A marine polysaccharide-based hydrogel and a preparation method and application thereof

Abstract

The present application relates to the technical field of medical devices, in particular to a marine polysaccharide-based hydrogel and a preparation method and application thereof. The present application utilizes chitosan, N-tris(hydroxymethyl)methyl glycine, carrageenan and sea cucumber protein peptide, and through a delicate three-step method of "chemical grafting-oxidation modification-protein peptide complexing", successfully prepares a marine polysaccharide-based hydrogel. The marine polysaccharide-based hydrogel obtained by the present application has superior self-healing properties, antibacterial properties and good biocompatibility, and has good application prospects in the field of preparing medical antibacterial and wound repair materials.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and in particular to a marine polysaccharide-based hydrogel, its preparation method, and its application. Background Technology

[0002] Wound healing is a complex physiological process. Chronic wounds caused by severe bacterial infection often fail to heal smoothly, requiring long-term treatment and causing significant physical and psychological suffering for patients. Wound infection is a challenging global problem that can lead to life-threatening complications, induce chronic inflammation, and impair wound repair.

[0003] The development of novel and efficient medical polymer materials has become a current research hotspot and an urgent industrial need. Hydrogels, due to their high water content, good biocompatibility, and three-dimensional network structure similar to the extracellular matrix, have shown great application potential in the fields of medical dressings and tissue engineering.

[0004] However, traditional synthetic polymer hydrogels (such as polyacrylic acid hydrogels) often have poor biocompatibility and biodegradability; while conventional hydrogels with physical or chemical cross-linking are prone to permanent damage to their network structure after mechanical injury, lacking self-healing ability. This may lead to functional failure in dynamically used wound environments and secondary damage due to frequent dressing changes. In addition, wounds are susceptible to bacterial infection during the healing process, and conventional hydrogels themselves usually do not have antibacterial functions, requiring the addition of antibiotics or silver ions, which may lead to problems such as bacterial resistance or biotoxicity.

[0005] Marine polysaccharides (such as chitosan and carrageenan) are considered ideal building blocks for medical polymer materials due to their wide availability, biodegradability, and good biocompatibility. However, the organic combination of self-healing and long-lasting antibacterial properties in marine polysaccharide-based hydrogel materials remains a significant technical challenge. Therefore, developing a safe, non-toxic marine polysaccharide-based hydrogel material that combines excellent self-healing properties with intrinsic antibacterial activity is of urgent practical need and significant application value. Summary of the Invention

[0006] Based on the above, the present invention provides a marine polysaccharide-based hydrogel, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] One of the technical solutions of this invention is a method for preparing a marine polysaccharide-based hydrogel, comprising the following steps:

[0009] Step 1: Dissolve chitosan in an aqueous acetic acid solution to obtain a chitosan solution;

[0010] N-Tris(hydroxymethyl)methylglycine was dissolved in water and an activator was added to obtain an activated solution of N-tris(hydroxymethyl)methylglycine.

[0011] An activated solution of N-tris(hydroxymethyl)methylglycine was added to a chitosan solution for reaction, followed by dialyzing and freeze-drying to obtain N-tris(hydroxymethyl)methylglycine-grafted chitosan.

[0012] Step 2: Dissolve carrageenan in water, add hydrogen peroxide solution and L-proline for oxidation, dialyze to obtain oxidized carrageenan solution;

[0013] Step 3: Dissolve N-tris(hydroxymethyl)methylglycine-grafted chitosan in water to obtain an aqueous solution of N-tris(hydroxymethyl)methylglycine-grafted chitosan. Add sea cucumber protein peptides and mix well. Then add oxidized carrageenan aqueous solution and mix well. Let stand until gel state to obtain marine polysaccharide-based hydrogel.

[0014] Step 1 and Step 2 are not in any particular order.

[0015] The second technical solution of the present invention is a marine polysaccharide-based hydrogel prepared by the above-mentioned preparation method.

[0016] The third technical solution of this invention is the application of the above-mentioned marine polysaccharide-based hydrogel in the preparation of medical antibacterial or wound repair materials.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The marine polysaccharide-based hydrogel of this invention exhibits unique comprehensive advantages. Its core lies in its use of all-marine biomass (chitosan, carrageenan, and sea cucumber protein peptides) as building blocks. Through a sophisticated three-step design of "chemical grafting-oxidative modification-protein peptide composite," it successfully integrates multiple core performance characteristics sought after by high-end medical devices, including biosafety, highly effective antibacterial properties, dynamic self-healing, and injectability. Dynamic self-healing stems from reversible imine bonds (-C=N-) and hydrogen bonds, greatly improving the reliability and safety of implanted devices. Antibacterial properties arise from the enhanced bactericidal effect of N-tris(hydroxymethyl)methylglycine grafted chitosan, which synergistically enhances the broad-spectrum antibacterial efficacy of the system with the composite sea cucumber protein peptides, effectively preventing implant infection. Furthermore, its injectable in-situ molding characteristics are suitable for minimally invasive surgery, and the introduced sea cucumber protein peptides further endow the material with bioactivity that actively promotes cell adhesion and tissue regeneration. The marine polysaccharide-based hydrogel material of this invention is not only an excellent example of the high-value utilization of marine biomass, but also a strong candidate for the next generation of high-end medical devices that meet the urgent clinical needs of the future. Moreover, the preparation process of this invention is simple and easy to industrialize. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The image shows the appearance and morphology of the marine polysaccharide-based hydrogel obtained in Example 1.

[0021] Figure 2 The image shows a scanning electron microscope (SEM) image of the freeze-dried sample obtained after freeze-drying the marine polysaccharide-based hydrogel obtained in Example 1.

[0022] Figure 3 The image shows a self-healing experiment of the marine polysaccharide-based hydrogel obtained in Example 1. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Unless otherwise specified, "room temperature" or "normal temperature" as used in this invention refers to 25±3℃.

[0029] The first aspect of this invention provides a method for preparing a marine polysaccharide-based hydrogel, comprising the following steps:

[0030] Step 1: Dissolve chitosan in an aqueous acetic acid solution to obtain a chitosan solution;

[0031] N-Tris(hydroxymethyl)methylglycine was dissolved in water and an activator was added to obtain an activated solution of N-tris(hydroxymethyl)methylglycine.

[0032] An activated solution of N-tris(hydroxymethyl)methylglycine was added to a chitosan solution for reaction, followed by dialyzing and freeze-drying to obtain N-tris(hydroxymethyl)methylglycine-grafted chitosan.

[0033] Step 2: Dissolve carrageenan in water, add hydrogen peroxide solution and L-proline for oxidation, dialyze to obtain oxidized carrageenan solution;

[0034] Step 3: Dissolve N-tris(hydroxymethyl)methylglycine-grafted chitosan in water to obtain an aqueous solution of N-tris(hydroxymethyl)methylglycine-grafted chitosan. Add sea cucumber protein peptides and mix well. Then add oxidized carrageenan aqueous solution and mix well. Let stand until gel state to obtain marine polysaccharide-based hydrogel.

[0035] Step 1 and Step 2 are not in any particular order.

[0036] The present invention also experimented with replacing the aqueous acetic acid solution with hydrochloric acid solution, and the results showed that hydrochloric acid solution degraded the chitosan molecular backbone and had a poor effect.

[0037] The present invention also experimented with replacing the aqueous hydrogen peroxide solution with sodium periodate, and the results showed that sodium periodate had no significant effect on the oxidation of carrageenan.

[0038] In a preferred embodiment of the present invention, in step 1, the mass fraction of acetic acid in the acetic acid aqueous solution is 1%-3%; the mass fraction of chitosan in the chitosan solution is 2%-3%; and the degree of deacetylation of chitosan is 85%-95%.

[0039] In a preferred embodiment of the present invention, in step 1, the activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine; the mass ratio of N-tris(hydroxymethyl)methylglycine to water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine in the N-tris(hydroxymethyl)methylglycine activation solution is 2:10:3:3; when preparing the N-tris(hydroxymethyl)methylglycine activation solution, the activator is added and the mixture is stirred at room temperature for 2 h.

[0040] In a preferred embodiment of the present invention, in step 1, the reaction conditions are set as follows: stirring at room temperature for 5 h.

[0041] In this invention, N-tris(hydroxymethyl)methylglycine-grafted chitosan exhibits the following superior effects: First, by modifying chitosan with N-tris(hydroxymethyl)methylglycine grafting, its water solubility is improved, and the viscosity of the solution is reduced, which is beneficial for the blending of polymeric polyelectrolytes. This allows for the preparation of structurally homogeneous polymeric biomaterials via a green approach. Second, the introduced N-tris(hydroxymethyl)methylglycine effectively enhances the antibacterial properties of chitosan itself and synergistically enhances the broad-spectrum antibacterial efficacy of the complexed sea cucumber protein peptides. Third, the chitosan-grafted N-tris(hydroxymethyl)methylglycine possesses a highly branched spatial structure, which facilitates the inclusion of sea cucumber protein peptide molecules. Simultaneously, it contains abundant terminal hydroxyl groups, which can stably bind to sea cucumber protein peptide molecules through hydrogen bonding, enhancing not only the stability of the sea cucumber protein peptides but also their bioavailability, thereby achieving a more durable antibacterial effect.

[0042] In a preferred embodiment of the present invention, in step 2, the carrageenan is 1-carrageenan; the mass fraction of the hydrogen peroxide aqueous solution is 25%; the mass ratio of carrageenan to water, hydrogen peroxide aqueous solution and L-proline is 2:(64~198):(16~20):1; the oxidation conditions are set as follows: oxidation at room temperature in the dark for 3 h.

[0043] This invention employs an L-proline-assisted hydrogen peroxide oxidation method to efficiently introduce aldehyde groups onto the carrageenan molecular chain. This method offers significant advantages: hydrogen peroxide acts as a green oxidant, producing only water as a byproduct with no harmful residues; the reaction conditions are mild under the promotion of L-proline, selectively oxidizing the hydroxyl groups on sugar units to generate aldehyde groups in high yield while effectively inhibiting excessive oxidation to carboxyl groups; simultaneously, it better preserves the molecular framework and gel properties of carrageenan, laying the foundation for constructing cross-linked materials via the Schiff base reaction. This method combines environmental friendliness with practical applicability.

[0044] In a preferred embodiment of the present invention, in step 3, the mass fraction of the N-tris(hydroxymethyl)methylglycine-grafted chitosan aqueous solution is 2% to 5%.

[0045] In a preferred embodiment of the present invention, the mass ratio of chitosan to N-tris(hydroxymethyl)methylglycine, carrageenan and sea cucumber protein peptide is (5~16):(6~24):(4~19):(0.2~0.5).

[0046] A second aspect of the present invention provides a marine polysaccharide-based hydrogel prepared using the above-described preparation method.

[0047] The third aspect of this invention provides the application of the above-mentioned marine polysaccharide-based hydrogel in the preparation of medical antibacterial or wound repair materials.

[0048] The marine polysaccharide-based hydrogel obtained by this invention has excellent self-healing properties, antibacterial properties, and good biocompatibility, and has good application prospects in the preparation of medical antibacterial and wound repair materials.

[0049] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0050] The sea cucumber protein peptides used in this embodiment of the invention are complex peptides prepared by enzymatic hydrolysis of sea cucumber. The enzymatic hydrolysis of sea cucumber protein peptides can be carried out according to conventional methods in the art, and the specific steps are as follows:

[0051] Fresh sea cucumbers (Stichopus horrens) were cleaned of mud and sand, viscera removed, washed, cut into small pieces, treated with liquid nitrogen, rapidly pulverized, and frozen at -20℃ for later use. The pre-treated sea cucumber powder was suspended in distilled water and sonicated for 15 min. The pH was then adjusted to the appropriate value for the enzymes using 1 mol / L NaOH or HCl solution. The mixture was then incubated with different enzymes (pepsin, papain, and alkaline protease, 3.0 kU / mL) at 50℃ for 6 h each. Finally, the mixture was placed in a boiling water bath for 10 min to inactivate the enzymes, centrifuged (10000 r / min, 10 min), and the supernatant was collected. Three volumes of pre-cooled anhydrous ethanol were added, and the mixture was placed at 4℃ for 12 h. After centrifugation (10000 r / min, 10 min), the supernatant was collected, concentrated under reduced pressure, ultrafiltered, and freeze-dried to obtain sea cucumber peptides. The molecular weight distribution range of the sea cucumber protein peptides of the present invention is as follows: the content of peptides with a molecular weight >3.0 kDa is 3.06%, the content of peptides with a molecular weight of 1.0~3.0 kDa is 11.52%, and the content of peptides with a molecular weight <1.0 kDa is 85.42%.

[0052] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] A method for preparing a marine polysaccharide-based hydrogel, comprising the following steps:

[0055] (1) 10 g of chitosan (with a degree of deacetylation of 90%) was added to a 2% acetic acid aqueous solution and stirred to dissolve, thus preparing a 2.5% chitosan solution (I); 15 g of N-tris(hydroxymethyl)methylglycine was dissolved in 75 g of distilled water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (22.5 g) and 4-dimethylaminopyridine (22.5 g) were added. The solution was stirred at room temperature for 2 h to obtain an activated solution of N-tris(hydroxymethyl)methylglycine (II).

[0056] (2) The activated solution (II) of N-tris(hydroxymethyl)methylglycine was added to the chitosan solution (I), stirred at room temperature for 5 h, dialyzed with distilled water (molecular weight cutoff 3500 Da), and freeze-dried to obtain N-tris(hydroxymethyl)methylglycine-grafted chitosan.

[0057] (3) Dissolve 11 g of ι-carrageenan in 539 g of distilled water, add 110 g of 25% hydrogen peroxide aqueous solution and 5.5 g of L-proline, oxidize at room temperature in the dark for 3 h, and dialyze with distilled water (molecular weight cutoff 3500 Da) to obtain oxidized carrageenan aqueous solution.

[0058] (4) Add the obtained N-tris(hydroxymethyl)methylglycine-grafted chitosan to distilled water to prepare a 3% N-tris(hydroxymethyl)methylglycine-grafted chitosan aqueous solution. Add sea cucumber protein peptide (0.3 g) and mix well. Then add the above oxidized carrageenan aqueous solution, mix well, and let stand until gel state to obtain marine polysaccharide hydrogel.

[0059] Figure 1 The image shows the morphology of the marine polysaccharide-based hydrogel obtained in Example 1. Figure 2 This is a scanning electron microscope cross-sectional image of the freeze-dried sample obtained after freeze-drying the marine polysaccharide-based hydrogel obtained in Example 1.

[0060] like Figure 1 As shown, the hydrogel has a smooth surface and high transparency. Scanning electron microscopy (SEM) images were obtained after freeze-drying. Figure 2 The structure exhibits a porous and layered structure. This structure possesses excellent permeability and high fluid absorption capacity, which helps maintain a moist microenvironment in the wound tissue, thereby promoting the wound healing process.

[0061] Example 2

[0062] (1) Add 5 g of chitosan (85% degree of deacetylation) to a 3% acetic acid aqueous solution and stir to dissolve to prepare a 2% chitosan solution (I); dissolve 6 g of N-tris(hydroxymethyl)methylglycine in 30 g of distilled water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (9 g) and 4-dimethylaminopyridine (9 g), stir at room temperature for 2 h to obtain an activated solution of N-tris(hydroxymethyl)methylglycine (II).

[0063] (2) The activated solution (II) of N-tris(hydroxymethyl)methylglycine was added to the chitosan solution (I), stirred at room temperature for 5 h, dialyzed with distilled water, and freeze-dried to obtain N-tris(hydroxymethyl)methylglycine-grafted chitosan.

[0064] (3) Dissolve 4 g of ι-carrageenan in 396 g of distilled water, add 32 g of 25% hydrogen peroxide aqueous solution and 2 g of L-proline, oxidize at room temperature in the dark for 3 h, and dialyze with distilled water to obtain oxidized carrageenan aqueous solution.

[0065] (4) Add the obtained N-tris(hydroxymethyl)methylglycine-grafted chitosan to distilled water to prepare a 2% N-tris(hydroxymethyl)methylglycine-grafted chitosan aqueous solution. Add sea cucumber protein peptide (0.2 g) and mix well. Then add oxidized carrageenan aqueous solution, mix well, and let stand until gel state to obtain marine polysaccharide hydrogel.

[0066] Example 3

[0067] (1) 16 g of chitosan (with a degree of deacetylation of 95%) was added to a 1% acetic acid aqueous solution and stirred to dissolve, thus preparing a 3% chitosan solution (I); 24 g of N-tris(hydroxymethyl)methylglycine was dissolved in 120 g of distilled water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (36 g) and 4-dimethylaminopyridine (36 g) were added. The solution was stirred at room temperature for 2 h to obtain an activated solution of N-tris(hydroxymethyl)methylglycine (II).

[0068] (2) The activated solution (II) of N-tris(hydroxymethyl)methylglycine was added to the chitosan solution (I), stirred at room temperature for 5 h, dialyzed with distilled water, and freeze-dried to obtain N-tris(hydroxymethyl)methylglycine-grafted chitosan.

[0069] (3) Dissolve 19 g of ι-carrageenan in 614 g of distilled water, add 152 g of 25% hydrogen peroxide aqueous solution and 9.5 g of L-proline, oxidize at room temperature in the dark for 3 h, and dialyze with distilled water to obtain oxidized carrageenan aqueous solution.

[0070] (4) Add the obtained N-tris(hydroxymethyl)methylglycine-grafted chitosan to distilled water to prepare a 5% N-tris(hydroxymethyl)methylglycine-grafted chitosan aqueous solution. Add sea cucumber protein peptide (0.5 g) and mix well. Then add oxidized carrageenan aqueous solution, mix well, and let stand until gel state to obtain marine polysaccharide hydrogel.

[0071] Comparative Example 1

[0072] A method for preparing a marine polysaccharide-based hydrogel differs from Example 1 only in that L-proline in step (3) is replaced with an equal mass of copper sulfate; the remaining process steps and parameters are the same as in Example 1.

[0073] Comparative Example 2

[0074] A method for preparing an antibacterial hydrogel differs from Example 1 only in that: the ι-carrageenan in step (3) is replaced with an equal mass of κ-carrageenan; the remaining process steps and parameters are the same as in Example 1.

[0075] Example 1

[0076] The self-healing properties of the prepared hydrogel samples were tested.

[0077] (1) Self-healing and injectability

[0078] The self-healing and injectability tests were conducted on the hydrogel prepared in Example 1. Two freshly prepared hydrogel samples were stained with Coomassie Brilliant Blue and Basic Fuchsin solutions, respectively. Each stained hydrogel was then cut into two segments with a scalpel. The cut surfaces of the two stained halves were brought into close contact and aligned, then placed in the same moistened petri dish and allowed to stand at room temperature for 30 minutes to allow for self-healing. The healing process was observed, and the macroscopic morphology was recorded using a digital camera. To evaluate injectability, a freshly prepared, ungelled hydrogel precursor solution was rapidly loaded into a disposable syringe equipped with a 32 mm long bevel needle. The syringe plunger was then slowly pushed, and the continuity and morphology of the extrudate were observed and recorded.

[0079] Hydrogel self-healing effect Figure 3As shown, the results indicate that the hydrogel can self-heal and reform into a complete hydrogel after being cut. The self-healed hydrogel can support its own weight and can be picked up with tweezers without breaking, indicating that the hydrogel of Example 1 has strong self-healing properties. In addition, the hydrogel of Example 1 can be smoothly extruded through a syringe needle and used to draw letter shapes, maintaining the pre-defined shape after extrusion, indicating that it has good injectability and shape retention capabilities.

[0080] (2) Self-healing efficiency

[0081] The self-healing efficiency of the hydrogels prepared in Examples 1-3 and Comparative Examples 1-2 was tested. The hydrogels were cut in half lengthwise with a scalpel, and the two halves were arranged along the cut. A slight external force was applied at room temperature to ensure complete contact between the cut surfaces. After 3 hours, the tensile strength of the samples was measured using a universal testing machine. The samples were stretched at a constant strain rate of 1 mm / min until complete tensile failure. Each test was repeated three times, and the average value was taken. The self-healing efficiency of the hydrogels was calculated according to formula (1). The results are shown in Table 1.

[0082] (1)

[0083] Table 1 Self-healing efficiency of hydrogels

[0084]

[0085] As shown in Table 1, Examples 1-3 exhibit good self-healing properties due to the reversibility of dynamic covalent / non-covalent bonds (imine bonds, electrostatic interactions, and multiple hydrogen bonds) in the hydrogels. However, in Comparative Examples 1-2, the self-healing efficiency of the hydrogels was significantly reduced and their self-healing properties weakened because the method of carrageenan oxidation in Comparative Example 1 was changed from hydrogen peroxide + L-proline to hydrogen peroxide + copper sulfate, and in Comparative Example 2, ι-carrageenan was replaced with κ-carrageenan.

[0086] (3) Rheological behavior

[0087] The hydrogel sample from Example 1 was stored at 4 °C for 6 h to ensure a uniform and stable gel state. Subsequently, it was tested using a rheometer.

[0088] Strain scanning results (strain range 0.1–1000%) showed that in the low-strain linear viscoelastic region, the storage modulus (G') remained stable and significantly higher than the loss modulus (G''), indicating that the hydrogel of Example 1 exhibits typical elastic-dominated gel behavior. The high G' value also indicates good support capacity. When the strain value approached the critical strain point (12.32%), G' decreased sharply and crossed with G'', indicating that the hydrogel crosslinking network dissociated or broke down, leading to liquefaction of the hydrogel.

[0089] Alternating strain scanning experiments (0.5%→30%→0.5%) further revealed the network reconstruction kinetics of the hydrogel in Example 1. At a strain of 30% (G>G'), the internal network of the hydrogel in Example 1 was disrupted, and the structure collapsed; however, at a strain of 0.5%, G' and G" immediately recovered to their initial state (G'>G"), indicating that the hydrogel structure of Example 1 possesses self-healing properties.

[0090] Example 2

[0091] The antibacterial effects of the hydrogel samples prepared in Examples 1-3 and Comparative Examples 1-2 were tested.

[0092] Gram-positive Staphylococcus aureus ( Staphylococcus aureus , S. aureus, ATCC 6538 ) and Gram-negative Escherichia coli ( Escherichia coli , E. coli, ATCC 25922 The evaluation of the antibacterial effect of the test bacteria sample includes the following steps:

[0093] The test bacteria in the logarithmic growth phase were diluted with broth to a concentration of 1.0 × 10⁻⁶. 6 CFU / mL, ready for use. Take 1 mL of sample solution (0.5 mg / mL) and add it to a sterile EP (Eppendorf) tube; add 1 mL of E. coli or S. aureus bacterial suspension (1.0 × 10⁻⁶). 6 CFU / mL), and cultured at 37℃ with shaking (100 r / min) for 18 h. A certain amount of bacterial culture was taken from the above EP tube and diluted to a suitable concentration with PBS buffer using the tenfold dilution method. 100 μL of the diluted bacterial suspension was dropped into an agar culture dish, spread evenly with a spreader, and cultured at 37℃ for 24 h. The number of colonies on the culture dish was recorded. A blank control group (using distilled water instead of sample solution) and an antibiotic positive control group (gentamicin sulfate, 0.1 mg / mL) were set up respectively. The antibacterial rate of the sample was calculated according to formula (2), and the results are shown in Table 2 (N-tris(hydroxymethyl)methylglycine-grafted chitosan in Table 2 is the N-tris(hydroxymethyl)methylglycine-grafted chitosan prepared in Example 1).

[0094] (2)

[0095] Where, N b and N s These represent the bacterial colony counts in the blank control group and the sample group, respectively.

[0096] Table 2 Antibacterial properties of the samples

[0097]

[0098] As shown in Table 2, at the experimental concentration of this invention, N-tris(hydroxymethyl)methylglycine exhibited weak antibacterial activity and did not show significant antibacterial activity; chitosan (90% deacetylation) had a certain antibacterial effect on the tested bacteria, but its antibacterial ability was low; compared with the raw chitosan, N-tris(hydroxymethyl)methylglycine-grafted chitosan showed a significantly enhanced antibacterial effect on the tested bacteria, indicating that the chemical modification of N-tris(hydroxymethyl)methylglycine-grafted chitosan improved the antibacterial ability of chitosan. Compared with the individual component samples, the hydrogel samples of Examples 1-3 showed strong antibacterial ability, indicating that the synergistic enhancement of antibacterial effect among the sample components. In contrast, in Comparative Examples 1-2, the antibacterial rate of the hydrogels was significantly reduced and the antibacterial ability was weakened because the method of carrageenan oxidation in Comparative Example 1 was changed from hydrogen peroxide + L-proline to hydrogen peroxide + copper sulfate, and in Comparative Example 2, ι-carrageenan was replaced with κ-carrageenan.

[0099] Example 3

[0100] Biocompatibility tests were performed on the hydrogel prepared in Example 1:

[0101] (1) Cell compatibility

[0102] The proliferation of immortalized human keratinocytes (HaCaT) was detected using the CCK-8 assay to investigate the effect of hydrogels on cell proliferation. The sample stock solution was serially diluted with fresh complete culture medium to prepare working solutions with concentrations of 25, 50, 100, 200, and 400 μg / mL using the two-fold dilution method, and then filtered through a 0.22 μm filter for sterilization. HaCaT cells in the logarithmic growth phase were collected, counted, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a concentration of 100 μL / well in a 96-well plate and incubated at (37±1)℃ in a 5% CO2 incubator for 24 h. The culture medium was then discarded, and DMEM culture medium or sample working solution (100 μL / well) was added. Six replicates were set up for each concentration. The plates were then incubated for another 24 h and 48 h, respectively. After incubation, the culture medium was discarded, and fresh culture medium was added. 10 μL of CCK-8 was added to each well, and blank control wells were set up. The plates were incubated at (37±1)℃ in the dark for 2 h. The optical density (OD) value of each well was read at 450 nm using a microplate reader, and cell viability was calculated.

[0103] The experimental results showed that after 24 hours of co-culture, the working solutions of the hydrogel samples obtained in Example 1 at different concentrations (25, 50, 100, 200, and 400 μg / mL) promoted cell growth, with cell viability of 104.89±2.09%, 108.52±2.16%, 108.35±2.14%, 106.76±2.11%, and 109.69±2.21%, respectively, all exceeding 100%. After 48 hours of co-culture, compared with 24 hours, the different gradient concentrations (25, 50, 100, 200, and 400 μg / mL) of the hydrogel samples obtained in Example 1 promoted cell growth. The cell viability (104.20±2.56%, 108.96±2.68%, 105.36±2.59%, 107.49±2.64%, and 102.71±2.53%, respectively) of the antibacterial hydrogel obtained in Example 1 showed no significant change; these results indicate that the antibacterial hydrogel obtained in Example 1 is non-cytotoxic and has good biocompatibility with HaCaT cells.

[0104] (2) Blood compatibility

[0105] Blood compatibility of the material was assessed using a erythrocyte hemolysis assay. Fresh rabbit blood was centrifuged (1000×g) for 10 min, the supernatant was discarded, and the blood was washed twice with physiological saline and gently resuspended to prepare a erythrocyte suspension. Simultaneously, the hydrogel stock solution obtained in Example 1 was sterilely filtered through a 0.22 μm filter membrane and serially diluted with phosphate-buffered saline (PBS) to working solutions of 1000, 500, 200, 100, 50, and 10 μg / mL. 1 mL of each concentration of working solution was placed in a 1.5 mL centrifuge tube and incubated at (37±1) °C for 30 min; then 20 μL of erythrocyte suspension was added, and incubation continued at (37±1) °C for 1 hour. 1 mL of physiological saline and 1 mL of deionized water were added to the negative control and positive control, respectively; all samples and controls were prepared in triplicate. After incubation, the mixture was centrifuged at 800×g for 5 min, and 0.2 mL of the supernatant was transferred to a 96-well plate. The absorbance at 545 nm was measured using an ELISA reader, and the hemolysis rate was calculated.

[0106] The experimental results showed that the hemolysis rate of hydrogel samples with different concentrations (1000, 500, 200, 100, 50 and 10 μg / mL) was less than 5% (0.46±0.17%, 0.80±0.19%, 0.42±0.15%, 0.52±0.21%, 0.48±0.34% and 0.94±0.23%, respectively), indicating that the hydrogel sample of Example 1 has good blood compatibility.

[0107] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a marine polysaccharide-based hydrogel, characterized in that, Includes the following steps: Step 1: Dissolve chitosan in an aqueous acetic acid solution to obtain a chitosan solution; N-Tris(hydroxymethyl)methylglycine was dissolved in water and an activator was added to obtain an activated solution of N-tris(hydroxymethyl)methylglycine. An activated solution of N-tris(hydroxymethyl)methylglycine was added to a chitosan solution for reaction, followed by dialyzing and freeze-drying to obtain N-tris(hydroxymethyl)methylglycine-grafted chitosan. Step 2: Dissolve carrageenan in water, add hydrogen peroxide solution and L-proline for oxidation, dialyze to obtain oxidized carrageenan solution; Step 3: Dissolve N-tris(hydroxymethyl)methylglycine-grafted chitosan in water to obtain an aqueous solution of N-tris(hydroxymethyl)methylglycine-grafted chitosan. Add sea cucumber protein peptides and mix well. Then add oxidized carrageenan aqueous solution and mix well. Let stand until gel state to obtain marine polysaccharide-based hydrogel. Step 1 and Step 2 are not in any particular order; In step 1, the activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine; in the activation solution of N-tris(hydroxymethyl)methylglycine, the mass ratio of N-tris(hydroxymethyl)methylglycine to water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 2∶10∶3∶3; when preparing the activation solution of N-tris(hydroxymethyl)methylglycine, the activator is added and the mixture is stirred at room temperature for 2 h. In step 2, the carrageenan is 1-carrageenan; the mass fraction of the hydrogen peroxide aqueous solution is 25%; the mass ratio of carrageenan to water, hydrogen peroxide aqueous solution and L-proline is 2:(64~198):(16~20):1; the oxidation conditions are set as follows: oxidation at room temperature in the dark for 3 h.

2. The preparation method according to claim 1, characterized in that, In step 1, the mass fraction of acetic acid in the acetic acid aqueous solution is 1%-3%; the mass fraction of chitosan in the chitosan solution is 2%-3%; and the degree of deacetylation of chitosan is 85%-95%.

3. The preparation method according to claim 1, characterized in that, In step 1, the reaction conditions are set as follows: stirring at room temperature for 5 hours.

4. The preparation method according to claim 1, characterized in that, In step 3, the mass fraction of the N-tris(hydroxymethyl)methylglycine-grafted chitosan aqueous solution is 2%~5%.

5. The preparation method according to claim 1, characterized in that, The mass ratio of chitosan to N-tris(hydroxymethyl)methylglycine, carrageenan, and sea cucumber protein peptide is (5~16):(6~24):(4~19):(0.2~0.5).

6. A marine polysaccharide-based hydrogel prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the marine polysaccharide-based hydrogel as described in claim 6 in the preparation of medical antibacterial or wound repair materials.

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