Oligosaccharide-based dynamic covalent bond cross-linked hydrogel modified with benzaldehyde, its preparation method and application

Abstract

The present invention discloses a benzaldehyde-modified oligosaccharide-based dynamic covalent bond cross-linked hydrogel, its preparation method and application. An oligosaccharide is esterified with p-carboxybenzaldehyde to obtain a benzaldehyde-modified oligosaccharide ester, and then it is cross-linked with a natural polymer containing primary amine through dynamic covalent bonds to form a hydrogel. The cross-linking points in the hydrogel of the present invention are formed by Schiff base bonds. The hydrogel has an adjustable gelation time, good responsiveness to environmental pH value, good mechanical properties and cell compatibility. After loading antibacterial peptides, it has good antibacterial properties, and the antibacterial property is enhanced with the decrease of environmental pH. This hydrogel can be used in tissue repair, drug delivery and other fields in the biomedical field.

Description

Technical Field:

[0001] The present invention belongs to the technical field of biomedical materials, and relates to a benzaldehyde-modified oligosaccharide-based dynamic covalent bond cross-linked hydrogel and a preparation method thereof. Background Art:

[0002] A hydrogel is a hydrophilic polymer network cross-linked by physical or covalent bonds, and has characteristics such as high water content, good swelling property, and strong water absorption. Due to its excellent water retention property and extracellular matrix-like network structure, it has been widely used in the biomedical field. The hydrogels prepared by traditional chemical cross-linking or physical cross-linking methods often have stable physicochemical properties and a fixed shape, and do not have a stimulus-responsive property to the surrounding environment; when used as a tissue filler, it will cause problems such as incomplete filling of tissue defects and loose adhesion to tissues. Therefore, it is of great significance to find high-performance hydrogels with environmental responsiveness and self-healing function for practical biomedical applications.

[0003] The dynamic covalent bond hydrogel is one of the self-healing materials developed in the past ten years. It is a hydrogel network composed of dynamic forces such as reversible covalent bonds (Diels-Alder reaction, disulfide bond, borate ester bond, acylhydrazone bond, Schiff base (also called imine bond), etc.). Under specific stimuli such as pH value, ionic strength, temperature, light, and magnetic force, bond cleavage and reversible recovery can be achieved. According to different requirements of the application environment, hydrogels with functions such as injectability, stimulus response, self-healing, or shear thinning can be prepared by dynamic covalent bond cross-linking.

[0004] The generalized Schiff base reaction includes the reactions of acylhydrazone bonds (from hydrazine) and imine bonds (from other primary amines), which are formed by the condensation of a carbonyl group (aldehyde or ketone) with a primary amine. When the pH value is low enough, the Schiff base bond is easily hydrolyzed; when the pH value gradually increases, the Schiff base bond can be restored again. Compared with other dynamic covalent bonds, the synthesis process of Schiff base is simple, the conditions are mild, and no additional initiator or cross-linking agent is required, and it is widely used in pH-responsive hydrogels based on natural polymers (such as polysaccharides and proteins). However, when polysaccharides are used as aldehyde donors for hydrogels, the polysaccharides are mainly polysaccharides linked by 1,4-glycosidic bonds oxidized by sodium periodate, while polysaccharides linked by 1,3-glycosidic bonds such as agarose, carrageenan, β-1,3-glucan, etc. cannot be oxidized. In addition, sodium periodate oxidation can also cause the destruction of the polysaccharide sugar ring, resulting in the loss of the biological function or biological activity of the polysaccharide. At present, the Schiff base hydrogels containing benzaldehyde mainly use benzaldehyde-modified tetra-armed polyethylene glycol as a cross-linking agent, and there are few studies on Schiff base hydrogels modified with benzaldehyde-containing saccharides.

[0005] Due to their short chains and oxidized sugar rings, natural oligosaccharides usually exhibit physicochemical and biological properties different from those of the corresponding polysaccharides, such as better biodegradability, bioavailability, and more biological activities such as immunomodulation, antioxidant, antibacterial, etc. However, due to the short chains and / or hydrophobicity after modification such as esterification, there are few reports on oligosaccharide-based hydrogels crosslinked only by Schiff base bonds. Summary of the Invention:

[0006] In view of the above reasons, the present invention provides a benzaldehyde-modified oligosaccharide-based dynamic covalent bond crosslinked hydrogel and its preparation method. This hydrogel is obtained by esterifying oligosaccharides with p-carboxybenzaldehyde (PCB) to get benzaldehyde-modified oligosaccharide esters, and then crosslinking them with natural polymers containing primary amines through dynamic covalent bonds to form a hydrogel. The crosslinking points in this hydrogel are formed by Schiff base bonding. The hydrogel has adjustable gelation time, good responsiveness to environmental pH, good mechanical properties and cell compatibility. After loading antibacterial peptides, it has good antibacterial properties, and the antibacterial property increases with the decrease of environmental pH. This hydrogel can be used in tissue repair and drug delivery in the biomedical field.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a benzaldehyde-modified oligosaccharide-based Schiff base hydrogel, which is obtained by esterifying oligosaccharides with p-carboxybenzaldehyde to get benzaldehyde-modified oligosaccharide esters, and then crosslinking them with natural polymers containing primary amines through dynamic covalent bonds to form a hydrogel.

[0009] Preferably, the oligosaccharides are selected from at least one of agar oligosaccharides (AO) and konjac glucomannan oligosaccharides (KOS). Further preferably, the oligosaccharides are prepared by acid degradation method or enzymatic degradation method.

[0010] Preferably, the molecular weight distribution range of the oligosaccharides is 1000 - 5000 Da.

[0011] Preferably, the method of esterifying oligosaccharides with p-carboxybenzaldehyde to get benzaldehyde-modified oligosaccharide esters is to add N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and p-carboxybenzaldehyde into DMSO, and at the same time add oligosaccharides to react. After the reaction is completed, first filter to remove dicyclohexylurea, and then add absolute ethanol to the filtrate to obtain oligosaccharide esters.

[0012] Preferably, the natural polymers containing primary amines are selected from at least one or several of carboxymethyl chitosan (CMCS) and polyglutamic acid substituted with adipic dihydrazide (HPGA).

[0013] Preferably, the hydrogel can also load antibacterial peptides, such as ε-polylysine (ε-PL).

[0014] Preferably, the oligosaccharide esterified product and the natural polymer containing primary amine are crosslinked by a dynamic covalent bond to form a hydrogel by dissolving the oligosaccharide esterified product in PBS buffer solution at a concentration of 4 g / 100 ml, and then mixing and oscillating with an equal volume of the natural polymer solution containing primary amine to form a hydrogel; the natural polymer solution containing primary amine is a 5 g / 100 ml carboxymethyl chitosan solution, a 10 g / 100 ml adipic dihydrazide-substituted polyglutamic acid solution, or a mixed solution formed by mixing a 10 g / 100 ml adipic dihydrazide-substituted polyglutamic acid solution and a 10 g / 100 ml ε-polylysine solution in a volume ratio of 9:1.

[0015] The present invention also provides the application of the benzaldehyde-modified oligosaccharide-based Schiff base hydrogel in the preparation of antibacterial drugs.

[0016] Preferably, the antibacterial drug is a drug that inhibits Escherichia coli or Staphylococcus aureus

[0017] The dynamic covalent bond in the present invention can be at least one of an imine bond or a hydrazone bond.

[0018] The hydrogel matrix of the present invention comprises a PCB-modified oligosaccharide esterified product and a water-soluble natural polysaccharide, natural protein or artificial polypeptide containing primary amine; the hydrogel is a polymer network structure formed by a Schiff base reaction.

[0019] The crosslinking points in the hydrogel of the present invention are formed by Schiff base bonding. The hydrogel has an adjustable gelation time, good responsiveness to environmental pH, good mechanical properties and cell compatibility. After loading antibacterial peptides, it has good antibacterial properties, and the antibacterial property is enhanced with the decrease of environmental pH. The hydrogel can be used in tissue repair and drug delivery and other fields in the biomedical field. Description of the drawings:

[0020] Figure 1 1H nuclear magnetic resonance spectrum of benzaldehyde-modified agar oligosaccharide esterified product (CBAO); 1 1H nuclear magnetic resonance spectrum;

[0021] Figure 2 1H nuclear magnetic resonance spectrum of benzaldehyde-modified konjac glucomannan oligosaccharide esterified product (BKOS); 1 1H nuclear magnetic resonance spectrum;

[0022] Figure 3 Photographs of CBAO / CMCS, CBAO / HPGA and CBAO / HPGA / ε-PL hydrogels;

[0023] Figure 4 Release behavior diagram of CBAO in CBAO / HPGA / ε-PL hydrogel at different pH values;

[0024] Figure 5 It is the cytotoxicity diagram of CBAO / CMCS hydrogel;

[0025] Figure 6 It is the antibacterial effect diagram of CBAO / CMCS and CBAO / HPGA / ε-PL hydrogels under different pH gelation conditions and different culture environment pH conditions. Specific implementation method:

[0026] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with the drawings and specific embodiments. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0027] Example 1 Preparation of CBAO Schiff base hydrogel

[0028] The specific operation steps of the method of the present invention are as follows:

[0029] 1. Preparation of AO: AO is prepared by acid degradation method

[0030] Dissolve agarose to obtain a 2% (m / v g / mL) agarose aqueous solution; after cooling, add HCl solution for degradation, the degradation temperature is 70 °C, the final concentration of HCl is 0.1 mol / L, and the degradation time is 1 h. The degradation product is dialyzed with pure water (the molecular weight cut-off of the dialysis bag is 1000 Da) for 3 d, and the dialysis solution is centrifuged at 6000 rpm to obtain the supernatant; the supernatant is freeze-dried to obtain AO freeze-dried powder.

[0031] 2. Preparation of CBAO:

[0032] Add N,N-dicyclohexylcarbodiimide (DCC, 2.45 mmol), 4-dimethylaminopyridine (DMAP, 0.41 mmol) and different amounts of p-carboxybenzaldehyde (PCB) (AO / PCB = 1:0.5, 1:1, 1:2, molar ratio) into DMSO (10 mL) respectively, and mix for 30 min; at the same time, dissolve the AO powder (1.63 mmol) in Example 1 in DMSO (15 mL), add it to the above system, and react at room temperature and N2 atmosphere for 24 h. After the reaction is completed, first filter to remove dicyclohexylurea (DCU), add absolute ethanol (250 mL) to the filtrate, and precipitate CBAO. The CBAO product is centrifuged (7000 rpm, 5 min) and freeze-dried to obtain a powdery product.

[0033] 3. Preparation of CBAO Schiff base hydrogel:

[0034] CBAO (degree of benzaldehyde substitution: 15.4%, initial concentration: 4% (m / v g / mL)) was dissolved in phosphate buffer (PBS, 0.01 mol / L, pH = 7.4), and was rapidly mixed with an equal volume of aqueous carboxymethyl chitosan (CMCS) solution (degree of amino substitution: 51.6%, initial concentration: 5% (m / v g / mL)) on a vortex oscillator (rotation speed: 1500 rpm) to form a hydrogel, obtaining the CBAO / CMCS hydrogel.

[0035] CBAO (degree of benzaldehyde substitution: 29.0%, initial concentration: 2% (m / v g / mL)) was dissolved in phosphate buffer (PBS, 0.01 mol / L, pH = 7.4), and was rapidly mixed with an equal volume of aqueous solution of polyglutamic acid substituted with adipic dihydrazide (HPGA, degree of hydrazide substitution: 12.8%, initial concentration: 10% (m / v g / mL)) on a vortex oscillator (rotation speed: 1500 rpm) to form a hydrogel, obtaining the CBAO / HPGA hydrogel.

[0036] CBAO (AO / PCB = 1:2, degree of benzaldehyde substitution: 27.8%, initial concentration: 2% (m / v g / mL)) was dissolved in phosphate buffer (PBS, 0.01 mol / L, pH = 7.4), and was rapidly mixed with an equal volume of the mixture of HPGA and ε-polylysine on a vortex oscillator (rotation speed: 1500 rpm) to form a hydrogel, obtaining the CBAO / HPGA / ε-PL hydrogel. The said mixture was prepared by mixing HPGA (degree of hydrazide substitution: 11.5%, initial concentration: 10% (m / v g / mL)) and ε-polylysine (ε-PL, initial concentration: 10% (m / v g / mL) aqueous solution) in a volume ratio of 9:1.

[0037] Preparation of the BKOS Schiff base hydrogel in Example 2

[0038] The specific operation steps of the method of the present invention are as follows:

[0039] 1. Preparation of KOS: KOS was prepared by an enzymatic degradation method

[0040] Weighed 1.5 g of purified konjac glucomannan (KGM) and dissolved it in 750 mL of pure water (0.2% m / v g / mL). Added 4.5 mg of β-mannanase (enzyme activity: 150 U / g), the reaction temperature was 50 °C, and the time was 2 h. After the reaction ended, boiled for 10 min, then filtered, and finally rotary evaporated to a volume of about 100 - 150 mL, and freeze-dried to obtain the freeze-dried powder of the degradation product KOS.

[0041] 2. Preparation of BKOS:

[0042] N,N'-Dicyclohexylcarbodiimide (DCC, 4.63 mmol), 4-dimethylaminopyridine (DMAP, 0.77 mmol) and different amounts of PCB (KOS / PCB = 1:1, 1:2, 1:3, molar ratio) were respectively added to DMSO (10 mL) and mixed for 30 min. Meanwhile, KOS powder (3.09 mmol) was dissolved in DMSO (15 mL) and added to the above system, and the reaction was carried out at room temperature and under N2 atmosphere for 24 h. After the reaction, DCU was first filtered off, and a large amount of absolute ethanol was added to the filtrate to precipitate BKOS. The product was centrifuged (7000 rpm, 5 min) and washed twice with ethanol, then dissolved in water and freeze-dried to obtain a powdery BKOS product.

[0043] 3. Preparation of BKOS Schiff base hydrogel:

[0044] BKOS (KOS / PCB = 1:2, benzaldehyde substitution degree 21.0%) was dissolved in phosphate buffer (PBS, 0.01 mol / L, pH = 7.4) at a concentration of 2% (m / v g / mL), and rapidly mixed with an equal volume of CMCS aqueous solution (amino substitution degree 51.6%, initial concentration 5% (m / v g / mL)) on a vortex oscillator (rotation speed 1500 rpm) to form a hydrogel, obtaining BKOS / CMCS hydrogel.

[0045] BKOS (KOS / PCB = 1:2, benzaldehyde substitution degree 21.0%) was dissolved in phosphate buffer (PBS, 0.01 mol / L, pH = 7.4) at a concentration of 2% (m / v g / mL), and rapidly mixed with an equal volume of HPGA (hydrazide substitution degree 11.5%, initial concentration 10% (m / v g / mL)) aqueous solution on a vortex oscillator (rotation speed 1500 rpm) to form a hydrogel, obtaining BKOS / HPGA hydrogel.

[0046] BKOS (KOS / PCB = 1:2, benzaldehyde substitution degree 21.0%) was dissolved in phosphate buffer (PBS, 0.01 mol / L, pH = 7.4) at a concentration of 2% (m / v g / mL), and rapidly mixed with an equal volume of the mixture of HPGA and ε-PL on a vortex oscillator (rotation speed 1500 rpm) to form a hydrogel, obtaining BKOS / HPGA / ε-PL hydrogel. The said mixture was prepared by mixing HPGA (hydrazide substitution degree 11.5%, initial concentration 10% (m / v g / mL)) and ε-polylysine (ε-PL, initial concentration 10% (m / v g / mL) aqueous solution) in a volume ratio of 9:1.

[0047] Example 4 Physicochemical Property Characterization of Oligosaccharide Ester Modified with Benzaldehyde Group and Its Crosslinked Hydrogel

[0048] 1. 1 1H nuclear magnetic resonance (NMR) spectroscopy

[0049] By 1 1H NMR was used to perform structural analysis and degree of substitution determination on CBAO of Example 1 and BKOS of Example 2. 15 - 20 mg of AO, KOS, PCB, CBAO, and BKOS samples were dissolved in 0.55 mL of DMSO-d6 for 1 1H spectrum analysis. The degree of substitution of CBAO was calculated from the integral area ratio of the aldehyde hydrogen on PCB (10.12 ppm) to the anomeric carbon hydrogen of 3,6-anhydrogalactose (A ring) in the AO unit (A1, 5.14 ppm) ( Figure 1 ); the degree of substitution of BKOS was calculated from the integral area ratio of the aldehyde hydrogen on PCB (10.12 ppm) to the seven hydrogens (3.5 - 5.5 ppm) on the glucose and mannose sugar rings in the KOS unit ( Figure 2 ). When the ratio of AO to PCB was AO / PCB = 1:0.5, 1:1, and 1:2, the degrees of benzaldehyde substitution of CBAO were approximately 5%, 15%, and 29%, respectively. When the ratio of KOS to PCB was KOS / PCB = 1:1, 1:2, and 1:3, the degrees of benzaldehyde substitution of BKOS were approximately 5%, 20%, and 29%, respectively.

[0050] 2. Gel time determination

[0051] The gel times of the hydrogels in Example 1 and Example 2 were accurately determined using a timer. The gel point was defined as the moment when the solution did not flow after the test tube was inverted and held for 1 min. CBAO / CMCS gelled within 7 minutes, CBAO / HPGA gelled within 4 minutes, and the CBAO / HPGA / ε-PL hydrogel gelled within 1 minute. BKOS / CMCS gelled within 3 minutes, the BKOS / HPGA hydrogel gelled within 1 minute, and the BKOS / HPGA / ε-PL hydrogel gelled within 30 seconds. The hydrogels formed by the two oligosaccharides with different components had similar appearances ( Figure 3 ).

[0052] 3. Release behavior of CBAO in the CBAO / HPGA / ε-PL hydrogel at different pH values

[0053] Take 400 μL of the CBAO / HPGA / ε-PL hydrogel (preparation method see Example 1), place it in 2 mL of PBS solution (pH values were 7.4, 6.5, and 5.6), soak at 37 °C for 24 h, and extract 100 μL of liquid at 2, 4, 8, 12, and 24 h, respectively. Then dilute the liquid and centrifuge it, and measure the absorbance (A 255 ) at a wavelength of 255 nm using a UV-visible spectrophotometer ( Figure 4)。In the first 2 h, the CBAO release amount of the hydrogel was the largest at pH = 5.6. After that, the release rate and disintegration rate slowed down, and there was a certain shrinkage. After 24 h, the CBAO released from the hydrogel at pH = 6.5 was the most (close to 12%). Since the release amount of CBAO was only 1-2% of the total mass, the remaining mass loss was provided by ε-PL and HPGA, with ε-PL being the main one. It can be seen that the CBAO / HPGA / ε-PL hydrogel can be partially disintegrated in an environment with a pH range of 5-7, thus having a sustained-release effect on the loaded substance.

[0054] 4. Cytotoxicity determination of CBAO / CMCS hydrogel

[0055] The freshly prepared CBAO (degree of benzaldehyde substitution 15.4%, 500 μg) and CMCS (500 μg) powders were separately dissolved in 1 mL of high-glucose DMEM medium (Gibco). A 4% (m / v g / mL) CBAO (DS = 15.4%, 50 μL, dissolved in pure water) and 5% (m / v g / mL) CMCS (50 μL, dissolved in pure water) aqueous solution were mixed to prepare a CBAO / CMCS hydrogel (100 mg) and soaked in 1 mL of high-glucose DMEM medium. After 48 h, it was centrifuged at 1000 rpm for 4 min, and the supernatant was serially diluted to obtain material extracts with different concentrations for cell proliferation assays.

[0056] The CCK8 method was used to determine the cell viability of NIH-3T3 ( Figure 5 A&5C) and RAW264.7 ( Figure 5 B&5D) at different culture times and solution / extract concentrations to evaluate the in vitro cytotoxicity of the CBAO hydrogel and its components. In a 96-well plate, they were respectively seeded at 4×10 3 / well and 3×10 3The number of wells was inoculated with 100 μL of NIH-3T3 and RAW264.7 cells. The culture medium used for NIH-3T3 and RAW264.7 cells was high-glucose DMEM (filtered through a 0.22 μm filter membrane) containing 10% (m / v g / mL) FBS. After the cells were completely adherent, the original cell culture medium was replaced with 100 μL of the test sample, and the cells were cultured in an incubator at 37 °C for 24, 48, and 72 h. Different concentrations of CBAO and CMCS solutions (500, 100, 50 μg / mL) and CBAO / CMCS hydrogel extracts (diluted 1, 10, 100, 200 times) were used as the experimental groups; cells cultured in high-glucose DMEM medium containing 10% FBS without samples were used as the control group. After incubation, 10 μL of CCK-8 solution was added, and the reaction was carried out at 37 °C for 1 h, and then the absorbance of the solution at 450 nm in each well was measured with an enzyme-linked immunosorbent assay reader. As can be seen from the figure, after treatment with different components and hydrogel extracts, the survival rates of NIH-3T3 and RAW264.7 cultured for 1 d and 3 d were both above 80%, reaching the grade I toxicity standard (survival rate > 75%).

[0057] 5. Determination of antibacterial activity of CBAO hydrogel

[0058] Escherichia coli and Staphylococcus aureus (1%, m / v g / mL) were inoculated into 10 mL of broth medium (21% (m / v g / mL), pH = 7.4) and activated at 37 °C for 12 h. Then, 1 mL of the activated bacterial solution was transferred to 10 mL of fresh broth medium and cultured at 37 °C for 8 h. When the OD value at 630 nm was 0.1, the bacterial concentration was approximately 10 7 CFU / mL; the bacterial solution was serially diluted to 10 3 CFU / mL as the working concentration.

[0059] The operating steps for testing the antibacterial activities of CBAO / CMCS and CBAO / HPGA / ε-PL hydrogels under different environmental pH conditions were as follows: The pH of PBS was adjusted to 7.4, 6.5, and 5.6 respectively, and then PBS was sterilized. Using the hydrogel raw material components described in Example 1 and Example 2, 200 μL of CBAO (benzaldehyde substitution degree 15.4%, 50 mg / mL), 200 μL of CMCS (amino substitution degree 51.6%, 50 mg / mL), or 200 μL of HPGA + ε-PL mixture (50 mg / mL, volume ratio of HPGA to ε-PL was 9:1) solution were mixed in a centrifuge tube, and 400 μL of CBAO / CMCS and CBAO / HPGA / ε-PL hydrogels were in-situ prepared respectively according to the conditions described in Example 1 and Example 2. Then, 4 mL of PBS with different pH values was added to extract the hydrogel (volume 400 μL). After soaking at 37 °C for 24 h, 400 μL of the extract from each well was taken and mixed with 1 mL of activated bacteria (103 CFU / mL) were co-cultured at 37 °C for 24 h. Meanwhile, 400 μL of PBS with different pH values was mixed with 1 mL of activated bacteria as a control group. After the culture ended, 100 μL of the co-culture solution was taken out and incubated on a sterilized LB agar plate at 37 °C for 24 h. The inhibition rate of the hydrogel was calculated according to the above method, and the control groups with pH = 6.5 and 5.6 were also compared with the control group with pH = 7.4 ( Figure 6 ). The mixed solution was prepared by mixing HPGA (acyl hydrazide substitution degree 11.5%, initial concentration 5% (m / v g / mL)) and ε-polylysine (ε-PL, initial concentration 5% (m / v g / mL) aqueous solution) in a volume ratio of 9:1.

[0060] The antibacterial activities of CBAO / CMCS and CBAO / HPGA / ε-PL hydrogels under different environmental pH conditions are as Figure 6 shown, where A - C are the antibacterial zones, and D - F are the colony counts at different acidities. As can be seen from the figure, with the increase in the acidity of the culture medium, the inhibition rate of the CBAO / CMCS hydrogel gradually increases: the inhibition rate against Escherichia coli increases from 52.9%, 54.6% to 69.2%, and the inhibition rate against Staphylococcus aureus increases from 57.0%, 59.4% to 62.1% ( Figure 6 A&6D). The antibacterial performance of the CBAO / HPGA / ε-PL hydrogel also improves with the increase in acidity. With the increase in the acidity of the gel-forming solution, the inhibition rates of the hydrogel against Escherichia coli are 78.9%, 81.3% and 83.7% in turn, and the inhibition rate against Staphylococcus aureus increases from 79.2%, 81.7% to 90.8% ( Figure 6 B&6E); and with the increase in the acidity of the culture medium, the inhibition rate of the hydrogel against bacteria further increases: for Escherichia coli, it increases from 79.2%, 82.8% to 91.4%, and for Staphylococcus aureus, it increases from 80.1%, 82.7% to 93.7% ( Figure 6 C&6F). This proves that the CBAO hydrogel has good pH responsiveness.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A preparation method of a benzaldehyde-modified oligosaccharide-based Schiff base hydrogel, characterized in that, The oligosaccharide is esterified with p-carboxybenzaldehyde to obtain a benzaldehyde-modified oligosaccharide ester, which is then crosslinked with a natural polymer containing primary amine through dynamic covalent bonds to form a hydrogel; The oligosaccharide is selected from at least one of agar oligosaccharide and konjac glucomannan oligosaccharide; The natural polymer containing primary amine is selected from at least one or several of carboxymethyl chitosan and polyglutamic acid substituted with adipic dihydrazide.

2. The preparation method according to claim 1, wherein The molecular weight distribution range of the oligosaccharide is 1000 - 5000 Da.

3. The preparation method according to claim 1, characterized in that, The process of esterifying the oligosaccharide with p-carboxybenzaldehyde to obtain the benzaldehyde-modified oligosaccharide ester is as follows: N,N-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and p-carboxybenzaldehyde are added to DMSO, and at the same time the oligosaccharide is added to the reaction. After the reaction is completed, dicyclohexylurea is first filtered off, and absolute ethanol is added to the filtrate to obtain the oligosaccharide ester.

4. The preparation method according to claim 1, characterized in that, The hydrogel also loads ε-polylysine.

5. The preparation method according to claim 1, characterized in that, The process of crosslinking the oligosaccharide ester with the natural polymer containing primary amine through dynamic covalent bonds to form a hydrogel is as follows: the oligosaccharide ester is dissolved in PBS buffer at a concentration of 4 g / 100 ml, and then mixed and shaken with an equal volume of the natural polymer solution containing primary amine to form a hydrogel; the natural polymer solution containing primary amine is a 5 g / 100 ml carboxymethyl chitosan solution, a 10 g / 100 ml polyglutamic acid solution substituted with adipic dihydrazide, or a mixture prepared by mixing a 10 g / 100 ml polyglutamic acid solution substituted with adipic dihydrazide and a 10 g / 100 ml ε-polylysine solution in a volume ratio of 9:

1.

6. A benzaldehyde-modified oligosaccharide-based Schiff base hydrogel prepared by the preparation method according to claim 1.

7. Use of the benzaldehyde-modified oligosaccharide-based Schiff base hydrogel according to claim 6 after loading an antibacterial peptide in the preparation of an antibacterial drug.

8. The application according to claim 7, wherein The antibacterial drug is a drug that inhibits Escherichia coli or Staphylococcus aureus.

Images