Functional double-network chitosan hydrogel, preparation method and application

Abstract

The present invention provides a functional double-network chitosan hydrogel, a preparation method and an application. The present invention utilizes acrylic acid and N,N-bis(acryloyl)cystamine as a cross-linking agent to simply polymerize and cross-link acryloyl chitosan to prepare a single-network hydrogel and optimize the mechanical properties of the product. Then, copper ions are introduced to carry out complexation of the carboxyl groups in the network to complete the double network construction of the hydrogel, further enhancing its mechanical properties. The use of copper ions has a role value in both structural optimization of the hydrogel and antibacterial application effect. Compared with the prior art, the preparation process of the double-network hydrogel of the present invention is simple and green, and the double-network structure of different forms provides its adjustable and excellent mechanical properties. The positive charge of the rich amino groups carried by chitosan gives the hydrogel an antibacterial function, and the metal ions involved in constructing the second network further enhance the antibacterial ability of the material, that is, the hydrogel has a dual antibacterial effect.

Description

Technical Field

[0001] The invention belongs to the technical field of functional hydrogels, and particularly relates to a functional double-network chitosan hydrogel, a preparation method and an application thereof. Background Art

[0002] Chitosan is the only alkaline polysaccharide in nature. It is a natural polymer material that is non-toxic, odorless, and has good biocompatibility and biodegradability. The chitosan molecular chain carries a wealth of active functional groups such as amino and hydroxyl groups, which are conducive to application-oriented functional modification or modification, and can be used to prepare functional materials for a variety of application scenarios. Therefore, hydrogels with a chitosan framework are widely used in wound dressings, tissue engineering, sustained release of drugs or growth factors, biosensors or actuators, heavy metal ion adsorption and other fields due to their advantages such as injectability, adhesion, degradability, biocompatibility, antibacterial activity, coagulation, antioxidant and positive charge.

[0003] Metal-loaded hydrogels are antibacterial materials that have emerged in recent years. The main preparation methods include loading metal particles or ions into the hydrogel through encapsulation, in situ generation, and electrostatic complexation. Based on the structure carrying antibacterial factors, large surface area contact of metal elements or stimulated release, the hydrogel can achieve efficient antibacterial effects.

[0004] In addition, the design of polymer network structures can endow hydrogels with various functional characteristics, such as redox, magnetic, optical, and thermal responsiveness. In addition to achieving intrinsic antibacterial properties, metal elements can also be used to implement more efficient antibacterial applications through photothermal or electrothermal conversion using their derivative structures. Commonly used metal elements include gold (Au), silver (Ag), copper (Cu), and iron (Fe). Metal elements loaded into hydrogels can not only improve antibacterial properties, but also maintain antibacterial activity for a long time, greatly reducing the emergence of bacterial resistance.

[0005] The patent with publication number CN 117116667A published on November 24, 2023 discloses a chitosan / polyacrylamide / MXene hybrid hydrogel electrolyte and its preparation method and application: a certain amount of single-layer Ti3C2Mxene aqueous dispersion and acrylamide monomer are added to the acetic acid solution of chitosan, and after stirring evenly, zinc salt, cross-linking agent and initiator are dissolved in sequence to obtain a mixed solution, which is reacted at a predetermined temperature for a preset time to obtain a hybrid hydrogel electrolyte. The prepared hybrid hydrogel electrolyte has high electrical conductivity, excellent mechanical tensile properties and self-healing properties. However, it uses Ti3C2Mxene, which is costly and does not have antibacterial properties. Summary of the Invention

[0006] The present invention aims to provide a functional double-network chitosan hydrogel and its preparation method. Acryloylated chitosan is cross-linked by simple polymerization of acrylic acid and N,N-bis(acryloyl)cystamine as a cross-linking agent to prepare a single-network hydrogel. The mechanical properties of the product can be optimized by structural control. The introduction of copper ions to complex the carboxyl groups within the network completes the double-network construction of the hydrogel, further enhancing its mechanical properties. The use of copper ions plays a valuable role in both optimizing the hydrogel's structure and achieving antibacterial effects. The preparation method is simple, the raw materials are readily available, and the cost is low.

[0007] Another object of the present invention is to provide an application of a functional double-network chitosan hydrogel for use as an antibacterial material.

[0008] The specific technical solutions of the present invention are as follows:

[0009] A method for preparing a functional double-network chitosan hydrogel comprises the following steps:

[0010] 1) preparing acryloyl chitosan;

[0011] 2) Preparation of single network chitosan hydrogel;

[0012] 3) Preparation of double-network chitosan hydrogel;

[0013] In step 1), chitosan is modified with acryloyl chloride;

[0014] In step 1), specifically, acryloyl chloride solution and sodium hydroxide solution are added dropwise to the water-soluble chitosan aqueous solution under ice bath conditions. After the addition is completed, the mixture is stirred in an ice bath and then at room temperature. The pH is then adjusted to neutral, and the mixture is separated and purified to obtain acryloyl chitosan.

[0015] In step 1), the concentration of the water-soluble chitosan aqueous solution is 0.05-0.1 g / mL; preferably 0.08 g / mL; the water-soluble chitosan is dissolved in deionized water to prepare a water-soluble chitosan aqueous solution;

[0016] In step 1), the acryloyl chloride solution is obtained by dissolving acryloyl chloride in dichloromethane; the volume ratio of acryloyl chloride dissolved in dichloromethane is 0.3-0.8:1, preferably 0.5:1;

[0017] In step 1), the sodium hydroxide solution refers to an aqueous sodium hydroxide solution, and the mass concentration of the sodium hydroxide solution is 25-30%, preferably 27%;

[0018] In step 1), the ratio of water-soluble chitosan in the water-soluble chitosan aqueous solution to acryloyl chloride in the acryloyl chloride solution is 3-5:1 g / mL, preferably 4:1 g / mL;

[0019] In step 1), the ratio of water-soluble chitosan to sodium hydroxide solution in the water-soluble chitosan aqueous solution is 1.5-3:1 g / mL, preferably 2:1 g / mL;

[0020] In step 1), the addition time is controlled within 10 minutes; the two solutions are added dropwise simultaneously);

[0021] In step 1), after the dropwise addition is completed, the stirring time in the ice bath is 2.5-3.5 hours, preferably 3 hours;

[0022] In step 1), the stirring time at room temperature is 15-18 h, preferably 16 h at 25 ° C;

[0023] In step 2), N,N-bis(acryloyl)cystamine and acrylic acid are polymerized and cross-linked to form acryloyl chitosan to obtain a single-network chitosan hydrogel;

[0024] Step 2) is specifically as follows: dissolving acryloyl chitosan in water, adding N,N-bis(acryloyl)cystamine and acrylic acid, stirring and mixing, adding potassium persulfate solution, bubbling nitrogen, placing the mixed solution in a mold, heating, and cooling to obtain a single network chitosan hydrogel.

[0025] In step 2), the ratio of acryloyl chitosan to water is 0.05-0.1 g / mL, preferably 0.0625 g / mL;

[0026] In step 2), the mass ratio of acryloyl chitosan to N,N-bis(acryloyl)cystamine is 8-12:1, preferably 10:1;

[0027] In step 2), the ratio of acryloyl chitosan to acrylic acid is 0.1-0.3 g / mL;

[0028] In step 2), the mass ratio of acryloyl chitosan to potassium persulfate in the potassium persulfate solution is 2-3:1;

[0029] The potassium persulfate solution is an aqueous solution of potassium persulfate, and the dosage ratio of potassium persulfate to solvent water is 0.1 g / mL;

[0030] In step 2), nitrogen was bubbled in for 30 min;

[0031] In step 2), the mold used is a polytetrafluoroethylene mold;

[0032] In step 2), heating refers to heating to 55-65°C for 3.5-4.5 hours, preferably 60°C for 4 hours;

[0033] In step 2), cooling refers to cooling to room temperature and maintaining for 1.5-2.5 hours, preferably cooling to room temperature and maintaining for 2.0 hours.

[0034] In step 3), a double-network chitosan hydrogel is prepared using metal cations; the metal cations are copper ions;

[0035] Step 3) is specifically as follows: freeze-drying the single-network chitosan hydrogel, immersing it in a copper ion aqueous solution, and taking it out to obtain the hydrogel.

[0036] In step 3), the freeze-drying refers to freeze-drying at (-40°C)-(-50°C) for 24 hours;

[0037] In step 3), the mass concentration of the copper ion aqueous solution is 9-11%, preferably 10%;

[0038] In step 3), the copper ion aqueous solution is selected from a copper chloride aqueous solution, a copper sulfate aqueous solution or a copper nitrate aqueous solution.

[0039] In step 3), the soaking refers to immersing the single network chitosan hydrogel in the copper ion solution at room temperature for 10-14 hours, preferably 10 hours;

[0040] The present invention provides a functional double-network chitosan hydrogel, which is prepared by adopting the above method.

[0041] The present invention provides an application of a functional double-network chitosan hydrogel for use as an antibacterial material.

[0042] The inventors discovered that by structurally modifying the positively charged chitosan to create a material with an internal network, they further utilized active groups (e.g., carboxyl groups) introduced into the network to complex with metal cations, thereby enhancing the mechanical strength of the material through a dual network. Simultaneously, the chitosan and the metal ions in the network structure imparted a synergistic dual antibacterial capability to the prepared hydrogel material. The metal cation model selected in the present invention is copper ion. Chitosan itself possesses an antibacterial effect, and the introduction of metal cations not only completes the dual network construction of the hydrogel, but also enables its dual-modal antibacterial function.

[0043] Compared to the prior art, the dual-network chitosan hydrogel provided by the present invention exhibits excellent antibacterial activity due to the abundant positively charged amino groups on the chitosan backbone chains, which can act on the negative charges on the surface of microorganisms. The positively charged copper ions introduced to construct the dual network further enhance the antibacterial function of the hydrogel, giving it great potential as an antibacterial functional material. During preparation, acrylic acid and N,N-bis(acryloyl)cystamine as a crosslinking agent are simply polymerized and cross-linked to form acryloyl chitosan, resulting in a single-network hydrogel. The mechanical properties of the product can be optimized by structural control. By introducing copper ions to complex the carboxyl groups within the network, the dual network of the hydrogel is constructed, the crosslinking density is increased, and its mechanical properties are further enhanced. Chitosan itself is antibacterial, and the introduction of copper ions with antibacterial properties also synergistically enhances its antibacterial properties. The use of copper ions plays a valuable role in both structural optimization of the hydrogel and its antibacterial application effect. The materials used in the present invention all have corresponding functionalities. The lack of one of them makes it difficult to obtain a dual network structure, which also leads to a reduction in antibacterial effect parameters. In addition, the change of acrylic acid dosage affects the mechanical properties of hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the H NMR spectrum of acryloyl modified chitosan;

[0045] Figure 2 The infrared spectra of chitosan, acryloyl chitosan, single-network chitosan hydrogel and double-network chitosan hydrogel are shown;

[0046] Figure 3 Stress-strain curves of single-network chitosan hydrogel and double-network chitosan hydrogel (3 ml acrylic acid dosage);

[0047] Figure 4 Comparison of frequency sweep rheological properties of functional single-network and double-network chitosan hydrogels (3 ml acrylic acid dosage);

[0048] Figure 5 Comparison of the swelling behavior of single-network chitosan hydrogel and double-network chitosan hydrogel (3 ml acrylic acid dosage);

[0049] Figure 6 The results of the antibacterial performance study of single-network and double-network chitosan hydrogels; (a) The left side is the control group with Staphylococcus aureus, and the right side is the single-network hydrogel (left) and double-network hydrogel (right); (b) The left side is the control group with Escherichia coli, and the right side is the single-network hydrogel (left) and double-network hydrogel (right); (c) Comparison of the inhibition zone values ​​of chitosan hydrogels. DETAILED DESCRIPTION

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0051] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0052] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0053] Example 1

[0054] A method for preparing a functional double-network chitosan hydrogel comprises the following steps:

[0055] 1) Preparation of acryloyl chitosan

[0056] 4 grams of water-soluble chitosan were dissolved in 50 milliliters of deionized water. 1 milliliter of acryloyl chloride was dissolved in 2 milliliters of dichloromethane and placed in a dropping funnel. 2 milliliters of a 27% (mass fraction) sodium hydroxide solution were placed in another dropping funnel. Under stirring in an ice-water bath, the solutions in the two funnels were simultaneously dripped into the chitosan aqueous solution over a period of 10 minutes. After stirring in an ice bath for 3 hours, stirring was continued at room temperature for 16 hours. Subsequently, the pH value of the mixture was adjusted to neutral with 1 mol / L sodium hydroxide solution, and the mixture was precipitated and washed three times with cold ethanol to remove impurities such as sodium acrylate and salts. After the solid was dried overnight in a vacuum at room temperature, hydrogen nuclear magnetic resonance spectroscopy confirmed that it was acryloyl chitosan. Figure 1 H NMR spectrum of acryloyl chitosan, 1 H NMR (D2O, δ, ppm): 4.36 (H a ) and 3.96-3.18(H b ,H c ,H d ,H e ,H f ), belonging to hydrogen at different positions of the chitosan skeleton structure; 6.19 (H g ) and 5.75(H h ) is the proton peak on the double bond of grafted acrylamide.

[0057] 2) Preparation of single-network chitosan hydrogel

[0058] 0.5 g of acryloyl-chitosan was dissolved in 8 ml of deionized water. 0.05 g of N,N-bis(acryloyl)cystamine and 2, 3, 4, or 5 ml of acrylic acid were added sequentially, and stirred at room temperature until the solution became clear. 2 ml of an aqueous solution containing 0.2 g of potassium persulfate was added to the mixture, mixed at room temperature, and after bubbling nitrogen for 30 minutes, the homogenized mixture was transferred and sealed in a polytetrafluoroethylene mold. The mold was heated to 60°C for 4 hours, then cooled to room temperature and held for 2 hours. The resulting dark yellow, transparent gel was a single-network chitosan hydrogel. Figure 2 The infrared spectra of acryloyl chitosan and single network chitosan hydrogel are shown. The hydrogel has an infrared spectrum of 1738 cm -1 A new peak at 40° is attributed to the C=O stretching vibration of the carboxyl group, indicating that acrylic acid cross-links chitosan through polymerization to form a network structure. In the figure, CS is water-soluble chitosan, AC-CS is acryloyl-acrylated chitosan, SN-CS-Gel is a single-network chitosan hydrogel, and DN--CS-Gel is a double-network chitosan hydrogel.

[0059] The amount of acrylic acid used during the experiment could be adjusted within the range of 2 ml, 3 ml, 4 ml, and 5 ml. Tensile tests showed that the tensile stresses of the resulting single-network hydrogels were 10.71 kPa, 48.53 kPa, 58.09 kPa, and 79.19 kPa, respectively, and the corresponding strains were 37.71%, 40.73%, 47.37%, and 74.82%, respectively. This suggests that increasing the polyacrylic acid component in the hydrogel network improves the material's mechanical properties.

[0060] 3) Preparation of double-network chitosan hydrogel

[0061] The single-network chitosan hydrogel prepared with 3 ml of acrylic acid in step 2) was selected, freeze-dried, and then immersed in a 10 wt% (mass fraction) copper chloride aqueous solution (20 mL) for 12 hours. Subsequently, the hydrogel was removed and rinsed to remove the ions adsorbed on the surface, thereby obtaining a green double-network chitosan hydrogel. Its infrared spectrum is shown in FIG. Figure 2 As shown in Figure 2, compared with the single network hydrogel, the double network hydrogel material has a -1 A new signal peak is generated, which belongs to the near-infrared characteristic absorption of metal copper ions. At the same time, the symmetrical stretching vibration peak of the carboxyl group increases from 1385 cm -1 Move to 1403cm -1This indicates that copper ions have entered the gel and complexed with carboxyl groups to form a second network structure. Tensile tests based on the national standard GB / T 16491-2008 show that the stress of the double-network hydrogel increased sharply from 48.53 kPa of its corresponding single-network hydrogel to 199.81 kPa, an increase of more than 4 times; while the elongation at break increased from 40.73% of its corresponding single-network hydrogel to 121.66%, an increase of nearly 3 times (e.g. Figure 3 As shown). The results show that the second network constructed by the metal ion complex carboxyl group dramatically improves the mechanical properties of the hydrogel from a structural perspective and expands the application space of the material. On the other hand, the rheological properties test of the hydrogel (the test method follows the industry standard JY / T0590-2020) further proves that the dual network structure improves the mechanical strength of the material. Figure 4 As shown in the figure, under the same feed rate, the storage modulus of the single-network hydrogel is as high as 10385Pa, while the storage modulus of the double-network hydrogel is as high as 45573Pa.

[0062] In terms of swelling performance, the prepared hydrogel (single network hydrogel or double network hydrogel) was immersed in 100 ml of deionized water for 48 h to allow the hydrogel to reach swelling equilibrium. The hydrogel before immersion was weighed and the mass was recorded as W. i During the soaking process, the hydrogel was weighed at certain time intervals. Before weighing, the surface water of the hydrogel was dried with absorbent paper. The obtained mass was recorded as W s The swelling rate is given by the formula: The result is as follows: Figure 5 As shown in the figure, the hydrogels were almost saturated after 24 hours of immersion. The swelling rates of the single-network hydrogel and the double-network hydrogel reached 191.8% and 208.1%, respectively, indicating that the incorporation and cross-linking of copper ions had little effect on the water absorption capacity of the hydrogel network.

[0063] Example 2

[0064] A functional double-network chitosan hydrogel is used as an antibacterial material, and the specific application method is as follows:

[0065] Prepare 300 ml of Luria-Bertani (LB) medium and incubate at 37°C for 12 hours before use. Sterilize the experimental materials at 120°C for 1 hour. Prepare 50 ml of LB medium to prepare bacterial suspensions of Escherichia coli and Staphylococcus aureus (both at a concentration of 1 × 10 8CFU / mL). Add 200 ml of LB liquid to agar to prepare a solid culture medium. The single-network chitosan hydrogel and double-network chitosan hydrogel samples and other supplies used in the experiment were sterilized under ultraviolet light in a clean bench for 1 hour. Take 100 microliters of each bacterial suspension on the surface of the LB solid culture medium, spread it evenly with a glass ball, and stick the sterilized single-network chitosan hydrogel and double-network chitosan hydrogel samples to be tested on the solid culture medium plate. Put 2 pieces of single-network and double-network hydrogel samples in each culture dish, and perform 3 parallel tests for each group of samples. Lightly press the sample to fully contact the solid culture medium. Keep it sterile throughout the process. At the same time, keep a group of blank samples of Escherichia coli and Staphylococcus aureus. Then turn the plate upside down and place it in a constant temperature incubator at 37°C for 12 hours, and calculate the area of ​​the inhibition zone. The results are as follows Figure 6 As shown in the figure, against Escherichia coli, the antibacterial rate of the double-network hydrogel was 61.98% higher than that of the single-network hydrogel; and against Staphylococcus aureus, the antibacterial rate of the double-network hydrogel was 53.29% higher than that of the single-network hydrogel, indicating that the antibacterial efficacy of the double-network hydrogel constructed with the participation of metal ions was greatly improved due to the synergistic effect of the two factors.

[0066] The present invention prepares a single-network chitosan hydrogel by simple free radical polymerization of a mixed aqueous solution of acryloyl-modified chitosan, N,N-bis(acryloyl)cystamine and acrylic acid; wherein, cystamine with two double bonds at the ends is used as a cross-linking agent, and after copolymerization with acrylic acid to cross-link chitosan to form a network, the carboxyl groups on the short chains of polyacrylic acid can synergistically enhance the mechanical properties of the hydrogel with the abundant amino groups on the chitosan chain in the form of multiple electrostatic interactions. In addition, metal ions are dispersed into the hydrogel by diffusion and assisted by the complexation promotion effect of the carboxyl groups in the hydrogel. Based on the complexation of the metal ions with the large number of carboxyl groups in the single-network hydrogel, a second network is constructed to prepare a double-network hydrogel with even better mechanical properties. The preparation process of the double-network hydrogel of the present invention is simple and green. The double-network structure of different forms provides its adjustable and excellent mechanical properties. The positive charge of the rich amino groups carried by chitosan gives the hydrogel an antibacterial function. The metal ions involved in constructing the second network further enhance the antibacterial ability of the material, that is, the hydrogel has dual antibacterial efficacy. The double-network chitosan hydrogel provided by the present invention is a functional material with structurally optimized mechanical properties and has great application potential in antibacterial and tissue repair.

[0067] The above embodiments are described to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a functional double-network chitosan hydrogel, characterized in that: The preparation method comprises the following steps: 1) Modification of chitosan by acryloyl chloride to prepare acryloyl chitosan; 2) Single-network chitosan hydrogel was prepared by cross-linking acryloyl chitosan with N,N-bis(acryloyl)cystamine and acrylic acid; 3) Preparation of double-network chitosan hydrogels using metal cations; In step 1), specifically, acryloyl chloride solution and sodium hydroxide solution are simultaneously added dropwise to the water-soluble chitosan aqueous solution under ice bath conditions, stirred in an ice bath after the addition is completed, and then stirred at room temperature, and then the pH is adjusted to neutral, and separated and purified to obtain acryloyl chitosan; the amount ratio of water-soluble chitosan in the water-soluble chitosan aqueous solution to acryloyl chloride in the acryloyl chloride solution is 3-5:1 g / mL; the amount ratio of water-soluble chitosan in the water-soluble chitosan aqueous solution to sodium hydroxide solution is 1.5-3:1 g / mL; Step 2) is specifically as follows: dissolving acryloyl chitosan in water, adding N,N-bis(acryloyl)cystamine and acrylic acid, stirring and mixing, adding potassium persulfate solution, bubbling with nitrogen, placing the mixed solution in a mold and heating, and cooling to obtain a single network chitosan hydrogel; the amount ratio of acryloyl chitosan to water is 0.05-0.1 g / mL; the mass ratio of acryloyl chitosan to N,N-bis(acryloyl)cystamine is 8-12:1; the amount ratio of acryloyl chitosan to acrylic acid is 0.1-0.3 g / mL; the mass ratio of acryloyl chitosan to potassium persulfate in the potassium persulfate solution is 2-3:1; in step 2), heating refers to heating to 55-65° C. for 3.5-4.5 hours; cooling refers to cooling to room temperature and maintaining for 1.5-2.5 hours; Step 3) is specifically as follows: freeze-dry the single network chitosan hydrogel, immerse it in a copper ion aqueous solution, and take it out to obtain the product.

2. A functional double-network chitosan hydrogel prepared by the preparation method according to claim 1.

3. An application of the functional double-network chitosan hydrogel according to claim 2, characterized in that: The functional double-network chitosan hydrogel is used for preparing antibacterial materials.

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