Preparation method and application of chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel

Abstract

The application discloses a preparation method and application of a chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel, and the injectable hydrogel is prepared by the following steps: first, preparing chitosan acetic acid solution, 3,4-dihydroxybenzaldehyde solution and sodium periodate solution respectively by using acetic acid solution and water; then, mixing the chitosan acetic acid solution and the 3,4-dihydroxybenzaldehyde solution, and adding the sodium periodate solution into the mixture and mixing; and finally, preparing the hydrogel by standing and cross-linking at 37 DEG C. The hydrogel is prepared by cross-linking chitosan and 3,4-dihydroxybenzaldehyde, the raw materials are simple and non-toxic, the hydrogel has good adhesion and slow release, the reaction condition is mild, no chemical cross-linking agent, photo initiator or enzyme needs to be introduced, no expensive and complex equipment is needed, the cost is low, the effect is remarkable, and the hydrogel can be used as an injectable gel drug delivery system for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to a method for preparing chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel and its application in cancer treatment. Background Technology

[0002] Currently, chemotherapy is one of the most effective treatments for cancer, ranking alongside surgery and radiotherapy as one of the three major cancer treatments. Surgery and radiotherapy are local treatments, effective only against tumors at the treatment site. They are less effective against potential metastatic lesions (cancer cells that have actually spread but cannot be detected clinically due to technological limitations) and cancers that have already clinically metastasized. Chemotherapy, on the other hand, is a systemic treatment. Regardless of the route of administration (oral, intravenous, or intracavitary), chemotherapeutic drugs circulate throughout most organs and tissues of the body via the bloodstream. Therefore, chemotherapy is a primary treatment for some tumors with a tendency to spread throughout the body and for metastatic, mid-to-late-stage tumors. However, chemotherapeutic drugs are cytotoxic, and some toxic side effects may occur during treatment. Furthermore, drug resistance and poor targeting severely limit their widespread application. To improve the effectiveness of chemotherapy and reduce drug costs, researchers have proposed many new drug delivery strategies in recent years, such as nanomedicine and drug-loaded hydrogels. Hydrogels are an effective drug delivery strategy, containing a large amount of water and a cross-linked polymer network. They have excellent biocompatibility, negligible cytotoxicity, and outstanding drug encapsulation capabilities, and have therefore been widely used in cancer treatment in recent years.

[0003] Taking bladder cancer as an example, treatment methods include transurethral resection followed by intravesical immunotherapy or chemotherapy, but recurrence is common after surgery. Intravesical BCG instillation is a recognized effective treatment, but currently, it can only be achieved through large, repeated instillations. This treatment method has many drawbacks, such as excessive BCG causing strong irritation to the bladder mucosa, leading to symptoms like hematuria, urinary frequency, urgency, and dysuria; the drug cannot be accurately released at the cancerous site; and poor adhesion means the drug is excreted during intermittent urination, resulting in low drug utilization. To alleviate patient suffering and improve treatment efficacy, it is essential to develop a drug delivery system that can adhere to the cancerous site and provide sustained release.

[0004] Chitosan and sodium alginate-based hydrogels are currently considered outstanding pharmaceutical excipients. Chitosan and sodium alginate possess excellent biocompatibility and many superior properties, making them highly promising for applications in drug encapsulation and delivery, tissue engineering, and other fields. Adding drugs to hydrogels can create a slow-release system, increasing the retention time of antitumor drugs in the body. Injectable hydrogels have attracted widespread attention due to their unique advantages, and natural polymers, compared to synthetic polymers, exhibit superior biocompatibility and biodegradability, making them a primary raw material for drug-loaded hydrogels. Chinese patent CN107118357B discloses a catechol-chitosan self-healing hydrogel material and its preparation method, which possesses a high storage modulus; the coordination of iron and catechol groups endows the hydrogel with self-healing properties. Chinese patent CN108159482A discloses an injectable natural hydrogel system with thermosensitive properties and high tissue adhesion, and its preparation method; this system exhibits a fast temperature response and can firmly adhere to the desired site even in a humid environment. In his study, "Research on Three Gel-Forming Mechanisms of Chitosan Based on Catechol Functionalization," Guo Zhongwei prepared a cadmium-based chitosan with a substitution degree as high as 70%. However, current methods for preparing hydrogels using chitosan and cadmium require reduction with sodium borohydride to obtain stable products. Sodium borohydride is corrosive, flammable, deliquescent, and exhibits acute toxicity, thus placing high demands on the hydrogel preparation process. Furthermore, residual sodium borohydride in the hydrogel poses cytotoxicity, affecting its application in drug delivery systems. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel, which improves the antibacterial properties of the hydrogel and reduces cytotoxicity.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel includes the following steps:

[0008] (1) Dissolve chitosan powder in 10-50 g / L acetic acid solution to obtain 5-30 g / L chitosan acetic acid solution;

[0009] (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 5-30 g / L 3,4-dihydroxybenzaldehyde solution;

[0010] (3) Dissolve sodium periodate in water to obtain a sodium periodate aqueous solution of 5-30 g / L;

[0011] (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous to obtain a mixed solution;

[0012] (5) Add sodium periodate aqueous solution to the mixed solution and stir evenly. Let stand at 37°C for 0.1h to 1h to crosslink and obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel.

[0013] Preferably, the volume ratio of 3,4-dihydroxybenzaldehyde solution to chitosan acetate solution in step (4) is 3:1 to 1:3.

[0014] Preferably, in step (5), the volume ratio of the sodium periodate solution to the chitosan solution and the 3,4-dihydroxybenzaldehyde solution is 1 to 5:40.

[0015] The present invention also provides a product prepared by the above preparation method.

[0016] The present invention also provides an application of the above-mentioned chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel in a targeted hydrogel drug delivery system for cancer treatment.

[0017] The process and mechanism of this invention are mainly as follows: After chitosan is mixed with 3,4-dihydroxybenzaldehyde, the amino groups in chitosan react with the aldehyde groups in 3,4-dihydroxybenzaldehyde in a Schiff base reaction to generate imine bonds. After the addition of sodium periodate solution, some of the catechol structures on 3,4-dihydroxybenzaldehyde are oxidized to catechol-diquinone structures, and the remaining 3,4-dihydroxybenzaldehyde forms oxidation products and 3,4-dihydroxybenzaldehyde oligomers. These catechol-diquinone-containing products, oxidation products, and oligomers are covalently crosslinked with the amino groups of chitosan through Schiff base and Michael addition reactions. These oligomers, 3,4-dihydroxybenzaldehyde monomers, oxidation products, and products grafted onto the chitosan backbone assemble into a stable hydrogel structure through hydrogen bonds and π-π conjugation. Figure 1 All of the aforementioned physical and chemical crosslinkings contribute to the gelation of the chitosan and 3,4-dihydroxybenzaldehyde mixture. Adding the model drug bovine serum albumin prior to hydrogel formation allows for premixing with the polymer solution, enabling simultaneous in-situ encapsulation into the hydrogel.

[0018] The beneficial effects of this invention are as follows:

[0019] 1. This invention can directly prepare injectable hydrogels through a one-step crosslinking method, without the need for reduction with sodium borohydride to form stable intermediate products. This overcomes the instability of carbon-nitrogen double bonds in intermediate products and reduces the cytotoxicity of sodium borohydride to hydrogels, while avoiding safety issues in the preparation process of injectable hydrogels. Furthermore, it eliminates the need for expensive crosslinking agents, photoinitiators, or enzymes during preparation, thus reducing costs.

[0020] 2. An injectable hydrogel can be prepared by a one-step crosslinking method, which eliminates intermediate synthesis steps and does not require expensive and complex equipment, making the preparation method simple, easy to operate, and easy to promote.

[0021] 3. The prepared injectable hydrogel has good adhesion, biodegradability, antibacterial properties, and biocompatibility. It can adhere to and release drugs at cancerous sites, solving the problem of low drug utilization rates in previous treatments. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the hydrogel formation principle in the method of the present invention.

[0023] Figure 2 The images show the microstructures of the injectable hydrogels prepared in Examples 1 and 5; the left image is for Example 1 and the right image is for Example 5.

[0024] Figure 3 The diagram shows the number of remaining adherent cells and the adhesion mechanism at different time points in the simulated adhesion experiment of the injectable hydrogels prepared in Examples 1 and 3 to the pig bladder; where a is a line graph of the number of remaining adherent cells of the hydrogel at different times, b is a photograph of the hydrogel adhering to the pig bladder mucosa, and c is a diagram of the adhesion mechanism.

[0025] Figure 4 Rheological properties of the injectable hydrogels prepared in Examples 1 and 5 are shown in the diagrams; where a is the change in storage modulus (G') of the injectable hydrogel, b is the change in loss modulus (G”) of the injectable hydrogel, and c is the change in the ratio of loss modulus to storage modulus (Tanδ) of the injectable hydrogel.

[0026] Figure 5 The graph shows the antibacterial properties of the injectable hydrogels prepared in Examples 1 to 5; where a is the inhibition rate of the injectable hydrogel against Escherichia coli, b is the inhibition rate of the injectable hydrogel against Staphylococcus aureus, and c is a bar graph showing the diameter of the antibacterial plaques of the injectable hydrogel against Escherichia coli and Staphylococcus aureus.

[0027] Figure 6 The bar charts show the cytotoxicity of the injectable hydrogels prepared in Examples 1 to 5 at different times; the left bar chart shows the cytotoxicity of the injectable hydrogel at 24h, and the right bar chart shows the cytotoxicity of the injectable hydrogel at 48h.

[0028] Figure 7The diagrams show the drug release rate and release mechanism of the injectable hydrogels prepared in Examples 1 and 5 at different pH values; where a is a line graph of the drug release rate of the injectable hydrogel at pH 7.4, b is a line graph of the drug release mechanism of the injectable hydrogel at pH 7.4, c is a line graph of the drug release rate of the injectable hydrogel at pH 4.0, and d is a line graph of the drug release mechanism of the injectable hydrogel at pH 4.0. Detailed Implementation

[0029] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0030] Example 1: Preparation of injectable hydrogel numbered CS / DBA5

[0031] like Figure 1 As shown, a Schiff base reaction occurs when the amino groups of chitosan are mixed with the aldehyde groups of 3,4-dihydroxybenzaldehyde, generating a mixture. Then, under the action of sodium periodate, the residual 3,4-dihydroxybenzaldehyde forms oxidation products and oligomers. Finally, these oligomers covalently crosslink with the amino groups of chitosan through Schiff base and Michael addition reactions. These oligomers, 3,4-dihydroxybenzaldehyde monomers, oxidation products, and products grafted onto the chitosan backbone assemble into a stable hydrogel structure through hydrogen bonds and π-π conjugation. The specific method is as follows:

[0032] (1) Dissolve 0.3g of chitosan in 20g / L acetic acid solution to obtain 30g / L chitosan acetic acid solution;

[0033] (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 5 g / L 3,4-dihydroxybenzaldehyde solution;

[0034] (3) Dissolve sodium periodate in water to obtain a 20 g / L sodium periodate solution;

[0035] (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous, with a volume ratio of 1:1, to obtain a mixed solution;

[0036] (5) Add sodium periodate solution to the mixed solution and stir. The volume ratio of sodium periodate solution to mixed solution is 3:40. Let stand at 37°C for 0.1 h to crosslink and obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel.

[0037] Example 2: Preparation of injectable hydrogel numbered CS / DBA10

[0038] (1) Dissolve 0.3g of chitosan in 20g / L acetic acid solution to obtain 30g / L chitosan acetic acid solution;

[0039] (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 10 g / L 3,4-dihydroxybenzaldehyde solution;

[0040] (3) Dissolve sodium periodate in water to obtain a 20 g / L sodium periodate solution;

[0041] (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous, with a volume ratio of 1:1, to obtain a mixed solution;

[0042] (5) Add sodium periodate solution to the mixed solution and stir. The volume ratio of sodium periodate solution to mixed solution is 3:40. After standing at 37°C for 0.5 h, crosslinking is obtained to obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel.

[0043] Example 3: Preparation of injectable hydrogel with part number CS / DBA15

[0044] (1) Dissolve 0.3g of chitosan in 20g / L acetic acid solution to obtain 30g / L chitosan acetic acid solution;

[0045] (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 15 g / L 3,4-dihydroxybenzaldehyde solution;

[0046] (3) Dissolve sodium periodate in water to obtain a 20 g / L sodium periodate solution;

[0047] (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous, with a volume ratio of 1:1, to obtain a mixed solution;

[0048] (5) Add sodium periodate solution to the mixed solution and stir. The volume ratio of sodium periodate solution to mixed solution is 3:40. After standing at 37°C for 0.5 h, crosslinking is obtained to obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel.

[0049] Example 4: Preparation of injectable hydrogel with serial number CS / DBA20

[0050] (1) Dissolve 0.3g of chitosan in 20g / L acetic acid solution to obtain 30g / L chitosan acetic acid solution;

[0051] (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 20 g / L 3,4-dihydroxybenzaldehyde solution;

[0052] (3) Dissolve sodium periodate in water to obtain a 20 g / L sodium periodate solution;

[0053] (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous, with a volume ratio of 1:1, to obtain a mixed solution;

[0054] (5) Add sodium periodate solution to the mixed solution and stir. The volume ratio of sodium periodate solution to mixed solution is 3:40. Let stand at 37°C for 1 hour to crosslink and obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel.

[0055] Example 5: Preparation of injectable hydrogel with part number CS / DBA25

[0056] (1) Dissolve 0.3g of chitosan in 20g / L acetic acid solution to obtain 30g / L chitosan acetic acid solution;

[0057] (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 25 g / L 3,4-dihydroxybenzaldehyde solution;

[0058] (3) Dissolve sodium periodate in water to obtain a 20 g / L sodium periodate solution;

[0059] (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous, with a volume ratio of 1:1, to obtain a mixed solution;

[0060] (5) Add sodium periodate solution to the mixed solution and stir. The volume ratio of sodium periodate solution to mixed solution is 3:40. Let stand at 37°C for 1 hour to crosslink and obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel.

[0061] Example 6

[0062] (1) Dissolve 0.2g of chitosan in 10g / L acetic acid solution to obtain 20g / L chitosan acetic acid solution;

[0063] (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 15 g / L 3,4-dihydroxybenzaldehyde solution;

[0064] (3) Dissolve sodium periodate in water to obtain a 5 g / L sodium periodate solution;

[0065] (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous. The volume ratio is 1:3 to obtain a mixed solution.

[0066] (5) Add sodium periodate solution to the mixed solution and stir. The volume ratio of sodium periodate solution to the mixed solution is 1:40. Let it stand at 37°C for 0.1 h to crosslink and obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel. Example 7

[0067] (1) Dissolve 0.1g of chitosan in 50g / L acetic acid solution to obtain 10g / L chitosan acetic acid solution;

[0068] (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 15 g / L 3,4-dihydroxybenzaldehyde solution;

[0069] (3) Dissolve sodium periodate in water to obtain a 30 g / L sodium periodate solution;

[0070] (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous. The volume ratio is 3:1 to obtain a mixed solution.

[0071] (5) Add sodium periodate solution to the mixed solution and stir. The volume ratio of sodium periodate solution to mixed solution is 5:40. After standing at 37°C for 0.5h, crosslinking is obtained to obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel.

[0072] Example 8

[0073] (1) Dissolve 0.05g of chitosan in 30g / L acetic acid solution to obtain 5g / L chitosan acetic acid solution;

[0074] (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 15 g / L 3,4-dihydroxybenzaldehyde solution;

[0075] (3) Dissolve sodium periodate in water to obtain a 10 g / L sodium periodate solution;

[0076] (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous, with a volume ratio of 1:1, to obtain a mixed solution;

[0077] (5) Add sodium periodate solution to the mixed solution and stir. The volume ratio of sodium periodate solution to mixed solution is 3:40. Let stand at 37°C for 1 hour to crosslink and obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel.

[0078] Result Validation

[0079] The chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogels obtained in Examples 1-8 were freeze-dried and then photographed under an electron microscope to determine the pore size of the prepared injectable hydrogels.

[0080] result( Figure 2The results showed that the pore sizes of chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogels prepared under different formulation ratios were inconsistent. Among them, the injectable hydrogel prepared by mixing equal volumes of 30 g / L chitosan acetate solution and 15 g / L 3,4-dihydroxybenzaldehyde solution, and then mixing it with 20 g / L sodium periodate solution (the volume ratio of sodium periodate solution to chitosan acetate solution and 3,4-dihydroxybenzaldehyde solution was 3:40) had moderate pore density and obvious antibacterial properties, making it suitable as a drug delivery system.

[0081] Example 9

[0082] The injectable hydrogels prepared in Examples 1 and 3 were used to conduct hydrogel adhesion experiments on pig bladders, and the results are as follows. Figure 3 As shown, with the extension of adhesion time, the remaining adhesion amount of the injectable hydrogel prepared in Example 1 (numbered CS / DBA5) gradually decreased, while the remaining adhesion amount of the injectable hydrogel prepared in Example 3 (numbered CS / DBA15) remained unchanged. This indicates that the denser the pores of the prepared injectable hydrogel, the lower its adhesion ability to pig bladder. Therefore, injectable hydrogels with relatively loose pores are more suitable as drug delivery systems.

[0083] Example 10

[0084] The internal interactions of injectable hydrogels are one of the key factors affecting drug release, and changes in the internal structure of injectable hydrogels are closely related to rheological properties such as viscosity. Therefore, studying the drug release performance of injectable hydrogels requires rheological experiments. The rheological properties of the injectable hydrogels prepared in Examples 1 and 5 were studied using a HAAKE MARS rheometer.

[0085] The results are as follows Figure 4 As shown in (a), the storage modulus of the injectable hydrogel is related to the degree of crosslinking. The storage modulus of the injectable hydrogel prepared in Example 1 is higher than that of the injectable hydrogel in Example 5, indicating that the injectable hydrogel in Example 1 has a higher degree of crosslinking and is relatively more stable. Figure 4 As shown in (b), the loss modulus (G”) can represent the viscosity of the injectable hydrogel. The injectable hydrogel of Example 5 has a relatively high loss modulus, which is consistent with the viscosity test results. The molding time of the injectable hydrogel can be reflected in the Tanδ value. Figure 4 (c) It can be seen that the injectable hydrogel with the serial number CS / BDA5 prepared in Example 1 can be rapidly molded. Therefore, when the storage modulus of the injectable hydrogel is high, the hydrogel can release drugs for a longer period of time, making it more suitable as a drug delivery system.

[0086] Example 11

[0087] The hydrogels prepared in Examples 1-5 were used to conduct antibacterial ability tests against Escherichia coli and Staphylococcus aureus, and the diameter of the inhibition plaques was counted. The method was as follows: a hydrogel with a diameter of about 6 mm and a thickness of about 1.5 mm was placed on a plate culture medium containing Escherichia coli or Staphylococcus aureus, and cultured at 37°C for 24 h. The diameter of the inhibition plaques was then counted.

[0088] Blank control (CS): 6 mm diameter circular filter paper discs were soaked in 30 g / L chitosan acetate solution for a period of time to allow them to fully absorb the chitosan acetate solution. They were then placed on agar plates containing Escherichia coli or Staphylococcus aureus and incubated at 37 °C for 24 h. The diameter of the inhibition plaques was then counted.

[0089] The results are as follows Figure 5 As shown, injectable hydrogels prepared with different formulations exhibit varying antibacterial abilities against *Escherichia coli* and *Staphylococcus aureus*. However, overall, with increasing concentrations of 3,4-dihydroxybenzaldehyde, the diameter of the inhibition zone against both *E. coli* and *Staphylococcus aureus* gradually increases, indicating a gradually strengthening antibacterial ability. Furthermore, when using lower concentrations of 3,4-dihydroxybenzaldehyde, the injectable hydrogel exhibits a larger inhibition zone diameter against *E. coli*; conversely, when using higher concentrations, the injectable hydrogel exhibits a larger inhibition zone diameter against *Staphylococcus aureus*. This suggests that injectable hydrogels prepared with low concentrations of 3,4-dihydroxybenzaldehyde possess stronger antibacterial ability against *E. coli*, while injectable hydrogels prepared with high concentrations of 3,4-dihydroxybenzaldehyde demonstrate stronger resistance to *Staphylococcus aureus*.

[0090] Example 12

[0091] When hydrogels are used as drug delivery systems, their cytotoxicity is one of the key factors affecting their application range. Therefore, hydrogels prepared in Examples 1-5 were used for cytotoxicity testing, and the specific methods are as follows:

[0092] Experimental group: Injectable hydrogel (20 mg) was added to 20 μL of sterile physiological saline, and the extract was obtained after 24 h. The extract was sterilized under UV irradiation for 30 min, and approximately 5 μL of the extract was added to each well containing 100 μL of HeLa cells. The plates were incubated for 24 h and 48 h. After each incubation period, 100 μL of thiazolyl blue (MTT) (5 mg / mL) solution was added to each well, and the plates were incubated for another 4 h. The cells were washed with phosphate-buffered saline (PBS). Then, 100 μL of DMSO was added to each well, and the absorbance at 490 nm was measured for each well.

[0093] Control group: The method is the same as the experimental group, except that the injectable hydrogel extract is replaced with ddH2O or a certain solution.

[0094] The results are as follows Figure 6 As shown, compared with the control group, cell viability decreased after treatment with injectable hydrogel extracts prepared using various formulations for 24 h and 48 h, but not significantly. In particular, the injectable hydrogel prepared in Example 4, designated CS / DBA20, showed increased cell viability after 48 h of treatment, indicating that the hydrogel has extremely low cytotoxicity and is suitable as a drug delivery system.

[0095] Example 13

[0096] The drug release rate of a drug-loaded system is also an important indicator for evaluating its suitability as a drug-loaded system. Therefore, the drug release rate and mechanism of the prepared injectable hydrogels were investigated experimentally. The method was as follows: The injectable hydrogels prepared in Examples 1 and 5 were placed in beakers containing 100 mL of PBS buffer solution at pH 7.4 and 4.0, respectively. The beakers were placed in a constant-temperature shaker and then shaken at 100 rpm in a 37°C water bath. At specified time intervals, 1 mL of PBS supernatant was collected and replaced with an equal volume of fresh PBS solution to maintain the volume. The collected solution was mixed thoroughly with 5 mL of prepared Coomassie Brilliant Blue solution. The UV-Vis absorption band of the bovine serum albumin (BSA) and Coomassie Brilliant Blue mixture was measured at 595 nm using UV-Vis spectroscopy. The amount of BSA released was determined using the standard curve method. In addition, the release kinetics of BSA were studied using the exponential Korsmeyer-Peppas equation.

[0097] The results are as follows Figure 7 As shown, at pH 7.4, the cumulative drug release of hydrogels with different formulations in PBS buffer solution showed a gradual increasing trend over time. The release rate of hydrogel CS / DBA5 prepared in Example 1 was significantly lower than that of hydrogel CS / DBA25 prepared in Example 5. Figure 7 (a). At pH 4.0, the cumulative drug release trend of hydrogels with different formulations was consistent with that at pH 7.4, but the cumulative drug release percentage of the hydrogel prepared in Example 5 (CS / DBA25) reached 100% at 480 min, while the cumulative drug release percentage of the hydrogel prepared in Example 1 (CS / DBA5) was only about 30% at this time. Figure 7Figure (c) shows that the drug release rate of the hydrogel is closely related to the degree of cross-linking. As the DBA concentration increases, the content of the cross-linking agent NaIO4 decreases relatively, the cross-linking density of the hydrogel decreases, and the drug release rate increases. The release kinetics of bovine serum albumin were studied based on the exponential Korsmeyer-Peppas equation. Figure (b) shows that the drug release mechanism is non-Fick diffusion, i.e., the combined effect of drug diffusion and skeletal dissolution. Figure 7 (d) It can be seen that the drug release mechanism is mainly the skeleton dissolution effect, mainly because the hydrogel skeleton is more corroded at a lower pH.

Claims

1. A method for preparing a chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel, characterized in that: Includes the following steps: (1) Dissolve chitosan powder in acetic acid solution to obtain chitosan acetic acid solution; (2) Dissolve 3,4-dihydroxybenzaldehyde in water to obtain a 3,4-dihydroxybenzaldehyde solution; (3) Dissolve sodium periodate in water to obtain an aqueous solution of sodium periodate; (4) Add the 3,4-dihydroxybenzaldehyde solution to the chitosan acetate solution and stir until homogeneous to obtain a mixed solution; (5) Add sodium periodate aqueous solution to the mixed solution and stir evenly. Let it stand at 37°C for 0.1-1h to crosslink and obtain chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel. The concentration of the chitosan acetate solution in step (1) is 5-30 g / L; In step (2), the concentration of the 3,4-dihydroxybenzaldehyde solution is 5–30 g / L; In step (4), the volume ratio of 3,4-dihydroxybenzaldehyde solution to chitosan acetate solution is 3:1 to 1:

3.

2. The method for preparing a chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel according to claim 1, characterized in that: The concentration of the acetic acid solution in step (1) is 10-50 g / L.

3. The method for preparing a chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel according to claim 1, characterized in that: In step (3), the concentration of sodium periodate aqueous solution is 5-30 g / L.

4. The method for preparing a chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel according to claim 1, characterized in that: In step (5), the volume ratio of the sodium periodate solution to the chitosan solution and the 3,4-dihydroxybenzaldehyde solution is 1 to 5:

40.

5. The chitosan / 3,4-dihydroxybenzaldehyde injectable hydrogel prepared by the preparation method according to any one of claims 1-4.

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