH3 is 1460 cm⁻¹. -1 The peak corresponds to the asymmetric variable-angle vibration of -CH3.

[0043] In this embodiment, 1 The procedure for HNMR characterization is as follows: Add 200 µL of deuterium oxide to 500 µL of the ultrafiltration concentrate, mix thoroughly, and then transfer to a 0.5 mm diameter NMR tube. Select the pressurized water peak pulse program for testing.

[0044] Figure 7 It is a modified product of 2-chloropropionic acid. 1 HNMR spectrum Figure 8 It is a carboxylated modified product of 2-bromopropionic acid. 1The HNMR spectra show that the peak positions of the two products are basically the same, indicating that the structures of the two products are basically the same. The peak near 1.2 ppm is the -CH3 proton peak of the carboxyethyl group; the peak near 2.0 ppm is the -CH3 proton peak of the acetamide group; the peak near 2.68 ppm is the -CH proton peak after the acetamide group on C2 is deacetylated; the peak signals near 3.2~4.0 ppm include the proton peaks on the repeating units C3, C4, C5, and C6, as well as the -CH proton peak of the carboxyethyl group.

[0045] Combined with FT-IR analysis, it was confirmed that both the modified products of 2-chloropropionic acid and 2-bromopropionic acid are 1-carboxyethyl chitin. Furthermore, within a similar peak signal intensity range of 3.2–4.0 ppm, the absorption peak intensity of the 2-bromopropionic acid modified product at 1.2 ppm is significantly greater than that of the 2-chloropropionic acid modified product, further demonstrating that 2-bromopropionic acid exhibits higher reactivity.

[0046] Example 2 Based on Example 1, this embodiment provides an orthogonal experiment to verify the optimal parameter selection of the above preparation method.

[0047] In this embodiment, the factors affecting the chitin carboxylation reaction conditions studied include: the amount of modifier added, temperature, amount of alkali added, reaction time, and the ratio of isopropanol to water in the reaction system (hereinafter referred to as alcohol-water ratio).

[0048] Orthogonal experiment parameter settings: In this embodiment, the increase in DS during carboxylation modification leads to the precipitation of more precipitates, which effectively prevents subsequent carboxylation reactions from proceeding. The influence of reaction time on the overall carboxylation reaction of chitin is gradually masked. Simultaneously, since β-chitin, as verified by existing technology, has higher reactivity than α-chitin, the alcohol-to-water ratio has a smaller impact compared to other reaction parameters. Therefore, in this embodiment, a three-factor, three-level orthogonal analysis will be conducted primarily using the modifier dosage, reaction temperature, and alkali dosage.

[0049] In this embodiment, the amount of modifier sampled is set as factor A, and the molar ratio of the amount of modifier sampled to the amount of raw material β-chitosan sampled in the reaction stage is used as the numerical difference. The three levels are A1 (1 times the amount of chitosan sampled), A2 (2.5 times the amount of chitosan sampled) and A3 (4 times the amount of chitosan sampled).

[0050] In this embodiment, the reaction temperature is set as factor B, and the main solvent of the reaction system is isopropanol, which has the characteristic of boiling violently above 80 °C. Excessive reaction temperature will cause the isopropanol to evaporate rapidly, affecting other parameters of the reaction system. In addition, the modified crude product is highly viscous and cannot be completely recovered in a flask reaction. Therefore, the highest temperature of the three levels of factor B is selected as 80 °C, and the three levels are set as B1 (50 °C), B2 (65 °C), and B3 (80 °C).

[0051] In this embodiment, the molar ratio of modifier to alkali in the reaction system is used as the C factor to measure the amount of alkali added. The three levels are set as C1 (alkali added is twice the amount of modifier), C2 (alkali added is three times the amount of modifier), and C3 (alkali added is four times the amount of modifier). The orthogonal conditions for the nine groups of reactions are shown in Table 4.

[0052] Table 2 Orthogonal Experimental Design Table Orthogonal experimental data analysis: The specific data of the orthogonal experiment are shown in Table 3. The nine groups of orthogonal data showed significant differences in the products DD and DS, indicating that the selected three sets of varying parameters had a significant impact on the carboxylation reaction of chitin. This demonstrates the rationality of the orthogonal experiment in terms of factor selection and numerical design at different levels of the same factor. The differences in products DD and DS among the orthogonal groups are shown in Table 3. Figure 9 The data for k-value (within-group mean) analysis and R-value (range) analysis, using DS / DD as the indicator, are listed in Table 4. Figure 9 Based on the differences in k and R values ​​in the range analysis of the DS / DD index for the orthogonal groups in Table 4, the optimal reaction conditions were determined to be A3, B1, and C1, namely, the molar ratio of chitosan, 2-bromopropionic acid, and base was set at 1:4:8, the reaction temperature was 50 ℃, and R... A >R B >>R C This proves that the amount of 2-bromopropionic acid added is the most critical influencing factor (A), while the molar ratio of the amount of base to the modifier added (C) has a relatively small impact on the carboxylation reaction aimed at preparing products with extremely low DD carboxylation.

[0053] Table 3. Differences in carboxylation modification effects among orthogonal experimental groups Table 4. K-value analysis and R-value analysis using DS / DD as the indicator. Tables 5 and 6 list the k-value and R-value analysis data of the orthogonal groups using products DD and DS as indicators, respectively. The ranking of influencing factors for DD is completely different from the other two. Factor B, reaction temperature, has the greatest impact on the DD value. However, there is some controversy regarding the ranking of the influence of factors A and C on DD. As the only factor among the three that is related to the amount of base sample, factor C ranks even lower in the R-value analysis than factor A, which is not directly related to the deacetylation reaction. There are two reasons for this result. One is that the carboxylation reaction products in the precipitate state have a significant interference effect on the deacetylation reaction. According to the R-value analysis of DS, factor A is the core factor of the nucleophilic substitution main reaction. Factor A indirectly affects the DD value by promoting the rapid progress of the carboxylation reaction and inhibiting the deacetylation reaction earlier. The other is the definition of factor C. The molar ratio of base to acid sample is not equivalent to the strength of alkalinity in the reaction solution. Only when the amount of modifier sample is consistent can factor C be expressed as the strength of alkalinity of the reaction system and show a correlation with the deacetylation reaction-related parameter DD.

[0054] Table 5. K-value analysis and R-value analysis using DD as an indicator Table 6. K-value analysis and R-value analysis using DS as an indicator In addition, k-value and R-value analyses were performed on the potential indicator C / N, and the data are shown in Table 7. In terms of the ranking of influencing factors, the ranking results of indicator C / N are consistent with those of indicators DS / DD and DS. However, in terms of R-value comparison, its ranking of influencing factors is closer to that of the DS indicator. A >>R B >R C Instead of the R of the DS / DD index A >R B >>R C Therefore, the C / N indicator is more often used as a substitute for the DS indicator.

[0055] Table 7. K-value analysis and R-value analysis using C / N as indicators Example 3 Based on the orthogonal experimental results of Example 2, this embodiment provides an optimization scheme for the preprocessing stage.

[0056] In this embodiment, based on the analysis of the above orthogonal experimental results, the DS of each group was high, with five of the nine experimental groups exceeding 100%. The excess DS originated from the carboxylation modification of the hydroxyl group at the C3 position or the carboxylation modification of the amino group at the C2 position generated by deacetylation. The reactions at these sterically hindered sites confirmed the problem of excessively long reaction times, thus masking the characterization data patterns of the pretreatment stage. Therefore, based on the experimental phenomena recorded in Table 8, the reaction time was initially optimized. The reaction conditions for the phenomenon recording group were set to react directly without alkali swelling to avoid the situation where the reaction was stopped prematurely before the rapid reaction stage was fully completed in other experimental groups. No precipitate was observed in the group without alkali swelling. Based on the differences in reaction phenomena, 40 min was used as the reaction time for subsequent studies.

[0057] Table 8. Comparison of Experimental Phenomena Data analysis of the room temperature alkalization time group experiment: The experimental group was subjected to the optimal conditions of orthogonal analysis, namely, the molar ratio of chitin, modifier and alkali was 1:4:8, the reaction temperature was 50 ℃, and the reaction time was 40 min. Only the swelling time after adding alkali solution in the pretreatment stage was set as a variable. The structural parameters of the prepared samples are shown in Table 9.

[0058] Table 9. Differences in carboxylation modification effects at different alkaline solution swelling times at room temperature The effect of alkaline soaking time at room temperature on the product's DD and DS is as follows: Figure 10 As shown, the DD value gradually increases with the extension of alkali swelling time, and the growth trend is relatively stable, which is consistent with the expected results. The DS value gradually increases with the extension of alkali swelling time in the range of 0~2 h. In the range of 2~5 h, the DS value decreases instead of increasing with the extension of swelling time, especially in the range of 2h~3 h, the DS value shows the greatest downward trend. Chitin disperses well when swollen in pure isopropanol; even a 24-hour swelling process does not cause chitin to agglomerate. However, the introduction of alkali rapidly increases the viscosity of the initially contacting molecular chain segments, leading to the formation of clumps that adhere to the beaker wall. The swelling effect of the molecular chain segments in the central region of these clumps is poor. A short swelling process does not result in a significant difference in swelling effect between the surface and central regions of the clumps. As the reaction system heats up, the viscosity of most clumps rapidly decreases and they disintegrate, causing little interference with the reaction process. Conversely, a prolonged swelling process causes the surface molecular chain segments of the clumps to become fully etherified and viscous. Upon heating, the clumps cannot disintegrate quickly, and the inner molecular chain segments come into contact with the modifier later. The central molecular chain segments are even encapsulated throughout the reaction and cannot undergo carboxylation. Figure 10The sharp decline in DS values ​​within the 2-3 h range indicates that the ideal alkalization and swelling time for chitin molecular chain segments is 2 h.

[0059] Data analysis of the low-temperature freezing alkalization group experiment: To address the issue of differential swelling of chain segments under alkaline solution at room temperature, this embodiment presents and verifies two solutions. One approach involves freezing at -20 °C, where water molecules in the reaction system condense into small ice crystals, creating a volume difference. Upon heating, the clumps disperse more easily, thus offsetting the negative impact of the difference in swelling degree. The other approach is to directly abandon the limitation of the alkaline environment, replacing the alkaline solution with water and freezing it at -20 °C to transform it into small ice crystals. This opens up the numerous non-crystalline, insoluble regions in β-chitosan, achieving a similar swelling effect.

[0060] In this embodiment, the experimental group also carried out the reaction under the optimal conditions of orthogonal analysis. The molar ratio of chitin, modifier and alkali was set to 1:4:8, the reaction temperature was 50 ℃, and the reaction time was 40 min. The time of freezing at -20 ℃ after adding alkali solution in the pretreatment stage was used as a variable. The differences in carboxylation modification effect of different freezing times are shown in Table 10.

[0061] Table 10. Differences in carboxylation modification effects with different alkaline solution freezing times. The experimental results of the three groups with freezing times > 0 h showed that the DS values ​​were all greater than 61.78% of those obtained by swelling in alkaline solution at room temperature for 3 h, proving that the idea of ​​creating a volume difference through freezing does indeed help alleviate the negative effect of clumping and thus affecting carboxylation. However, as... Figure 11 The curves showing the effects of different alkali freezing times on the product's DD and DS values ​​indicate that the DS and DD values ​​do not show a clear pattern. There are two reasons for this: firstly, the water content in the reaction system is low, resulting in a small volume difference during freezing, which is insufficient to penetrate the interior of the clump, and the clump still plays a dominant role in inhibiting the reaction; secondly, the amount of alkali dissolved in the solution at -20 ℃ is too small, which greatly reduces the promoting effect on swelling and makes it impossible to reflect the changing trend of the swelling effect.

[0062] Data analysis of the low-temperature freezing without alkalization group experiment: In this embodiment, the experimental group was set with a molar ratio of chitin, modifier, and alkali of 1:4:8, a reaction temperature of 50 °C, and a reaction time of 40 min. Water was used instead of alkali to assist in the swelling of β-chitin. The mixture was stirred at room temperature for 10 min and then frozen at -20 °C for 24 h. The amount of water added was used as a variable. The data from this group are compared with the best data from Tables 9 and 10 in Table 11. The bar chart of the experimental results is shown below. Figure 12 As shown.

[0063] Table 11 Differences in carboxylation modification effects of different pretreatment methods Under the same 40-minute reaction time, the dissolved solids (DS) of all three products in the pure freezing group showed a significant increase, while the dissolved diastolic solids (DD) of the product increased slightly compared to the unfrozen group, but the increase was small. The swelling process essentially expands the non-crystalline, insoluble region of β-chitin, making the increase in DD inevitable. In the two groups with excess water (1:1 alcohol-water and 1:2 alcohol-water groups), the introduced water molecules have penetrated into the non-crystalline region and cannot be removed by a simple alcohol washing step. The increased water content in the reaction system, based on the experimental results, is more conducive to the deacetylation reaction.

[0064] In this embodiment, as shown in Table 12 and Figure 13 As shown, by introducing a no-pretreatment group (no freezing) with extended reaction time and a pure freezing group with different alcohol-to-water ratios for comparison, the no-pretreatment group even had a higher product DS after extending the reaction time than the freezing group. In terms of product DD value, the no-pretreatment group was higher than the pure freezing group. This is related to the slower rate of aggregation into small sticky spheres in the no-pretreatment group. All molecular chain segments in the no-pretreatment group were gradually carboxylated, and there was no problem of rapid carboxylation in the early stage of the reaction forming a large number of small sticky spheres, which led to the encapsulation of a small number of chain segments with a low degree of carboxylation and termination of the reaction. Similarly, the product morphology that hinders the deacetylation reaction appeared later, which also increased the product DD value.

[0065] Table 12 Differences in carboxylation modification effects between the pure freezing group and the untreated group In conclusion, if the goal is to obtain chitin with extremely low DD carboxylation, then the best raw material pretreatment method is one that does not involve freezing or alkali soaking.

[0066] Example 4 This embodiment is based on the experimental results of Embodiments 2 and 3, and provides analytical experiments for different single factors.

[0067] In this embodiment, the order of single-factor analysis is based on the influence ranking obtained from the orthogonal experiment, and is carried out in the order of modifier dosage → temperature → alkali dosage. The study of the reaction time parameter is placed after the three factors of the orthogonal analysis.

[0068] In this embodiment, when the amount of modifier added is a variable, the reaction raw material is set as β-chitosan, and β-chitosan only participates in the reaction after being stirred and swollen in isopropanol for 24 h. The reaction temperature is set at 50 ℃, the reaction time is 40 min, the amount of alkali added is set to twice the amount of modifier added, the amount of alkali solution is kept constant at 6.35 mL, and the remaining sodium hydroxide is added to the reaction system in solid form. The specific product structure data are shown in Table 13.

[0069] In this embodiment, the effect of the modifier dosage on the product DD and DS is as follows: Figure 13 As shown in the figure, the differences in DD values ​​of the products in this experimental group were small, showing no obvious pattern; DS showed an M-shaped change pattern with the change in modifier dosage. The first turning point was in the 4.6:1 modifier dosage group, where the constant alkali solution of 6.35 mL in each group corresponded exactly to 9 times the β-chitosan dosage, while the alkali dosage in the 4.6:1 group was 9.2 times; in the experimental groups after the 4.6:1 group, there was a significant amount of alkali in solid form. Compared to the optimal alcohol-water ratio in the 4.6:1 group, the solubility of alkali increased during the reaction heating process, and some solid alkali dissolved and competed with the molecules related to the main reaction for water molecules. When the reaction system contained only an alcohol-water ratio of 10:0, the carboxylation substitution efficiency of chloroacetic acid was greatly reduced, and the excess solid alkali led to a decrease in the carboxylation rate, which competed with the carboxylation rate increase brought about by the modifier dosage. Figure 14 The initial decrease followed by an increase in the DS values ​​of the products from the 5.2:1 group to the 7.6:1 group reflects the competitive result of the two effects; after the 7.6:1 group, the DS values ​​of the products dropped sharply and then stabilized.

[0070] Table 13 Differences in carboxylation modification effects with different acid dosages In this embodiment, with reaction temperature as the variable, the reaction was set such that the raw material β-chitin only swelled in isopropanol for 24 h before participating in the reaction. The molar ratio of chitin, modifier, and alkali was 1:7.6:15.2, the reaction time was 40 min, the amount of alkali solution was kept constant at 6.35 mL, and the remaining sodium hydroxide was added to the reaction system in solid form. The differences in structural parameters of the products from each experimental group are shown in Table 14. The effect of reaction temperature on the DD and DS of the products is as follows. Figure 15As shown, within the temperature range of 30–50 °C, the product DS gradually increases with increasing temperature. However, within the temperature range of 50–60 °C, the product DS gradually decreases with increasing temperature. This contradicts the orthogonal analysis results of B1 < B2 < B3 (in terms of carboxylation effect, 50 °C < 65 °C < 80 °C). The reason for this is that higher temperatures cause a large number of molecular chain segments to carboxylate more rapidly in the initial stage and aggregate into sticky balls or clumps, greatly hindering the carboxylation reaction. Within the temperature range of 30–60 °C, the product DD value continues to increase, and the increase in DD value is significantly greater in the temperature range of 50–60 °C. This is consistent with the experimental results of orthogonal analysis that temperature is the primary influencing factor for the deacetylation reaction.

[0071] Table 14 Differences in carboxylation modification effects at different reaction temperatures In this embodiment, with the amount of alkali added as a variable, the reaction was set such that the raw material β-chitin only participated in the reaction after swelling in isopropanol for 24 h. The molar ratio of chitin to modifier was set to 1:7.6, the reaction temperature was 50 °C, and the reaction time was 40 min. The alkali solution was kept constant at 6.35 mL, and the remaining sodium hydroxide was added to the reaction system in solid form. The differences in structural parameters of the products from each experimental group are shown in Table 15. The effect of the amount of alkali added on the DD and DS of the products is as follows. Figure 16 As shown, the product DS increases with the increase of alkali dosage, then stabilizes and finally slowly decreases; the DD difference is small and the regularity is not obvious; based on the DS / DD values, within the stable range of 1.6:1 to 2.2:1, 1.6:1 is established as the optimal reaction condition, that is, the molar ratio of β-chitoxin, 2-bromopropionic acid and alkali is 1:7.6:12.16.

[0072] Table 15 Differences in carboxylation modification effects with different alkali dosages In this embodiment, with reaction time as the variable, the reaction was set such that the raw material β-chitin only participated in the reaction after swelling in isopropanol for 24 h. The molar ratio of chitin, modifier, and alkali was set to 1:7.6:12.16, and the reaction temperature was 50 °C. The differences in structural parameters of the products from each experimental group are shown in Table 16. The effect of reaction time on the products DD and DS is as follows. Figure 17 As shown, the product DS and DD values ​​gradually increased with the extension of reaction time and then tended to stabilize. The DS value stabilized after 40 min of reaction, while the DD value stabilized after 60 min. Therefore, the optimal reaction time was set to 40 min.

[0073] Table 16 Differences in carboxylation modification effects at different reaction times In summary, groups with higher leaving activity can pass through the initial reaction stage more quickly, thus enabling the preparation of carboxylated chitin with extremely low DD. Preliminary experiments were conducted on the 2-bromopropionic acid carboxylation modification of β-chitin. Under the same experimental conditions, the DS of the 2-bromopropionic acid product was as high as 121.26±0.83%, compared to only 54.32±0.45% for the 2-chloropropionic acid product, demonstrating higher reactivity. Furthermore, the differences in experimental phenomena among chloroacetic acid, 2-chloropropionic acid, and 2-bromopropionic acid also confirm that the substitution activity of 2-bromopropionic acid is much greater than that of 2-chloropropionic acid and only slightly lower than that of chloroacetic acid.

[0074] Based on the characteristic that the carboxylation modified product of 2-bromopropionic acid interferes with subsequent reactions after agglomeration into precipitates, a rigorous orthogonal analysis → pretreatment optimization → single-factor analysis process was established to study the carboxylation modification with extremely low DD. Using the values ​​of DS and DD as indicators, the optimal reaction conditions for the extremely low DD carboxylation reaction were determined to be: β-chitin only participates in the reaction after swelling in isopropanol at room temperature for 24 h; the molar ratio of chitin, modifier and base was set to 1:7.6:12.16; the reaction temperature was 50 ℃; and the reaction time was 40 min.

[0075] In the raw material pretreatment stage, by comparing the effects of four swelling methods—alkaline solution-free room temperature swelling, alkaline solution-free room temperature swelling, alkaline solution-free freezing swelling, and alkaline solution-free freezing swelling—the pretreatment condition for β-chitoxin based on either higher reactivity or lower DD was determined to be alkaline solution-free room temperature swelling.

[0076] Example 5 This embodiment provides a performance characterization experiment for carboxylated chitin.

[0077] In this embodiment, the carboxylated chitin is a very low degree of deacetylation carboxylated chitin obtained by the preparation method based on the preferred parameters in the above embodiment. The carboxylated chitin is 1-carboxyethyl chitin (1-CECT), and the degree of deacetylation of 1-carboxyethyl chitin is DD≤10%.

[0078] In this embodiment, the performance characterization experiments for 1-carboxyethyl chitin include: viscoelasticity test, thermal stability test, hygroscopicity and moisture retention test, in vitro enzyme degradation performance test, in vitro cytotoxicity test, and heavy metal content test.

[0079] In this embodiment, the viscoelasticity test is divided into viscosity test and elasticity test. The direct ductility (DD) and loss modulus (DS) of the carboxylated chitin samples used for viscoelasticity comparison should be as close as possible. For the evaluation of the elastic modulus and loss modulus of 1-carboxyethyl chitin samples, carboxymethyl chitin (CMCT) samples with similar DD and DS are used as controls, and characterization is performed within the same frequency range. The detailed modification conditions for the samples are shown in Table 1-1. The structural parameters of the 1-CECT sample are DD=14.53% and DS=115.38%. The modification parameters are: chitin is first frozen in a 1:2 solution of isopropanol and water for 24 h; the molar ratio of chitin, modifier (2-bromopropionic acid), and base is 1:4:8; the reaction temperature is 50 ℃; and the reaction time is 40 min. The structural parameters of the CMCT sample are DD=17.51% and DS=118.22%. The molar ratio of chitin, modifier chloroacetic acid, and base is 1:4:9; the reaction temperature is 60 ℃; and the reaction time is 10 min.

[0080] Table 1-1 Sample Modification Conditions The specific procedure is as follows: First, prepare a 3% (w / w) solution of the lyophilized 1-CECT and CMCT samples. Accurately measure 280 µL of this solution onto the 25 mm diameter rheometer sample plate, forming a large circular droplet. Then, add a ring of silicone oil around the droplet to seal the edge and prevent moisture evaporation. The distance between the upper plate and the sample plate is set to 0.4 mm, and the corresponding tests are performed. For the elasticity test, the dynamic scan uses frequency as the variable, with a test range of 1–10 Hz, recording the elastic modulus (G′) and loss modulus (G″) at different frequencies. For the viscosity test, the dynamic scan uses shear rate as the variable, with a test range of 0.1–100 s. -1 The scanning strain was 0.1%, and the shear viscosity η at different shear rates was recorded.

[0081] The elastic modulus-frequency curves of the two carboxylated chitins are as follows: Figure 2-1 As shown, the loss modulus-frequency curve is as follows: Figure 2-2 As shown, the shear viscosity-shear rate is as follows: Figure 2-3 As shown. In the 1–10 Hz range, the loss modulus G″ of the 1-CECT group is lower than that of the CMCT group; however, in terms of elastic modulus G′, the 1-CECT group exceeds the CMCT group around 5 Hz, and is slightly lower than the CMCT group in the remaining range. In addition, [the following text is missing]. Figure 1 -2 shows a comparison of viscosity between the 1-CECT group and the CMCT group, in the range of 0.1~100s. -1 Within the test range, the shear viscosity η of the 1-CECT group was lower than that of the CMCT group.

[0082] The elastic modulus G′, loss modulus G″, and shear viscosity η of 1-CECT and CMCT samples are comparable, but there are some differences. This difference stems from the interaction between the methyl and carboxyl groups in 1-CECT. The methyl and carboxyl groups are separated by only one carbon atom. Besides being affected by the combined effects of carboxyl induction and conjugation, the methyl group also faces steric hindrance with the carboxyl group, resulting in insignificant hydrophobicity and hydrogen bonding crosslinking in 1-CECT. Although the viscoelasticity of uncrosslinked 1-CECT and CMCT is not as good as that of HA, the crosslinking methods used in HA can be learned to improve viscoelasticity through chemical crosslinking to meet the requirements of biomedical applications.

[0083] In this embodiment, the thermal stability test was conducted on 1-CECT samples with a DS not exceeding 100% and a very low DD value. Detailed modification conditions are shown in Appendix Table 2-1. The structural parameters of the 1-CECT samples were DD=7.10% and DS=82.89%. The modification parameters were: isopropanol swelling at room temperature for 24 h, a molar ratio of chitin, modifier, and alkali of 1:7.6:12.16, a reaction temperature of 50℃, and a reaction time of 40 min. The control group was the raw material β-chitin.

[0084] Appendix Table 2-1 Modification conditions for 1-CECT samples The specific procedure is as follows: Weigh 2-10 mg of sample and place it in the alumina crucible of the simultaneous thermal analyzer. Then, calcine the sample gradually under nitrogen atmosphere at a heating rate of 10 °C / min, with a temperature range of 25-800 °C. After the test, the TG thermogravimetric curve and DSC heat flow curve can be obtained, such as... Figure 3-1 and Figure 3-2 As shown.

[0085] Figure 3-1The thermogravimetric curves (TGA) of 1-CECT and β-chitosan are compared. The TGA curves of the sample are similar to those of the raw material β-chitosan, and can be roughly divided into three stages: the evaporation stage of free and bound water, the stage of rapid decomposition and mass decrease due to heating, and the stage where the sample has been largely decomposed and the mass gradually stabilizes. For 1-carboxyethyl chitosan, the first stage of water evaporation is from 25 to 131 °C, with a mass loss rate of about 15%. Compared with β-chitosan, the end temperature of the first stage is lower and the mass loss rate is higher, reflecting the stronger hygroscopic and moisturizing ability of 1-carboxyethyl chitosan. The second stage of rapid decomposition of the sample has a turning point at 336 °C, so it can be further divided into two stages: 240–336 °C and 336–497 °C. The mass loss rate decreases from 15% to 58% at the turning point and then gradually decreases to 76%. The starting temperature of the second stage is also lower than that of β-chitosan. The turning point at 336 °C corresponds to the decomposition of the chitosan side chains (carboxyethyl and acetamide groups) and the breakage of the main chain, respectively.

[0086] Figure 3-2 The comparison of the heat flow curves of 1-CECT and the raw material β-chitosan shows that for 1-carboxyethylchitosan, the endothermic peak at 72℃ corresponds to the water evaporation stage of the first phase of the thermogravimetric curve (25~131℃), the inflection point at 210℃ roughly corresponds to the first inflection point of 240℃ in the rapid decomposition stage of the sample in the thermogravimetric curve, and the exothermic peak at 336℃ corresponds exactly to the inflection point of 336℃ in the thermogravimetric curve.

[0087] In this embodiment, in the hygroscopicity and moisture retention test, 1-CECT samples with different DS and HA samples with different molecular weights were compared under the same environment and for the same time. The detailed modification conditions for 1-CECT are shown in Table 3-1. For DS56, the DS value is 56.37%, and the modification parameters are: swelling in isopropanol at room temperature for 24 h, a molar ratio of chitosan, modifier, and alkali of 1:7.6:12.16, a reaction temperature of 50 °C, and a reaction time of 15 min. For DS83, the DS value is 82.89%, and the modification parameters are: swelling in isopropanol at room temperature for 24 h, a molar ratio of chitosan, modifier, and alkali of 1:7.6:12.16, a reaction temperature of 50 °C, and a reaction time of 40 min. For DS126, the DS value is 125.75%, and the modification parameters are: chitosan first stirred in an isopropanol solution of alkali at room temperature for 1 h, a molar ratio of chitosan, modifier, and alkali of 1:2.5:7.5, a reaction temperature of 50 °C, and a reaction time of 1 min. h; DS163 has a DS=162.90%, and the modification parameters are as follows: chitosan is first placed in a solution of isopropanol and water in a 5.5:1 ratio at room temperature for 24 h; the molar ratio of chitosan, modifier, and alkali is 1:4:8; the reaction temperature is 50 °C; and the reaction time is 2 h. Molecular weight M wHA values ​​of 80–100 k, 800–900 k, and 1,800–2,000 k were denoted as HA(LMw), HA(MMw), and HA(HMw) respectively, serving as controls.

[0088] Table 3-1 Sample Modification Conditions The specific procedure for the hygroscopicity test is as follows: Weigh 0.3~0.5 g of freeze-dried sponge sample into a 200 mL beaker, then transfer the sample to a vacuum drying oven and dry it thoroughly at 50 ℃ under vacuum for 5 days. After drying, weigh the sample and record the initial mass. W 0 The sample was then transferred to a silica gel desiccator with appropriate humidity. After the sample absorbed moisture for a preset time, its mass was weighed again and recorded as follows. W t The moisture absorption rate is calculated according to formula (5). R a The details are as follows: (5).

[0089] The humidity options include a relative humidity of 43% and a relative humidity of 81%. The former involves simulating an environment for at least 72 hours by placing a silica gel desiccator containing 400 mL of saturated potassium carbonate aqueous solution, and after confirming that the ambient humidity meets the standard with a hygrometer, the preset moisture absorption time is 5 days. The latter involves simulating an environment for 72 hours by placing a silica gel desiccator containing 400 mL of saturated ammonium sulfate aqueous solution, and after confirming that the ambient humidity meets the standard with a hygrometer, the preset moisture absorption time is 3 days.

[0090] The specific procedure for the moisture retention test is as follows: Weigh 0.3~0.5 g of freeze-dried sponge sample into a 100 mL beaker, then transfer the sample to a vacuum drying oven and dry it thoroughly at 50 ℃ under vacuum for 5 days. After drying, transfer the sample to a silica gel desiccator with a relative humidity of 43% (RH) for 4 hours to absorb moisture. Weigh the sample and record the mass. W 0 The samples were then transferred to a thoroughly dried silica gel desiccator containing dried silica gel particles. After 24 hours, the samples were weighed and their mass recorded. W t The moisture retention rate is calculated according to formula (6). R r The details are as follows: (6) Figure 4-1 and Figure 4-2This reflects the difference in hygroscopicity between the 1-CECT group with different DS and the HA group with different molecular weights. For the 1-CECT group, all samples had a hygroscopicity greater than 100% under both humidity conditions. Unlike the hygroscopicity pattern of carboxymethyl chitin, the hygroscopicity value of 1-carboxyethyl chitin did not show a positive correlation with DS. This is because of the differences in DD among the samples. The amine group formed after the deacetylation reaction participates in a certain degree of hydrogen bond cross-linking while assisting in improving water solubility. As for HA, the hygroscopicity R... a The hygroscopic rate decreases with increasing molecular weight because HA becomes viscous after absorbing water, thus encapsulating some of the chain segments that did not fully absorb water. This phenomenon is more pronounced in high molecular weight HA, reflected in the hygroscopic rate R. a The trend of decreasing as molecular weight increases.

[0091] Figure 4-3 This reflects the difference in moisturizing rate between the 1-CECT group with different DS and the HA group with different molecular weights. HA with different molecular weights showed almost identical moisturizing rates. The moisturizing rates of all four samples in the 1-CECT group were lower than those of HA, and the moisturizing rate R... r It shows a clear positive correlation with DS.

[0092] In this embodiment, HA can be considered as a polysaccharide with a DS of approximately 50% at the C6 position. A 1-CECT sample with a DS close to 50% and a very low DD value was selected for thermal stability testing. Detailed modification conditions are shown in Table 4-1. The structural parameters of the 1-CECT sample were DD=5.29% and DS=56.37%. The modification parameters were: isopropanol swelling at room temperature for 24 h; a molar ratio of chitin, modifier, and alkali of 1:7.6:12.16; a reaction temperature of 50 °C; and a reaction time of 15 min.

[0093] Table 4-1 Sample Modification Conditions Specific procedures: The lyophilized 1-CECT sample was fully dissolved in 0.1 mol / L phosphate buffer at pH 7.4 to prepare a 0.5% (w / w) carboxylated chitin solution. Lysozyme was then added to the above solution to prepare an initial in vitro enzymatic degradation solution with a lysozyme content of 1 mg / mL. The enzymatic reaction was carried out in a water bath at a constant temperature of 37 ℃. The OD value of the solution at 218 nm was measured at 1 h, 2 h, 3 h, 4 h and 5 h after the start of the reaction.

[0094] UV full-spectrum scans of 0.5% (w / w) samples at different degradation times, for example... Figure 5-2As shown, the samples did not exhibit significant spectral differences across the entire UV spectrum within 0–5 h after the addition of lysozyme. Therefore, the OD value at 218 nm was used as the standard for in vitro enzymatic hydrolysis. The OD value-lysozyme degradation time curve at 218 nm for the 0.5% (w / w) sample is shown in the figure. Figure 5-3 The results show that as the degradation time of lysozyme increases, the OD value at 218 nm gradually decreases, but the decrease is small, proving that the sample can be biodegraded and has a certain degree of degradation resistance.

[0095] The main relevant parameter for in vitro cytotoxicity is DD, and the detailed modification conditions of the tested samples are shown in Table 5-1. Among them, the structural parameters of 2-bromopropionic acid carboxylation sample 1-CECT (DD 5) are DD=5.29% and DS=56.37%, and the modification parameters are: swelling in isopropanol at room temperature for 24 h, the molar ratio of chitin, modifier and alkali is 1:7.6:12.16, the reaction temperature is 50 ℃, and the reaction time is 15 min; the structural parameters of chloroacetic acid carboxylation sample CMCT (DD 18) are DD=17.51% and DS=118.22%, and the modification parameters are: stirring in isopropanol solution in alkali at room temperature for 1 h, the molar ratio of chitin, modifier and alkali is 1:4:9, the reaction temperature is 60 ℃, and the reaction time is 10 min; the structural parameters of 2-chloropropionic acid carboxylation sample 1-CECT (DD 22) are DD=22.41% and DS=50.05%, and the modification parameters are: stirring in isopropanol solution in alkali at room temperature for 1 h. The molar ratio of chitin, modifier, and alkali was 1:4.5:9, the reaction temperature was 60 °C, and the reaction time was 1 h.

[0096] Table 5-1 Sample Modification Conditions The specific operation is based on the industry standard YY / T 0953-2020 "Medical Carboxymethyl Chitosan". The in vitro cytotoxicity of carboxylated chitosan is tested according to the national standard GB / T 16886.1 "Biological Evaluation of Medical Devices". The MTT method, also known as the MTT colorimetric method, determines the cell viability by characterizing the activity of succinate dehydrogenase in mitochondria of living cells. Only living cells can reduce the added MTT (thiazolyl blue) into water-insoluble blue-purple crystals, while dead cells cannot perform this life activity. The precipitated blue-purple crystals have a significant absorption peak at 490 nm after being dissolved in DMSO (Dimethyl Sulfoxide). The difference in OD (Optical Density) values ​​between the experimental group and the control group at 490 nm is detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability can be inferred according to formula (7).

[0097] (7).

[0098] The specific procedure is as follows: Add sterile phosphate buffer solution to the outermost ring of a 96-well plate, adding 100 µL to each well. Seed L929 mouse fibroblasts into the inner ring of the 96-well plate, approximately 6 × 10⁶ cells per well. 3 Cells were seeded into wells and incubated at 37 °C with 5% CO2 for 24 h. Once the cells adhered, initial concentrations of 2% (w / w), 1% (w / w), and 0.5% (w / w) culture medium were added to the wells of the experimental groups, while an equal volume of PBS buffer was added to the wells of the control group. Five replicates were set up for each group, and incubation was performed for 24 h, 48 h, and 72 h, respectively. After incubation, the cells were initially observed using a fluorescence microscope. The cytotoxicity grading of cell morphology is shown in Table 5-2. After observation and recording, 25 µL of 5 mg / mL MTT sterile phosphate buffer was added to each well for a second incubation of 3 h. The culture medium was then removed, and 150 µL of DMSO was added to each well to dissolve the blue-purple crystals. After shaking, the OD value at 490 nm was measured using a microplate reader.

[0099] Table 5-2 Qualitative Classification of Cytotoxicity Based on Cell Morphology The in vitro cytotoxicity of L929 in the three samples is shown in Table 5-3. The differences in in vitro cytotoxicity between the two 1-CECT samples at different cell culture times are shown in Table 5-3. Figure 6-1 As shown, the DD5 group had higher cell viability than the DD22 group at both 24 h and 72 h of cell culture, and the standard deviation of the data was smaller. Figures 6-2a to 6-2d As shown, cell morphology images of two 1-CECT sample cells cultured for 48 h and 72 h were compared. Figure 6-2a and Figure 6-2c At 48 h of cell culture, both the DD5 and DD22 groups showed cells that had essentially covered the surface, with fewer dead round cells; in fact, the DD22 group had a denser cell distribution. Figure 6-2d As shown, however, at 72 h of cell culture, the number of cells in the DD 22 group decreased significantly, and the fine dots on the vacant sites were traces left by the complete apoptosis of dead cells; as shown Figure 6-2bAs shown, at 72 h of cell culture, the number of cells in the DD 5 group was still relatively large, the proportion of dead cells did not change significantly, and the cell distribution was even denser than at 48 h. This further demonstrates the weaker cytotoxicity of the DD 5 group. Given that the molecular structures of the two experimental groups are consistent and the DS values ​​of the products are similar, this difference in cytotoxicity can be mainly attributed to the difference in DD values.

[0100] Table 5-3 Cytotoxicity of different carboxylic acid modified products at different culture times and concentrations In addition, the 2% (w / w) DD 5 group samples also showed a significant decrease in cell viability after 72 h of cell culture compared to 48 h. In contrast, the 1% (w / w) and 0.5% (w / w) samples had higher cell viability, proving that the lower cell viability of the 2% (w / w) DD 5 group samples was not due to the cytotoxicity of the samples, but rather to the high viscosity of the samples.

[0101] The cytotoxicity comparison between the 1-CECT (DD 5) group and the CMCT (DD 18) group at different culture times is as follows: Figure 6-3 As shown, the cytotoxicity of the two carboxylated chitosans was compared at two suitable concentrations of 1% (w / w) and 0.5% (w / w). The cell viability of the DD5 group did not show a significant difference with increasing cell culture time, and the standard deviation was also small. Conversely, the cell viability of the DD18 group gradually decreased with increasing cell culture time, dropping to near the 75% cytotoxicity threshold at 72 h of culture. The results for each concentration of DD5 and DD18 samples at 48 h and 72 h of cell culture are shown below. Figures 6-4a to 6-4d as well as Figures 6-5a to 6 As shown in ~5d, both concentrations of carboxylated modified product samples grew well at 48 h, with dense cell distribution and few dead cells; while at 72 h, Figure 6-5 c is significantly higher than Figure 6-5 a has more dead cells, Figure 6-5 d is significantly less distributed in cells than Figure 6-5 b. Tight. Combining the viscoelastic differences between 1-carboxyethyl chitosan and carboxymethyl chitosan mentioned above, the decrease in cell viability is a combined result of the viscoelastic differences and DD differences.

[0102] In this embodiment, the heavy metal content was tested using 1-CECT samples with a DS of no more than 100% and a very low DD value. Detailed modification conditions are shown in Table 6-1. The structural parameters of the 1-CECT samples were DD = 7.10% and DS = 82.89%. The modification parameters were: isopropanol swelling at room temperature for 24 h; a molar ratio of chitin, modifier, and alkali of 1:7.6:12.16; a reaction temperature of 50 ℃; and a reaction time of 40 min.

[0103] Table 6-1 Sample Modification Conditions Specific procedures: The lyophilized 1-CECT sample was placed in a vacuum drying oven and dried under vacuum at 50 °C for 5 days. Then, it was attached to the sample stage with conductive adhesive, and gold was sputtered onto the sample surface three times. EDS energy dispersive spectroscopy analysis was performed using a scanning electron microscope at an accelerating voltage of 15 kV.

[0104] Figure 7-1 These are elemental mapping scans of 1-CECT samples for some common elements. The outlines, lines, and details of the carbon (C) and oxygen (O) elemental mapping scans are compared with the original images. Figure 7-1 The top left corner is almost identical; nitrogen (N) and sodium (Na) are distributed one per carboxylated chitin repeating unit on average, with relatively low content but very uniform dispersion. Therefore, the outline of the original image can also be roughly seen in the elemental distribution diagrams of nitrogen and sodium, but the lines are noisy and the details are unclear. As shown in Table 6-2, heavy metals such as mercury (Hg), lead (Pb), chromium (Cr), and cadmium (Cd) have extremely low signal intensity and cannot map the original appearance of the sample. Heavy metals are generally present with the raw materials and are easily chelated by chitin / chitosan, making them difficult to remove. This proves that the 1-carboxyethyl chitin material prepared from squid cartilage β-chitin is almost free of heavy metals and is suitable for subsequent research and development of related products.

[0105] Table 6-2 Elemental content of 1-carboxyethyl chitosan samples The above is a detailed description of the preferred embodiments of the present invention. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. A method for preparing carboxylated chitin with extremely low degree of deacetylation, characterized in that, Includes the following steps: S1. Raw material pretreatment: Chitin raw material is dispersed in isopropanol and subjected to swelling pretreatment to obtain swollen chitin; S2, Carboxylation reaction: The swollen chitin is mixed with bromoic acid and alkali, and carboxylation reaction is carried out at a reaction temperature of 45℃~55℃ for 30min~50min to obtain the carboxylation reaction product; wherein, the molar ratio of chitin, bromoic acid and alkali is 1.0:7.0~8.2:11.5~13.

0. S3. Product purification: The carboxylation reaction product is post-processed to obtain carboxylated chitosan with a degree of deacetylation DD≤10%.

2. The method for preparing extremely low degree of deacetylation carboxylated chitin according to claim 1, characterized in that, The chitin raw material is β-chitin; the bromoacid is 2-bromopropionic acid.

3. The method for preparing extremely low degree of deacetylation carboxylated chitin according to claim 2, characterized in that, In step S1, the swelling pretreatment time is 20h~28h.

4. The method for preparing carboxylated chitin with extremely low deacetylation degree according to claim 2, characterized in that, In step S1, the swelling pretreatment is performed at room temperature.

5. The method for preparing extremely low degree of deacetylation carboxylated chitin according to claim 3, characterized in that, In step S2, the molar ratio of β-chitosan, 2-bromopropionic acid and alkali is 1:7.6:12.

16.

6. The method for preparing carboxylated chitin with extremely low degree of deacetylation according to claim 3, characterized in that, In step S2, the carboxylation reaction is carried out at a temperature of 50°C.

7. The method for preparing carboxylated chitin with extremely low deacetylation degree according to claim 2, characterized in that, In step S2, the carboxylation reaction takes 40 minutes.

8. The method for preparing carboxylated chitin with extremely low deacetylation degree according to claim 2, characterized in that, In step S3, the post-processing includes: terminating the reaction, washing with alcohol, dissolving, adjusting pH, filtering, ultrafiltration, and lyophilizing.

9. The method for preparing carboxylated chitin with extremely low deacetylation degree according to claim 8, characterized in that, The alcohol washing process involves a first alcohol washing using a mixture of ethanol and 5M acetic acid aqueous solution, followed by a subsequent alcohol washing using a mixture of ethanol and purified water.

10. A 1-carboxyethyl chitosan prepared by the method for preparing extremely low deacetylation carboxylated chitosan according to any one of claims 1 to 9, characterized in that, The degree of deacetylation of the 1-carboxyethyl chitosan is ≤10%.

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