Novel antibacterial peptide molecule reinforced biomass antibacterial hydrogel and preparation method thereof

Abstract

The invention relates to the technical field of biomass materials and antibacterial materials, and discloses novel antibacterial peptide molecule reinforced biomass antibacterial hydrogel and a preparation method thereof.The preparation method comprises the steps that a, aldehyde hyaluronic acid AHA is synthesized, specifically, aldehyde modification is conducted on hyaluronic acid molecules so as to facilitate a cross-linking reaction between the hyaluronic acid molecules and chitosan quaternary ammonium salt QCS and antibacterial polypeptide molecules; step b, synthesis of chitosan quaternary ammonium salt / hydroformylated hyaluronic acid / antibacterial polypeptide AHA / QCS / epsilon-LP composite antibacterial hydrogel: preparing the biomass antibacterial hydrogel through interaction and cross-linking reaction between molecules; according to the preparation method, the antibacterial peptide and biomass molecules are effectively connected by utilizing intermolecular interaction and cross-linking reaction; the formed antibacterial hydrogel can regulate and control the release rate of antibacterial peptide molecules to realize a continuous antibacterial effect; meanwhile, the antibacterial hydrogel can form a protective film on a wound part, so that a moist environment is kept while bacteria are killed, and cell regeneration and wound repair are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of biomass materials and antibacterial materials, and in particular to a novel antibacterial peptide molecule-reinforced biomass antibacterial hydrogel and a preparation method thereof. Background Art

[0002] Antimicrobial materials refer to materials that have the ability to inhibit or kill microorganisms such as bacteria, viruses, and fungi. These materials are widely used in medicine, environment, food packaging, textiles, construction, and daily life. According to the antibacterial mechanism of antimicrobial materials, they can be divided into three types: physical antimicrobial materials, chemical antimicrobial materials, and biological antimicrobial materials. According to the type of material, they can be divided into metals, nanoparticles, polymers, biomass antimicrobial materials, etc.

[0003] Biomass refers to organic matter directly or indirectly produced by organisms such as plants, animals and microorganisms through photosynthesis, including crops, trees and their residues, animal feces, industrial organic waste and domestic organic waste. As a renewable energy source, biomass has the advantages of abundant resources, wide distribution and environmental friendliness, and can be effectively used in energy, construction, materials and chemical industries. Through physical, chemical and biological cross-linking, biomass hydrogels with excellent biological properties and biological activity can be prepared based on natural biomass. Compared with ordinary polymer hydrogels, biomass hydrogels have some advantages: first, biomass materials are non-toxic to organisms and can be used in the biomedical field. Second, biomass hydrogels can be degraded in the natural environment or in organisms without polluting the environment. Moreover, biomass hydrogels have good mechanical properties and can be used in different scenarios.

[0004] By embedding antimicrobial agents into the hydrogel network to form antimicrobial hydrogels, bacteria and other microorganisms can be effectively inhibited or killed, and can be further applied to wound dressings and tissue engineering. Commonly used antimicrobial agents include metal ions (such as silver ions, copper ions, zinc ions), nanoparticles (nanosilver, titanium dioxide, zinc oxide, etc.) and organic compounds with antimicrobial activity. These antimicrobial agents have good antibacterial effects, but have certain biological toxicity, are difficult to degrade in organisms, and pose potential hazards to organisms and the environment. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems and to design a novel biomass antibacterial hydrogel reinforced with antibacterial peptide molecules and a preparation method thereof.

[0006] The first aspect of the present invention provides a method for preparing a novel biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules, the method comprising the following steps:

[0007] Step a, synthesis of aldehyde-modified hyaluronic acid AHA: aldehyde-modifying the hyaluronic acid molecules to facilitate the cross-linking reaction with chitosan quaternary ammonium salt QCS and antibacterial polypeptide molecules;

[0008] Step b, synthesis of chitosan quaternary ammonium salt / aldehyded hyaluronic acid / antimicrobial polypeptide AHA / QCS / ε-LP composite antimicrobial hydrogel: the biomass antimicrobial hydrogel is prepared through the interaction and cross-linking reaction between molecules.

[0009] Optionally, in a first implementation of the first aspect of the present invention, step a specifically includes the following steps:

[0010] Step a1, weigh 1 g of hyaluronic acid powder with a purity of 95% in a beaker, dissolve it in 100 mL of phosphate buffer under stirring, adjust the pH to 5, and set aside;

[0011] Step a2, adding 0.5 g of sodium periodate with a purity of 95% to the hyaluronic acid solution obtained in step a1, stirring and reacting at room temperature in the dark for 5 hours, and setting aside;

[0012] Step a3, adding 1 mL of ethylene glycol to the hyaluronic acid / sodium periodate solution obtained in step a2 to stop the reaction;

[0013] Step a4: dialyze the mixture obtained in step a3 for three days using a dialysis bag to remove unreacted substances, and then freeze-dry to obtain aldehyde-modified hyaluronic acid AHA.

[0014] Optionally, in a second implementation of the first aspect of the present invention, step b specifically includes the following steps:

[0015] Step b1, dissolving the aldehyded hyaluronic acid AHA synthesized in step a in a phosphate buffer solution under magnetic stirring at a concentration of 40 mg / mL;

[0016] Step b2: dissolving chitosan quaternary ammonium salt QCS in 1.8% CaCl2 solution under magnetic stirring, wherein the concentration of chitosan quaternary ammonium salt QCS is 40 mg / mL.

[0017] Step b3, under magnetic stirring, adding the antibacterial polypeptide ε-LP to the mixed solution of aldehyded hyaluronic acid AHA and chitosan quaternary ammonium salt QCS to adjust the concentration of the antibacterial polypeptide ε-LP;

[0018] Step b4: Take 5 mL of the mixed solution obtained in step b3 and shake it vigorously by hand for 2 minutes to form an AHA / QCS / ε-LP hydrogel.

[0019] Optionally, in a third implementation of the first aspect of the present invention, in step b3, the concentration of the antimicrobial polypeptide ε-LP in the hydrogel is 0-10 mg / mL.

[0020] Optionally, in a fourth implementation of the first aspect of the present invention, in step b3, the concentration of the antimicrobial polypeptide ε-LP in the hydrogel is 2-4 mg / mL.

[0021] Optionally, in a fifth implementation of the first aspect of the present invention, the biomass molecule AHA reacts with QCS and ε-PL through a Schiff base reaction to form imine bonds, and the Schiff base bonds are dynamically cross-linked to form a hydrogel.

[0022] The second aspect of the present invention provides a novel biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules. The novel biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules is an antibacterial hydrogel that can be directly injected and covered on the wound surface.

[0023] Optionally, in an implementation of the second aspect of the present invention, the novel antimicrobial peptide molecule-reinforced biomass antimicrobial hydrogel is an antimicrobial hydrogel suitable for covering irregular wounds to form a moist and closed wound healing environment.

[0024] Compared with the existing known technology, the technical solution provided by the present invention has the following beneficial effects:

[0025] 1. A novel antimicrobial peptide molecule reinforced biomass antimicrobial hydrogel and a preparation method thereof, wherein the biomass molecule AHA forms an imine bond with QCS and ε-PL through a Schiff base reaction, and the Schiff base bond is dynamically cross-linked to form a hydrogel, which exhibits excellent self-healing and injectability;

[0026] 2. The novel biomass antibacterial hydrogel reinforced with antibacterial peptide molecules and its preparation method described in the present invention can be directly injected to cover the wound surface, and can be suitable for covering irregular wounds to form a moist and closed wound healing environment, which is conducive to cell regeneration and wound repair.

[0027] 3. The novel antimicrobial peptide molecule-reinforced biomass antimicrobial hydrogel and its preparation method described in the present invention are simple and convenient, can be formed by mixing and shaking the solution, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0029] Figure 1Spectral analysis of the materials of Example 2, Example 3 and Example 4 of the present invention;

[0030] Figure 2 It is a reaction mechanism diagram between AHA, QCS and ε-PL of the present invention;

[0031] Figure 3 This is a solid image of the hydrogel obtained in Example 2 of the present invention;

[0032] Figure 4 It is a scanning electron microscope image of the hydrogel obtained in Example 2, Example 3 and Example 4 of the present invention;

[0033] Figure 5 The rheological and self-healing properties of the hydrogel obtained in Example 2 of the present invention;

[0034] Figure 6 This is a diagram showing the antibacterial effect of the antibacterial hydrogel obtained in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific embodiments.

[0036] The technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] In the description of the present invention, unless otherwise specified, the raw materials used are all commercially available products in the art.

[0038] In the present invention, the main experimental equipment includes: Fourier transform infrared spectrometer (Thermo Scientific Nicolet iS20): used to analyze chemical bonds and functional groups in samples;

[0039] Mainly used for chemical bond identification, purity assessment, polymer analysis, and biological sample analysis. Chemical bond identification: Determine the functional groups in the compound by detecting the vibration frequency of specific chemical bonds; Purity assessment: Detect the presence of impurities and evaluate the purity of the sample; Polymer analysis: Used for polymer research, such as structure, crystallinity, etc.; Biological sample analysis: Analyze the structural characteristics of biological macromolecules such as proteins and nucleic acids;

[0040] Nuclear Magnetic Resonance Spectroscopy (BRUKERAVANCE 400): Used to analyze the electronic environment around the nucleus and thus determine the structure of a compound.

[0041] Mainly used for structure identification, kinetics research, purity analysis, and drug development. Structural identification: Determine the structure of the compound through hydrogen spectrum (1H-NMR) and carbon spectrum (13C-NMR); Kinetics research: Study the kinetic process of intermolecular interaction; Purity analysis: Evaluate the purity of the sample through NMR spectra; Drug development: Used for structure confirmation in the process of drug discovery and optimization;

[0042] Regulus 8100 scanning electron microscope (TESCAN MIRALMS, Czech Republic): used to observe the microscopic morphology and structure of the sample surface; mainly used for surface morphology analysis, particle size analysis, defect detection, and material science research. Surface morphology analysis: observe the micro-nanoscale morphology of the sample surface; particle size analysis: determine the size distribution of particles; defect detection: discover defects and cracks in the material; material science research: widely used in the fields of nanotechnology and material science;

[0043] Rheometer (MCR302, Anton Paar): used to measure the flow and deformation characteristics of materials; mainly used for viscosity measurement, elastic modulus determination, thixotropy research, and processing performance evaluation. Viscosity measurement: measuring the viscosity of liquids or semi-solids; elastic modulus determination: determining the elastic modulus and loss modulus of solids or gels; thixotropy research: analyzing the thixotropic behavior of materials, that is, the response of materials after being subjected to stress; processing performance evaluation: evaluating the flow behavior of materials during processing.

[0044] The hyaluronic acid powder, sodium periodate, ethylene glycol, CaCl2 solution, and phosphate buffer used in the examples of the present invention were purchased from Sinopharm Chemical Reagent Co., Ltd., and the antibacterial polypeptide ε-LP was purchased from Nanjing Jiepeptide Biotechnology Co., Ltd.

[0045] Example 1

[0046] This embodiment provides a novel method for preparing a biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules, comprising the following steps:

[0047] Step a, synthesis of aldehyde-modified hyaluronic acid AHA: aldehyde-modifying the hyaluronic acid molecule, one of the main components of the synthetic hydrogel, to facilitate the cross-linking reaction with the chitosan quaternary ammonium salt QCS and the antibacterial polypeptide molecule;

[0048] Step b, synthesis of chitosan quaternary ammonium salt / aldehyded hyaluronic acid / antimicrobial polypeptide AHA / QCS / ε-LP composite antimicrobial hydrogel: the biomass antimicrobial hydrogel is prepared through the interaction and cross-linking reaction between molecules.

[0049] In this embodiment, the biomass molecule AHA used forms an imine bond with QCS and ε-PL through Schiff base reaction, and the Schiff base bond is dynamically cross-linked to form a hydrogel; the Schiff base reaction is a chemical reaction in which the carbonyl group of an aldehyde or ketone reacts with a primary amine or a secondary amine to form an imine intermediate. In this process, the aldehyde group in the aldehyde-modified hyaluronic acid AHA can react with the amino group in the chitosan quaternary ammonium salt QCS or the antibacterial polypeptide ε-PL to form an imine bond; the imine bond is unstable and easily hydrolyzed in an aqueous solution to reform the initial aldehyde and amine. This reversibility makes the Schiff base bond part of the dynamic cross-linking network, thereby allowing the formation of a hydrogel; when the aldehyde-modified hyaluronic acid AHA is mixed with the chitosan quaternary ammonium salt QCS or the antibacterial polypeptide ε-PL, the aldehyde group in the aldehyde-modified hyaluronic acid AHA and the amino group in the chitosan quaternary ammonium salt QCS or the antibacterial polypeptide ε-PL form an imine bond through Schiff base reaction; the imine bond is reversible, which means that they can be hydrolyzed in an aqueous environment to reform the initial aldehyde and amine. This dynamic nature helps to form a dynamically balanced cross-linked network; as more and more Schiff base bonds are formed, these cross-linking points gradually establish a three-dimensional network structure, and when this network is dense enough, a hydrogel is formed.

[0050] Example 2

[0051] This embodiment provides a novel method for preparing a biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules, comprising the following steps:

[0052] Step a1, weigh 1 g of hyaluronic acid powder with a purity of 95% in a beaker, dissolve it in 100 mL of phosphate buffer at a stirring speed of 200 rpm, adjust the pH to 5, and set aside;

[0053] Step a2, adding 0.5 g of sodium periodate (purity 99%) with a purity of 95% to the hyaluronic acid solution obtained in step a1, stirring the reaction at room temperature in the dark (stirring speed is 200 rpm) for 5 hours, and setting aside;

[0054] Step a3, adding 1 mL of ethylene glycol (purity 95%) to the hyaluronic acid / sodium periodate solution obtained in step a2 to stop the reaction;

[0055] Step a4: dialyze the mixture obtained in step a3 for three days using a dialysis bag to remove unreacted substances, and then freeze-dry to obtain aldehyde-modified hyaluronic acid AHA.

[0056] In this example, the purpose of the test is to verify the successful synthesis of the aldehyded hyaluronic acid (AHA) prepared by the above-mentioned chemical modification method and to determine its structural characteristics.

[0057] Testing steps:

[0058] Sample preparation: Take an appropriate amount of the final product (freeze-dried aldehyde-modified hyaluronic acid AHA) from the synthesis process and further process the sample according to the requirements of the selected spectral technique;

[0059] Infrared spectroscopy (IR) analysis: The samples were scanned using a Fourier transform infrared spectrometer (FTIR) to record the infrared absorption spectrum of the samples, focusing on the absorption peak associated with the aldehyde group (C=O), located at 1700-1750cm -1 In this range, attention is paid to the characteristic absorption peaks of other important functional groups (such as hydroxyl-OH, etc.) in order to confirm the basic skeleton structure of hyaluronic acid;

[0060] Nuclear magnetic resonance spectroscopy (NMR) analysis: The samples were tested using a nuclear magnetic resonance spectrometer, using 1H-NMR and 13C-NMR to record the hydrogen and carbon spectra of the samples, analyze the changes in the chemical environment of each functional group, especially the hydrogen and carbon signals adjacent to the aldehyde group, and compare the nuclear magnetic resonance spectra of the original hyaluronic acid and the modified aldehyde-modified hyaluronic acid AHA to confirm the introduction of the aldehyde group and the changes in the original structure;

[0061] Data processing and analysis: Analyze the new absorption peaks appearing in the infrared spectrum to confirm the existence of aldehyde groups. Through the changes in the nuclear magnetic resonance spectrum, confirm the introduction of aldehyde groups and the retention of the original structure. Combined with theoretical calculations or known standard spectra for comparison, confirm the successful synthesis of aldehyde-modified hyaluronic acid AHA.

[0062] Interpretation of results: If a clear absorption peak is observed in the 1700-1750 cm-1 region in the infrared spectrum, it indicates that the aldehyde group has been successfully introduced. If the NMR spectrum shows that the hydrogen and carbon signals adjacent to the aldehyde group have changed and the basic skeleton characteristics of hyaluronic acid have been retained, it further confirms the successful synthesis of AHA;

[0063] Conclusion: If Figure 1 As shown, through the above spectral analysis, we can determine that the synthesized AHA has the expected chemical structure, that is, the aldehyde functional group is successfully introduced into the hyaluronic acid molecule. From this data analysis, it can be found that AHA is successfully synthesized.

[0064] Step b1, under the condition of magnetic stirring (stirring speed is 200 rpm), dissolving the AHA synthesized in step a in a phosphate buffer solution at a concentration of 40 mg / mL;

[0065] Step b2: Under magnetic stirring (stirring speed of 200 rpm), chitosan quaternary ammonium salt QCS was dissolved in 1.8% CaCl2 solution (purity 95%), and the concentration of QCS was 40 mg / mL.

[0066] Step b3, under the condition of magnetic stirring (stirring speed of 200 rpm), add antimicrobial polypeptide ε-LP (purity 95%) to the mixed solution of AHA and QCS, and adjust the final antimicrobial polypeptide concentration to 1 mg / mL;

[0067] Step b4: Take 5 mL of the mixed solution obtained in step b3 and shake it vigorously by hand for 2 minutes to form an AHA / QCS / ε-LP hydrogel.

[0068] In this example, the purpose of the test is to illustrate the interaction mechanism of aldehyded hyaluronic acid AHA, chitosan quaternary ammonium salt QCS and antibacterial polypeptide ε-PL in the process of hydrogel formation;

[0069] Detection principle: Use chemical theory combined with experimental results to analyze the interaction between components, including electrostatic interaction, hydrogen bonding, covalent bonding, etc.

[0070] Interaction mechanism of biomass molecules in hydrogels and molecular cross-linking Figure 2 As shown in the figure, the interaction between aldehyde-hydrated hyaluronic acid AHA and chitosan quaternary ammonium salt QCS: the aldehyde group on the aldehyde-hydrated hyaluronic acid AHA can react with the amino group on the chitosan quaternary ammonium salt QCS through Schiff base to form a covalent bond, which is the basis for forming the hydrogel network structure; the interaction between the antibacterial polypeptide ε-PL and the hydrogel network: the antibacterial polypeptide ε-PL can interact with the negatively charged aldehyde-hydrated hyaluronic acid AHA or chitosan quaternary ammonium salt QCS through electrostatic interaction, and can also interact with other molecules through hydrogen bonds to enhance the stability of the hydrogel; the formation of the hydrogel network: through the covalent cross-linking between the aldehyde-hydrated hyaluronic acid AHA and the chitosan quaternary ammonium salt QCS and the embedding of the antibacterial polypeptide ε-PL, a three-dimensional network structure is formed, which has good mechanical properties and biocompatibility.

[0071] In this embodiment, the obtained solid hydrogel is as follows Figure 3 As shown, the purpose of the test is to visually display the appearance characteristics of the prepared hydrogel;

[0072] Detection principle: The hydrogel image obtained by ordinary camera is used to evaluate its physical properties such as shape, color and texture;

[0073] Appearance of hydrogel: The hydrogel is uniform and transparent, without obvious particles or bubbles;

[0074] Shape of hydrogel: It can be observed that the hydrogel has good molding ability and maintains the specific shape formed by hand shaking.

[0075] In this example, the purpose of the test is to observe the internal microstructure of the hydrogel using a scanning electron microscope to evaluate its porosity, surface roughness, and continuity of the network structure;

[0076] Detection principle: Scanning electron microscope can magnify thousands to tens of thousands of times and is used to observe the surface morphology and microstructure of materials;

[0077] Testing steps:

[0078] Sample preparation: The hydrogel was quickly frozen and freeze-dried to keep its internal structure intact.

[0079] Sample coating: In order to improve conductivity, a layer of gold or platinum is usually plated on the surface of the sample;

[0080] Scanning electron microscope imaging: The processed samples were imaged using a scanning electron microscope;

[0081] Conclusion: The SEM characterization of the obtained solid hydrogel is as follows Figure 4 As shown in b, the scanning electron microscope image shows that the hydrogel has an open porous structure with uniform pore distribution and moderate pore size, indicating that the hydrogel network structure has good continuity without breaks or defects.

[0082] In this example, the purpose of the test is to evaluate the rheological properties of the hydrogel and its self-healing ability;

[0083] Detection principle: The shear modulus, storage modulus and loss modulus of the hydrogel are measured by a rheometer, and its recovery after damage is monitored;

[0084] Testing steps:

[0085] Rheological test: Dynamic shear test was performed using a rheometer to measure the storage modulus (G') and loss modulus (G") of the hydrogel at different frequencies;

[0086] Self-healing test: Apply external force to the hydrogel to cause damage, and then observe whether it can recover to its original state within a certain period of time;

[0087] Conclusion: Rheological and self-healing properties are as follows Figure 5 As shown, the data show that the hydrogel has a higher storage modulus G', indicating that it has good elasticity. The hydrogel can self-repair and restore its original mechanical properties within a short time after injury.

[0088] Example 3

[0089] This embodiment provides a novel method for preparing a biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules, comprising the following steps:

[0090] Step a1, weigh 1 g of hyaluronic acid powder with a purity of 95% in a beaker, dissolve it in 100 mL of phosphate buffer (purity 99%) under stirring (stirring speed is 200 rpm), adjust the pH to 5, and set aside;

[0091] Step a2, add 0.5 g of 95% pure sodium periodate (purity 99%) to the hyaluronic acid solution obtained in step a1, stir (stirring speed is 200 rpm) at room temperature in the dark for 5 hours, and set aside;

[0092] Step a3, adding 1 mL of ethylene glycol (purity 95%) to the hyaluronic acid / sodium periodate solution obtained in step a2 to stop the reaction;

[0093] Step a4: dialyze the mixture obtained in step a3 for three days using a dialysis bag to remove unreacted substances, and then freeze-dry to obtain aldehyde-modified hyaluronic acid AHA.

[0094] In this embodiment, Figure 1 As shown, through the above spectral analysis, we can determine that the synthesized AHA has the expected chemical structure, that is, the aldehyde functional group is successfully introduced into the hyaluronic acid molecule. From this data analysis, it can be found that AHA is successfully synthesized.

[0095] Step b1, under the condition of magnetic stirring (stirring speed is 200 rpm), dissolving the AHA synthesized in step a in a phosphate buffer solution at a concentration of 40 mg / mL;

[0096] Step b2, under the condition of magnetic stirring (stirring speed is 200rpm), chitosan quaternary ammonium salt QCS is dissolved in 1.8% CaCl2 solution (Sinopharm Chemical Reagent Co., Ltd., purity 95%), and the concentration of QCS is 40mg / mL.

[0097] Step b3, under the condition of magnetic stirring (stirring speed of 200 rpm), add antimicrobial peptide ε-LP (Nanjing Jiepeptide Biotechnology Co., Ltd., purity 95%) to the mixed solution of AHA and QCS, and adjust the final antimicrobial peptide concentration to 2 mg / mL;

[0098] In this example, the internal microstructure of the hydrogel was observed using a scanning electron microscope to evaluate its porosity, surface roughness, and continuity of the network structure. The scanning electron microscope characterization of the obtained solid hydrogel is as follows: Figure 4 As shown in c.

[0099] Example 4

[0100] This embodiment provides a novel method for preparing a biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules, comprising the following steps:

[0101] Step a1, weigh 1 g of hyaluronic acid powder with a purity of 95% in a beaker, dissolve it in 100 mL of phosphate buffer (purity 99%) under stirring (stirring speed is 200 rpm), adjust the pH to 5, and set aside;

[0102] Step a2, adding 0.5 g of sodium periodate (purity 99%) with a purity of 95% to the hyaluronic acid solution obtained in step a1, stirring the reaction at room temperature in the dark (stirring speed is 200 rpm) for 5 hours, and setting aside;

[0103] Step a3, adding 1 mL of ethylene glycol (purity 95%) to the hyaluronic acid / sodium periodate solution obtained in step a2 to stop the reaction;

[0104] Step a4: dialyze the mixture obtained in step a3 for three days using a dialysis bag to remove unreacted substances, and then freeze-dry to obtain aldehyde-modified hyaluronic acid AHA.

[0105] In this embodiment, Figure 1 As shown, through the above spectral analysis, we can determine that the synthesized AHA has the expected chemical structure, that is, the aldehyde functional group is successfully introduced into the hyaluronic acid molecule. From this data analysis, it can be found that AHA is successfully synthesized.

[0106] Step b1, under the condition of magnetic stirring (stirring speed is 200 rpm), dissolving the AHA synthesized in step a in a phosphate buffer solution at a concentration of 40 mg / mL;

[0107] Step b2, under the condition of magnetic stirring (stirring speed is 200rpm), chitosan quaternary ammonium salt QCS is dissolved in 1.8% CaCl2 solution (Sinopharm Chemical Reagent Co., Ltd., purity 95%), and the concentration of QCS is 40mg / mL.

[0108] Step b3, under the condition of magnetic stirring (stirring speed of 200 rpm), add antimicrobial polypeptide ε-LP (purity 95%) to the mixed solution of AHA and QCS, and adjust the final antimicrobial polypeptide concentration to 4 mg / mL;

[0109] In this example, the internal microstructure of the hydrogel was observed using a scanning electron microscope to evaluate its porosity, surface roughness, and continuity of the network structure. The scanning electron microscope characterization of the obtained solid hydrogel is as follows: Figure 4 As shown in d.

[0110] In an embodiment of the present invention, hydrogel samples containing different concentrations of ε-PL are prepared. This experiment includes at least two concentrations: 2 mg / mL and 4 mg / mL; the prepared hydrogel samples are contacted with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacterial solutions of known concentrations for co-culture for 2 hours. This co-culture is to allow the ε-PL in the hydrogel to contact the bacteria and exert its antibacterial effect. After the co-culture, a certain amount of the mixed solution (for example, 20 μL) is taken from each sample and added to a 48-well plate containing 100 μL PBS (phosphate buffer) for dilution. The diluted sample is taken out from the 48-well plate and evenly spread on a solid culture medium; cultured for 24 hours under appropriate conditions to allow bacteria to grow and form colonies; the antibacterial properties of hydrogels with different ε-PL contents are evaluated using co-culture and plate spread counting tests, and it is observed after 24 hours of culture on the solid culture medium. Figure 6 The antibacterial results showed that the hydrogels containing 2 mg / mL and 4 mg / mL ε-PL had a killing effect of more than 99.9% on Escherichia coli and Staphylococcus aureus.

[0111] ε-PL is a naturally occurring polypeptide with strong antibacterial activity. It can kill bacteria by destroying their cell membranes. The co-culture step allows the ε-PL in the hydrogel to come into direct contact with the bacteria in order to observe its antibacterial effect. The plate spread count estimates the number of live bacteria in the original sample by counting the number of colonies on the plate.

[0112] Through the implementation of the above scheme, antimicrobial peptides are a class of naturally occurring or artificially synthesized short-chain polypeptides with broad-spectrum antimicrobial activity, which can effectively inhibit the growth of bacteria, fungi, viruses and parasites; the present invention introduces antimicrobial peptides into biomass hydrogels to prepare biomass antimicrobial hydrogels with excellent antimicrobial properties; utilizes intermolecular interactions and cross-linking to effectively connect antimicrobial peptides with biomass molecules; the formed antimicrobial hydrogel can regulate the release rate of antimicrobial peptide molecules to achieve a sustained antimicrobial effect; at the same time, the antimicrobial hydrogel can form a protective film at the wound site, while killing bacteria, maintaining a moist environment, which is conducive to cell regeneration and wound repair.

[0113] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a novel biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules, characterized in that: The preparation method of the novel antimicrobial peptide molecule reinforced biomass antimicrobial hydrogel comprises the following steps: Step a, synthesis of aldehyde-modified hyaluronic acid AHA: aldehyde-modifying the hyaluronic acid molecules to facilitate the cross-linking reaction with chitosan quaternary ammonium salt QCS and antibacterial polypeptide molecules; Step b, synthesis of chitosan quaternary ammonium salt / aldehyded hyaluronic acid / antimicrobial polypeptide AHA / QCS / ε-LP composite antimicrobial hydrogel: the biomass antimicrobial hydrogel is prepared through the interaction and cross-linking reaction between molecules.

2. The method for preparing a novel antimicrobial peptide molecule reinforced biomass antimicrobial hydrogel according to claim 1, characterized in that: Step a specifically includes the following steps: Step a1, weigh 1 g of hyaluronic acid powder with a purity of 95% in a beaker, dissolve it in 100 mL of phosphate buffer under stirring, adjust the pH to 5, and set aside; Step a2, adding 0.5 g of sodium periodate with a purity of 95% to the hyaluronic acid solution obtained in step a1, stirring and reacting at room temperature in the dark for 5 hours, and setting aside; Step a3, adding 1 mL of ethylene glycol to the hyaluronic acid / sodium periodate solution obtained in step a2 to stop the reaction; Step a4: dialyze the mixture obtained in step a3 for three days using a dialysis bag to remove unreacted substances, and then freeze-dry to obtain aldehyde-modified hyaluronic acid AHA.

3. The method for preparing a novel antimicrobial peptide molecule reinforced biomass antimicrobial hydrogel according to claim 1, characterized in that: Step b specifically includes the following steps: Step b1, dissolving the aldehyded hyaluronic acid AHA synthesized in step a in a phosphate buffer solution under magnetic stirring at a concentration of 40 mg / mL; Step b2: dissolving chitosan quaternary ammonium salt QCS in 1.8% CaCl2 solution under magnetic stirring, wherein the concentration of chitosan quaternary ammonium salt QCS is 40 mg / mL. Step b3, under magnetic stirring, adding the antibacterial polypeptide ε-LP to the mixed solution of aldehyded hyaluronic acid AHA and chitosan quaternary ammonium salt QCS to adjust the concentration of the antibacterial polypeptide ε-LP; Step b4: Take 5 mL of the mixed solution obtained in step b3 and shake it vigorously by hand for 2 minutes to form an AHA / QCS / ε-LP hydrogel.

4. The method for preparing a novel antimicrobial peptide molecule reinforced biomass antimicrobial hydrogel according to claim 3, characterized in that: In step b3, the concentration of the antimicrobial polypeptide ε-LP in the hydrogel is 0-10 mg / mL.

5. The method for preparing a novel antimicrobial peptide molecule reinforced biomass antimicrobial hydrogel according to claim 4, characterized in that: In step b3, the concentration of the antimicrobial polypeptide ε-LP in the hydrogel is 2-4 mg / mL.

6. The method for preparing a novel biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules as claimed in claim 1, characterized in that: The biomass molecule AHA reacts with QCS and ε-PL through Schiff base reaction to form imine bonds, and Schiff base bonds dynamically cross-link to form hydrogel.

7. A novel biomass antibacterial hydrogel reinforced with antimicrobial peptide molecules, characterized in that: The novel antimicrobial peptide molecule-reinforced biomass antimicrobial hydrogel is an antimicrobial hydrogel that can be directly injected and covered on the wound surface.

8. The novel antimicrobial peptide molecule-reinforced biomass antimicrobial hydrogel according to claim 7, characterized in that: The novel antimicrobial peptide molecule-reinforced biomass antimicrobial hydrogel is an antimicrobial hydrogel suitable for covering irregular wounds and forming a moist and closed wound healing environment.

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