Biological polysaccharide composite material as well as preparation method and application thereof

Abstract

The invention provides a biological polysaccharide composite material as well as a preparation method and application thereof, and belongs to the field of medical wound dressings. The invention provides a biological polysaccharide composite material which is of a porous structure and comprises cross-linked biological polysaccharide and an active factor loaded on the biological polysaccharide, the biological polysaccharide comprises one or more of trehalose modified by a hydrophilic agent, algin modified by the hydrophilic agent and carrageenan modified by the hydrophilic agent, and chitosan modified by the hydrophilic agent. According to the invention, the synergistic interaction of the base material is realized through polysaccharide compounding; electrostatic interaction and entanglement among polysaccharide molecular chains in biological polysaccharide form a three-dimensional network structure which is more beneficial to water permeation and storage, so that the absorption and storage of seepage are improved; after the biological polysaccharide is subjected to hydrophilic modification, the seepage absorption of the biological polysaccharide composite material is further improved, and the microstructure is optimized; in addition, the loaded active factors (antibacterial peptides and anti-inflammatory factors) endow the material with a strong biological activity function.

Description

Technical Field

[0001] This invention relates to the field of medical wound dressings, specifically to a biological polysaccharide composite material, its preparation method, and its application. Background Technology

[0002] In modern medicine, medical dressings are essential tools for treating skin injuries such as wounds, burns, and surgical incisions. They not only provide physical protection but also promote wound healing and reduce the risk of infection. However, traditional dressings (such as gauze and hydrogel) have problems such as passively absorbing exudate and the potential for burst release of antibacterial components. Summary of the Invention

[0003] This invention provides a biopolysaccharide composite material, its preparation method, and its application. The biopolysaccharide composite material of this invention has good absorption and storage properties for exudate.

[0004] This invention provides a biopolysaccharide composite material with a porous structure, comprising cross-linked biopolysaccharides and active factors loaded on the biopolysaccharides; The biopolysaccharide includes one or more of the following: hydrophilic-modified trehalose, hydrophilic-modified alginate, and hydrophilic-modified carrageenan, along with hydrophilic-modified chitosan.

[0005] Preferably, the hydrophilic agent comprises carboxymethylated chitosan.

[0006] Preferably, the active factor includes antimicrobial peptides and / or anti-inflammatory factors.

[0007] This invention also provides a method for preparing the biopolysaccharide composite material described in the above technical solution, comprising the following steps: A solution of biopolysaccharide raw material is modified by mixing it with a hydrophilic agent to obtain a solution of biopolysaccharide; the biopolysaccharide raw material includes one or more of trehalose, alginate and carrageenan, and chitosan; The solution of the biopolysaccharide, the solution of the pH-sensitive crosslinking agent, and the pH adjuster are mixed to carry out the first crosslinking reaction to generate the crosslinked biopolysaccharide, thus obtaining the crosslinking reaction system; The cross-linking reaction system is mixed with a solution of active factors for loading to generate biopolysaccharides loaded with active factors. The resulting loaded system is then subjected to pre-freezing and freeze-drying to obtain the biopolysaccharide composite material.

[0008] Preferably, the mass of the hydrophilic agent is 1-5% of the mass of the solution of the biopolysaccharide raw material; The modification was performed at room temperature for 1 hour, and the modification was carried out under stirring conditions at a speed of 300-600 rpm.

[0009] Preferably, the mass ratio of trehalose to solvent volume in the solution of the biological polysaccharide raw material is 1~5g:100mL, the mass ratio of chitosan to solvent volume is 0.5~2g:100mL, the mass ratio of alginate to solvent volume is 0.5~2g:100mL, and the mass ratio of carrageenan to solvent volume is 0.5~2g:100mL. The pH-sensitive crosslinking agent contains an aldehyde group; the pH-sensitive crosslinking agent includes citral; The mass ratio of the pH-sensitive crosslinking agent to the volume of the solvent in the solution is 5~10g:100mL; The ratio of the amount of aldehyde group in the pH-sensitive crosslinking agent to the amount of amino group in chitosan is 0.2~0.8:1; The pH adjuster includes a sodium hydroxide solution, and the concentration of the pH adjuster is 1M; The pH value of the system obtained by mixing the solution of the biopolysaccharide, the solution of the pH-sensitive cross-linking agent, and the pH adjuster is 5-6.

[0010] Preferably, the mass ratio of the active factor to the volume of the solvent in the solution of the active factor is 0.05~1.5g:100mL; when the active factor includes antimicrobial peptides and / or anti-inflammatory factors, the concentration of the antimicrobial peptides in the solution of the active factor is 0.1~1g:100mL, and the mass ratio of the anti-inflammatory factors to the volume of the solvent is 0.05~0.5g:100mL. The loading was carried out under light-proof and stirring conditions, with the temperature of the loading being <4℃ and the time being 30min.

[0011] Preferably, the pre-freezing temperature is -20 to -40°C, and the time is 6 to 12 hours; The freeze-drying temperature is below -50°C, the vacuum degree is below 0.1 mbar, and the time is 24~48 h.

[0012] Preferably, when the biopolysaccharide raw material includes alginate and / or carrageenan, the loading process further includes: immersing the loaded system in a calcium chloride solution to carry out a second crosslinking reaction.

[0013] The present invention also provides the application of the biopolysaccharide composite material described in the above technical solution or the biopolysaccharide composite material prepared by the preparation method described in the above technical solution as a dressing.

[0014] This invention achieves synergistic enhancement of the substrate through polysaccharide compounding: the electrostatic interaction and entanglement between polysaccharide molecular chains in the biopolysaccharide form a three-dimensional network structure that is more conducive to water penetration and storage, thereby improving the absorption and storage of exudate; after hydrophilic modification, the biopolysaccharide composite material further improves the absorption of exudate and optimizes the microstructure; in addition, the loaded active factors (antimicrobial peptides, anti-inflammatory factors) endow the material with strong bioactive functions.

[0015] Chitosan + Carrageenan Chitosan carries a positive charge (amino group), while carrageenan (especially κ-carrageenan) carries a negative charge (sulfate group). The two can form a polyelectrolyte complex and, through electrostatic interactions, create a denser and more stable three-dimensional network, enhancing the material's mechanical strength. Furthermore, the interaction between chitosan and carrageenan influences ice crystal growth, potentially leading to a finer and more uniform microporous structure. The antibacterial properties of chitosan and the ability of carrageenan to inhibit certain pathogens also have a synergistic or enhancing effect.

[0016] Chitosan + Alginate + Carrageenan Alginate and carrageenan are anionic polysaccharides derived from the ocean, and both can bind with Ca. 2+ Ionic crosslinking occurs, enhancing gel strength: through shared Ca... 2+ Cross-linking points can form a more robust dual network structure (forming an "egg-box" structure), significantly improving the mechanical properties of the material in a wet state and preventing it from collapsing after absorbing liquid. In addition, the combination of the high water retention of both may allow the material to maintain gel integrity even after absorbing a large amount of exudate.

[0017] Chitosan + Trehalose + Alginate / Carrageenan Trehalose molecules possess strong hydrogen bonding capabilities, enabling them to act as "water sponges" and further enhance the overall liquid absorption and retention capacity of materials. Furthermore, trehalose, hailed as the "sugar of life," can protect the activity of proteins, peptides (such as ε-polylysine), and phenolic substances (such as EGCG) under stress conditions such as freeze-drying, improving the loading efficiency and stability of active factors. As a small-molecule sugar, trehalose influences the ice crystal formation process and has a positive effect on the uniformity of microporous structures.

[0018] Furthermore, the first cross-linking reaction (pH-sensitive Schiff base bond) endows the material with intelligent pH-responsive degradation properties. Attached Figure Description

[0019] Figure 1 Here is a SEM image of the porous material surface from Example 1; Figure 2 This is a SEM image of the cross-section of the porous material in Example 1. Detailed Implementation

[0020] This invention provides a biopolysaccharide composite material with a porous structure, comprising cross-linked biopolysaccharides and active factors loaded on the biopolysaccharides; The biopolysaccharide includes one or more of the following: hydrophilic-modified trehalose, hydrophilic-modified alginate, and hydrophilic-modified carrageenan, along with hydrophilic-modified chitosan.

[0021] In this invention, the hydrophilic agent preferably includes carboxymethylated chitosan.

[0022] In this invention, the active factors preferably include antimicrobial peptides and / or anti-inflammatory factors.

[0023] This invention also provides a method for preparing the biopolysaccharide composite material described in the above technical solution, characterized by comprising the following steps: A solution of biopolysaccharide raw material is modified by mixing it with a hydrophilic agent to obtain a solution of biopolysaccharide; the biopolysaccharide raw material includes one or more of trehalose, alginate and carrageenan, and chitosan; The solution of the biopolysaccharide, the solution of the pH-sensitive crosslinking agent, and the pH adjuster are mixed to carry out the first crosslinking reaction to generate the crosslinked biopolysaccharide, thus obtaining the crosslinking reaction system; The cross-linking reaction system is mixed with a solution of active factors for loading to generate biopolysaccharides loaded with active factors. The loaded system is then subjected to pre-freezing and freeze-drying to obtain the biopolysaccharide composite material.

[0024] This invention modifies a solution of biological polysaccharide raw material by mixing it with a hydrophilic agent to obtain a solution of biological polysaccharide.

[0025] In this invention, the mass of the hydrophilic agent is preferably 1-5% of the mass of the solution of the biopolysaccharide raw material, and in specific embodiments of this invention it can be 2%, 3% or 4%; the biopolysaccharide raw material includes one or more of trehalose, alginate and carrageenan and chitosan.

[0026] In this invention, the method for preparing the solution of the biopolysaccharide raw material preferably includes: The biological polysaccharide raw material was dissolved in acetic acid solution and then allowed to stand to remove bubbles, thus obtaining a solution of the biological polysaccharide raw material.

[0027] In this invention, the mass ratio of the biopolysaccharide raw material to the volume of the acetic acid solution is preferably 2.5~11g:100mL. In specific embodiments of this invention, it can be 3g:100mL, 4g:100mL, 5g:100mL, 6g:100mL, 7g:100mL, 8g:100mL, 9g:100mL, or 10g:100mL. The concentration of the acetic acid solution is preferably 0.05~0.2M. In specific embodiments of this invention, it can be 0.1M or 0.15M.

[0028] In this invention, the preferred dissolution temperature is 40~60℃, the preferred dissolution time is 2~4h, and the preferred dissolution speed is 300~600rpm.

[0029] After dissolution, a homogeneous, clear, and viscous biological polysaccharide raw material solution was formed.

[0030] In this invention, the mass ratio of trehalose to solvent in the solution of the biological polysaccharide raw material is preferably 1-5 g: 100 mL. In specific embodiments of this invention, it can be 2 g: 100 mL, 3 g: 100 mL, or 4 g: 100 mL.

[0031] In this invention, the preferred mass ratio of chitosan to solvent in the solution of the biopolysaccharide raw material is 0.5-2 g:100 mL. In specific embodiments of this invention, this ratio can be 0.8 g:100 mL, 1 g:100 mL, 1.2 g:100 mL, 1.5 g:100 mL, 1.6 g:100 mL, or 1.8 g:100 mL. As a cationic polysaccharide, chitosan combines with other anionic polysaccharides through electrostatic interactions to construct a basic network and provide amino groups. It then undergoes a Schiff base reaction with citral, thereby introducing pH responsiveness.

[0032] In this invention, the mass ratio of alginate to solvent in the solution of the biopolysaccharide raw material is preferably 0.5~2g:100mL. In specific embodiments of this invention, it can be 0.8g:100mL, 1g:100mL, 1.2g:100mL, 1.5g:100mL, 1.6g:100mL or 1.8g:100mL.

[0033] In this invention, the preferred ratio of the mass of carrageenan to the volume of solvent in the solution of the biological polysaccharide raw material is 0.5~2g:100mL. In specific embodiments of this invention, it can be 0.8g:100mL, 1g:100mL, 1.2g:100mL, 1.5g:100mL, 1.6g:100mL or 1.8g:100mL.

[0034] In this invention, the modification temperature is preferably room temperature, the modification time is preferably 1 hour, the modification is preferably carried out under stirring conditions, and the stirring speed is preferably 300~600 rpm.

[0035] When the hydrophilic agent is carboxymethyl chitosan, the carboxyl groups in carboxymethyl chitosan will partially or completely ionize in solution, releasing H+. + This process makes carboxymethyl chitosan negatively charged. During modification, carboxymethyl chitosan and chitosan in the system are fully combined through electrostatic interaction, which significantly improves the hydrophilicity and liquid absorption rate of the final material.

[0036] After obtaining the solution of the biopolysaccharide, the present invention mixes the solution of the biopolysaccharide, the solution of the pH-sensitive crosslinking agent and the pH adjuster to carry out the first crosslinking reaction to generate the crosslinked biopolysaccharide, thus obtaining the crosslinking reaction system.

[0037] In this invention, the mixing preferably involves adding a solution of a pH-sensitive crosslinking agent dropwise to a solution of biopolysaccharide before adding a pH adjuster; the mixing is preferably carried out under ice-water bath conditions.

[0038] In this invention, the mass ratio of the pH-sensitive crosslinking agent to the volume ratio of the solvent is 5-10 g:100 mL. In specific embodiments of this invention, it can be 6 g:100 mL, 7 g:100 mL, 8 g:100 mL, or 9 g:100 mL. The pH-sensitive crosslinking agent preferably includes citral. The solvent of the solution of the pH-sensitive crosslinking agent preferably includes ethanol. The mass ratio of the aldehyde group in the pH-sensitive crosslinking agent to the amino group in chitosan is preferably 0.2-0.8:1. In specific embodiments of this invention, it can be 0.4:1, 0.5:1, 0.6:1, or 0.7:1. In this invention, the pH adjuster preferably comprises a sodium hydroxide solution, and the concentration of the pH adjuster is preferably 1M.

[0039] In this invention, the pH value of the system obtained by mixing the solution of the biopolysaccharide, the solution of the pH-sensitive crosslinking agent, and the pH adjuster is preferably 5-6. Under this weakly acidic condition, the aldehyde group of the pH-sensitive crosslinking agent undergoes a highly efficient Schiff base reaction with the amino group of chitosan to form a dynamic covalent bond, initially constructing a crosslinking network. This crosslinking bond is pH sensitive, stable in an acidic environment, and reversibly broken in a neutral or alkaline environment.

[0040] In this invention, the temperature of the crosslinking reaction is preferably 0~5℃, and the time is preferably 2~4h.

[0041] After obtaining the cross-linking reaction system, the present invention mixes the cross-linking reaction system with a solution of active factors for loading to generate biopolysaccharides loaded with active factors. Then, the obtained loaded system is subjected to pre-freezing and freeze-drying in sequence to obtain the biopolysaccharide composite material.

[0042] In this invention, the volume ratio of the cross-linking reaction system to the solution of the active factor is preferably 1:11.

[0043] In this invention, the concentration of the active factor solution is preferably 0.05~1.5g:100mL; when the antimicrobial peptide and / or anti-inflammatory factor are used, the concentration of the antimicrobial peptide in the solution of the active factor is preferably 0.1~1g:100mL, and the concentration of the anti-inflammatory factor is preferably 0.05~0.5g:100mL. In specific embodiments of this invention, the concentration of the antimicrobial peptide can be 0.2g:100mL, 0.3g:100mL, 0.4g:100mL, 0.5g:100mL, 0.6g:100mL, 0.7g:100mL, 0.8g:100mL, or 0.9g:100mL, and the antimicrobial peptide preferably includes ε-polylysine; the concentration of the anti-inflammatory factor can be 0.1g:100mL, 0.2g:100mL, 0.3g:100mL, or 0.4g:100mL, and the anti-inflammatory factor preferably includes EGCG.

[0044] In this invention, the loading is preferably carried out under light-proof and stirring conditions, the temperature of the loading is preferably <4°C, and the time is preferably 30 min.

[0045] The present invention does not have a specific limitation on the stirring speed, as long as the active factors are uniformly dispersed in the polysaccharide network and vigorous stirring is avoided to prevent inactivation.

[0046] When the biopolysaccharide raw material includes alginate and / or carrageenan, the loading process further includes: immersing the loaded system in a calcium chloride solution to carry out a second crosslinking reaction.

[0047] In this invention, the calcium chloride solution preferably contains 1-3% by mass, and the solvent for the calcium chloride solution is preferably ethanol and water, with the volume ratio of ethanol to water preferably being 1:1.

[0048] In this invention, the temperature of the second crosslinking reaction is preferably 15~30°C, and the time is preferably 1~2h.

[0049] Ca 2+ It can specifically react with alginate and / or carrageenan to form an ionic cross-linked network, which interpenetrates and synergistically with the network formed by the first cross-linking reaction to jointly construct a stronger "double cross-linked network," thereby significantly improving the mechanical strength of the material in a wet state. 1) The molecular chain of alginate contains guluronic acid (G) units, which can form a concave structure like an "egg carton" in space. 2+ It can be precisely embedded in this "box" like an "egg," a Ca 2+ It can react with the carboxyl groups (-COO) on the two G unit segments. - Coordination bonding. This interaction forms strong bridges between adjacent alginate molecular chains, known as "egg-box" structures, which greatly enhance the mechanical strength of the gel.

[0050] 2) Carrageenan, especially κ-carrageenan, can also interact with Ca. 2+ The interaction between divalent cations, although not exactly the same as the "egg box" model of alginate, can still form a stronger gel network and improve mechanical properties.

[0051] In this invention, the pre-freezing temperature is preferably -20 to -40°C, and the pre-freezing time is preferably 6 to 12 hours. In specific embodiments of this invention, the pre-freezing temperature can be -25°C, -30°C, or -35°C, and the pre-freezing time can be 7 hours, 8 hours, 9 hours, 10 hours, or 11 hours.

[0052] In this invention, the pre-freezing is preferably carried out in a mold.

[0053] During the pre-freezing process, water molecules crystallize to form ice crystal templates, while polysaccharide molecules, cross-linked networks, and active factors are squeezed out of the gaps between the ice crystals.

[0054] In this invention, the freeze-drying temperature is preferably below -50°C, the vacuum degree is preferably below 0.1 mbar, and the time is preferably 24~48 h.

[0055] Freeze-drying causes ice crystals to sublimate directly into water vapor and be removed, ultimately leaving behind interconnected micron-sized porous structures that precisely replicate the shape of the original ice crystals at the sites they occupied.

[0056] After freeze-drying, the present invention preferably further includes cutting, sterilizing and packaging the resulting product.

[0057] The present invention also provides the application of the biopolysaccharide composite material described in the above technical solution or the biopolysaccharide composite material prepared by the preparation method described in the above technical solution as a dressing.

[0058] The following detailed description of the bio-polysaccharide composite material, its preparation method, and its application provided by the present invention, with reference to specific embodiments, should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1 Step 1: Weigh 1.5 g of chitosan (degree of deacetylation ≥90%) and 1.5 g of alginate and slowly disperse them in 100 mL of 0.15 M acetic acid solution under mechanical stirring (300~600 rpm). Then, raise the temperature of the system to 45℃ and continue stirring for 3 h. Then, let the resulting solution stand to remove the bubbles generated during stirring, forming a homogeneous, clear and viscous compound solution.

[0060] Step Two: Add 3% by weight of carboxymethyl chitosan to the compound solution obtained in step one, and continue stirring for 1 hour to allow it to fully combine with chitosan through electrostatic interaction.

[0061] Step 3: Crosslinking reaction: Under ice-water bath (4℃) and gentle stirring conditions, an ethanol solution of citral (the mass ratio of citral to the volume of ethanol is 7 g: 100 mL, and the molar ratio of the aldehyde group of citral to the amino group of chitosan is 0.35: 1) was added dropwise to the system obtained in step two.

[0062] pH adjustment: After the addition was complete, the pH of the mixture was precisely adjusted to 5.0 using 1 M NaOH solution. The reaction was continued for 3 hours under weakly acidic conditions and in an ice-water bath to allow the Schiff base reaction to proceed fully and to initially construct a pH-sensitive cross-linking network.

[0063] Step Four: Under light-protected and low-temperature (<4℃) conditions, the active factor solution (the mass ratio of ε-polylysine to water is 0.3g:100mL, and the mass ratio of EGCG to water is 0.23g:100mL) is slowly added to the cross-linking precursor solution obtained in step three (the volume ratio of active factor solution to cross-linking precursor solution is 1:11). The mixture is gently stirred for 30 minutes to ensure that the active factor is uniformly dispersed in the polysaccharide network and to avoid deactivation caused by vigorous stirring.

[0064] Step 5: Cryogenic Construction and Casting of Microporous Structures: The final functionalized sol solution is injected into the desired mold. Pre-freezing: The mold is placed in a -20°C environment for 10 hours of pre-freezing. During this time, water forms ice crystals, which act as a pore-forming template.

[0065] Freeze-drying: The completely frozen sample is rapidly transferred to a freeze dryer. Freeze-drying is carried out for 24 hours at a condenser temperature of -50°C and a vacuum of 0.1 mbar to sublimate the ice crystals and leave a porous structure.

[0066] Step Six: Post-processing Removal and Cutting: Remove the dried porous material from the freeze dryer, cut it into shape as needed, sterilize it (using Co-60 γ-ray irradiation to sterilize the material), and package it (sealed packaging) to obtain the final product.

[0067] Example 2 The only difference from Example 1 is: Step 1: The mass of chitosan is 1g, the mass of carrageenan (κ-type) is 2g, and no alginate is added; Step 2: The mass of carboxymethyl chitosan is 2% of the total mass of the compound solution obtained in Step 1; Step 3: The molar ratio of the aldehyde group in citral to the amino group in chitosan is 0.5:1; Step 4: Active factor solution (the mass ratio of ε-polylysine to water is 0.3g:100mL, and the mass ratio of EGCG to water is 0.1g:100mL). The rest is the same as in Example 1.

[0068] Simulated performance data: Liquid uptake rate (pH 6.5): ~3550 g / g. The rapid hydration properties of carrageenan result in an initial liquid uptake rate that is approximately 20% faster than in Example 1.

[0069] Elongation at break in wet conditions: ~45% (approximately 30% in Example 1), exhibiting better flexibility.

[0070] Antibacterial properties (diameter of inhibition zone): Staphylococcus aureus: 18mm; Escherichia coli: 16mm.

[0071] pH-responsive degradation: Similar to Example 1, the degradation rate was 83% within 7 days at pH 7.4.

[0072] Example 3 The only difference from Example 1 is: Step 1: The mass of chitosan is 1g, the mass of alginate is 1g, and the mass of carrageenan (κ-type) is 1g; Step 2: The mass of carboxymethyl chitosan is 4% of the total mass of the compound solution obtained in Step 1; Step 3: The molar ratio of the aldehyde group in citral to the amino group in chitosan is 0.4:1; Step 4: The mass ratio of ε-polylysine to water in the active factor solution was 0.5 g: 100 mL, with no EGCG added; The rest is the same as in Example 1.

[0073] Step Seven: The dried porous material was placed in an ethanol / water solution of calcium chloride (calcium chloride mass fraction of 2%, ethanol and water volume ratio of 1:1) and soaked for 2 hours to allow the alginate to react with the calcium chloride. 2+ Ionic cross-linking occurs, forming a dual network.

[0074] Simulated performance data: Liquid absorption rate (pH 6.5): ~3650 g / g. Due to the highest cross-linking density, the liquid absorption rate is slightly lower, but the shape retention is excellent.

[0075] Wet compressive strength: approximately 150% higher than that of Example 1. It can still resist external forces after fully absorbing liquid and is not easily collapsed.

[0076] Antibacterial properties (diameter of inhibition zone): Staphylococcus aureus: 22mm; Escherichia coli: 20mm.

[0077] pH-responsive degradation: Due to the double crosslinking, the degradation rate at pH 7.4 is slower than in Example 1, and complete degradation occurs on day 10, providing a longer protection period.

[0078] Example 4 The only difference from Example 1 is: Step 1: The mass of chitosan is 1g, and the mass of trehalose is 3g; Step 3: The molar ratio of the aldehyde group in citral to the amino group in chitosan is 0.3:1; Step 4: Active factor solution (without ε-polylysine, the mass ratio of EGCG to water is 0.3g:100mL); The rest is the same as in Example 1.

[0079] Degradability: Completely degrades within 5 days at pH 7.4, achieving rapid and residue-free degradation.

[0080] Simulated performance data: Liquid absorption rate (pH 6.5): ~2950 g / g. It exhibits excellent liquid absorption and water retention capabilities.

[0081] Cell migration assay (scratch assay): Compared with the control group, the migration rate of fibroblasts cultured with the extract of Example 4 increased by 35% (25% increase in Example 1), indicating that trehalose and a mild cross-linking environment are more conducive to cell repair.

[0082] Cell migration assay (scratch assay): Plating: ① Prepare low-serum culture medium (99% MEM + 1% serum + 0.1% penicillin-dextrose antibody); ② Digest adherent cells (L929, fifth generation) in logarithmic growth phase using 0.25% trypsin to prepare a single-cell suspension and count the cells; ③ After cell counting, seed the cells into six-well plates at 0.158 mL per well and 5.69 × 10⁶ cells per well. 5Add culture medium to each well to bring the final total culture medium volume to 2 mL. Gently shake to mix, and incubate at 37°C with 5% CO2 for 24 hours.

[0083] Scratching: ① Using a ruler as a guide, use a 200 µL pipette to create cell scratches (the flat surface of the pipette tip should be perpendicular to the culture plate and scrape across the cell layer); aspirate the culture medium, gently wash twice with PBS to thoroughly remove floating cells; ② Set up a blank group, an H2O2 group, an example group, and a commercially available similar group, with two parallel runs (i.e., two wells) for each group; after changing the medium, place the cells in a 37°C, 5% CO2 incubator for culture.

[0084] Add sample: Use a pipette to add 200 μL of test solution to the scratch, and add low serum culture medium to a total volume of 2 mL.

[0085] Observation: Cells were observed and photographed at 0h, 12h and 24h respectively, and the results were recorded.

[0086] Example 5 The only difference from Example 1 is that in step four, the mass ratio of ε-polylysine to water is 0.5 g: 100 mL, and the mass ratio of EGCG to water is 0.2 g: 100 mL.

[0087] Example 6 The only difference from Example 5 is that 2g of trehalose was added to the acetic acid solution.

[0088] Retention rate of active ingredients: Accelerated experiments showed that after 4 weeks of storage, the retention rate of EGCG in Example 6 was 15% higher than that in Example 5, proving that trehalose has a significant protective effect on active ingredients.

[0089] Basis for testing the retention rate of active factors: 1) National Standard of the People's Republic of China GB / T 37973-2019 High Performance Liquid Chromatography.

[0090] 2) General Chapter 0512 of the Pharmacopoeia of the People's Republic of China (2020 Edition).

[0091] Test principle: The actual content of active factors in the sample is accurately determined by high performance liquid chromatography (HPLC), and the retention rate after preparation is calculated by comparing it with the theoretical loading.

[0092] Test steps Sample pretreatment: Accurately weigh 20.0 mg of each group of dried sample, pulverize, and then perform ultrasonic extraction with 10 mL of methanol-water (1:1, v / v) solution (30 min, 25℃). The extract was filtered through a 0.22 μm microporous membrane and then tested.

[0093] Chromatographic conditions: Chromatographic column: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm) Mobile phase: Phase A is an aqueous solution of 0.1% trifluoroacetic acid by mass, and Phase B is acetonitrile.

[0094] Gradient elution program: 0-10 min, phase B increases linearly from 5% to 25%; 10-15 min, phase B is maintained at 25%.

[0095] Flow rate: 1.0 mL / min Column temperature: 30℃ Detection wavelength: EGCG at 280 nm; ε-polylysine was detected by derivative method or by mass spectrometry.

[0096] Injection volume: 10 μL Standard curve plotting: A series of concentration solutions were prepared using EGCG and ε-polylysine standards, and the solutions were injected for analysis. A linear regression was performed on the peak area (Y) against the concentration (X) to plot the standard curve.

[0097] Content calculation and retention rate: Based on the chromatographic peak area of ​​the sample, substitute it into the standard curve equation to calculate the measured content (C) of the active factor in the sample. 实测 ).

[0098] The formula for calculating the Retention Rate of Active Ingredient (RRA) is as follows: RRA (%) = (C 实测 / C 理论 )×100% Among them, C 理论 This is the theoretical load content calculated based on the amount of material fed.

[0099] Comparative Example 1 The only differences from Example 1 are: no alginate was added; and step two was not performed.

[0100] Comparative Example 2 The only differences from Example 1 are: no chitosan was added; and step two was not performed. Step seven follows step six: The dried porous material was placed in an ethanol / water solution of calcium chloride (calcium chloride mass fraction of 2%, ethanol and water volume ratio of 1:1) and soaked for 2 hours to allow the alginate to react with the calcium chloride. 2+ Ionic cross-linking occurs.

[0101] Comparative Example 3 The only difference from Example 1 is that step two was not performed.

[0102] Comparative Example 4 Take a commercially available brand of sodium alginate hydrocolloid dressing.

[0103] Liquid absorption performance test Test solutions: To simulate the application performance of materials under different environments, two test solutions were selected: Solution A (pH≈6.5): Prepared with phosphate-buffered saline (PBS) to simulate the slightly acidic environment of chronic wound exudate.

[0104] Solution B (pH≈7.4): Prepared with PBS to simulate a normal physiological environment.

[0105] Test steps: Each group of dried samples was precisely cut into pieces with a mass of M. dry (Approximately 20 mg, accurate to 0.1 mg) cubes.

[0106] Place the sample into a pre-weighed clean nylon mesh bag (100 mesh), accurately weigh the total mass of the mesh bag and the dry sample, and record it as W1.

[0107] Completely immerse the mesh bag containing the sample in a beaker containing sufficient test solution (solution A or B) and let it stand at room temperature.

[0108] After soaking for 30 minutes, gently lift the mesh bag with tweezers and hang it for 30 seconds to drain any unabsorbed free liquid.

[0109] Quickly weigh the total mass of the mesh bag and the sample after liquid aspiration, and record it as W2.

[0110] Take a blank nylon mesh bag, repeat steps 3-5, and measure the mass of the blank mesh bag after draining, denoted as W. empty .

[0111] Calculation formula: The swelling ratio (SR) is calculated using the following formula, with units of grams of liquid per gram of dry material (g / g): SR=(W2-W1-W empty ) / M dry Test Results Table 1: Liquid uptake rate (SR, g / g) of each sample group in solutions with different pH values

[0112] Note: At pH 7.4, the sample in Example 1 swelled rather than dissolved due to the reversible breakage of the Schiff base bonds, and the structure remained intact, but the liquid absorption rate decreased, demonstrating pH response characteristics.

[0113] Results analysis: Synergistic effect of compounding: The liquid absorption rate of Comparative Example 3 (unmodified compound) (2450 g / g) was significantly higher than that of Comparative Example 1 (chitosan alone, 1050 g / g) and Comparative Example 2 (alginate alone, 1850 g / g). This demonstrates that the compounding of chitosan and alginate produces a significant synergistic effect, and the electrostatic interaction and entanglement between their molecular chains form a three-dimensional network structure that is more conducive to water penetration and storage.

[0114] The key role of hydrophilic modification: The liquid absorption rate of Example 1 (compound modified with carboxymethyl chitosan) (3850 g / g) was increased by approximately 57% compared to Comparative Example 3 (compound unmodified, 2450 g / g). This fully demonstrates that the introduction of carboxymethyl chitosan greatly improves the hydrophilicity of the material, and the additional carboxyl and hydroxyl groups introduced enhance the overall hydration capacity of the material, thereby achieving a leap in liquid absorption rate.

[0115] Advantages compared to commercial products: The liquid absorption rate of Example 1 (3850 g / g) is significantly higher than that of the commercially available hydrocolloid dressing of Comparative Example 4 (2050 g / g), representing a performance improvement of nearly 88%. This demonstrates that the material of the present invention has a significant competitive advantage in core physical properties.

[0116] pH Response Characteristics: The material in Example 1 exhibited a lower liquid absorption rate at pH 7.4 (2850 g / g) than at pH 6.5 (3850 g / g). This phenomenon is attributed to the pH sensitivity of the citral-Schiff base bond: the cross-linked network is stable and exhibits high liquid absorption in a slightly acidic environment (pH 6.5); in a neutral to slightly alkaline environment (pH 7.4), the Schiff base bond undergoes partial hydrolysis, resulting in a relatively loose network structure and decreased water retention capacity. This characteristic is well-suited for wound dressings: they can absorb large amounts of liquid in the slightly acidic environment of a high-exudate wound, while potentially separating more easily from the wound during dressing changes (neutral environment).

[0117] Antibacterial rate test Strain selection: Staphylococcus aureus (ATCC 6538) was selected as a representative of Gram-positive bacteria; Escherichia coli (ATCC 25922) was selected as a representative of Gram-negative bacteria.

[0118] Sample preparation: After sterilizing the samples of Example 1 and Comparative Example 3, they were cut into round pieces with a diameter of 5 mm.

[0119] Procedure: Inoculate the bacterial strain into liquid culture medium, activate it to the logarithmic growth phase at 37°C, and dilute with PBS to a concentration of 1×10⁻⁶. 6A bacterial suspension of CFU / mL was prepared. 0.2 mL of the bacterial suspension was evenly spread onto a solid agar medium. The sample discs were then firmly attached to the agar surface. The culture dishes were placed in a 4°C refrigerator for diffusion for 4 hours, and then transferred to a 37°C incubator for 18–24 hours. The diameter of the zone of inhibition around the sample (including the sample diameter) was observed and measured.

[0120] Evaluation index: The larger the diameter (D) of the inhibition zone, the stronger the antibacterial activity of the material. D < 1 mm indicates no inhibition zone.

[0121] Test Results Table 2: Antibacterial properties of each group of samples (diameter of inhibition zone, unit: mm)

[0122] Results analysis: Example 1 showed that it produced large and clear inhibition zones (>20 mm) against two common pathogens, demonstrating its potent and broad-spectrum antibacterial ability.

[0123] Comparative Example 3 produced only a small inhibition zone, primarily due to the weak antibacterial properties of chitosan itself. The significant difference from Example 1 demonstrates the successful loading of the antimicrobial peptide ε-polylysine and its dominant antimicrobial contribution.

[0124] This invention utilizes physical encapsulation and intermolecular forces to stably load ε-polylysine into a porous network, enabling its continuous release and providing a long-lasting antibacterial effect.

[0125] pH response degradation curve Test solutions: Phosphate-buffered saline (PBS) solutions with pH 5.5 and pH 7.4 were prepared to simulate the environment at different stages of wound healing.

[0126] Sample preparation: Example 1 (containing Schiff bases) and Comparative Example 2 (single alginate, Ca) were prepared. 2+ The dried sample (crosslinked) was accurately weighed (W0).

[0127] Operating steps: The samples were immersed in 10 mL of PBS solutions at pH 5.5 and pH 7.4, respectively, and placed in a constant temperature shaker at 37°C and 50 rpm.

[0128] At preset time points (1, 3, 5, 7, 14 days), the samples were taken out, quickly rinsed with deionized water, freeze-dried, and then weighed accurately again (Wt).

[0129] Three parallel samples were set up at each time point.

[0130] Calculation formula: Remaining mass percentage (%) = (Wt / W0) × 100%. Plot the curve of remaining mass percentage over time, i.e., the degradation curve.

[0131] Test Results Example 1 (pH 5.5): After 14 days, the remaining mass percentage remained above 85%, indicating slow degradation. This demonstrates that the material structure is stable during the wound inflammation phase (slightly acidic environment), providing sustained mechanical protection and functional release.

[0132] Example 1 (pH 7.4): By day 7, the remaining mass percentage decreased to approximately 40%; by day 14, it was completely degraded. This exhibits a clear pH-responsive rapid degradation behavior.

[0133] Comparative Example 2 (pH 5.5 & 7.4): At both pH values, the degradation rate was very slow, and the remaining mass percentage was >90% after 14 days, with no obvious pH response.

[0134] Results Analysis: The degradation behavior of Example 1 was significantly dependent on the pH of the environment, which perfectly validates the pH sensitivity of the citral-Schiff base bond. In a neutral environment (pH 7.4), the Schiff base bond hydrolyzes and breaks, leading to network collapse; in an acidic environment (pH 5.5), the bond remains stable.

[0135] This intelligent degradation property is extremely valuable for wound dressing applications: when the wound is in the acidic inflammatory phase, the dressing remains intact and continues to work; when the wound heals and becomes neutral, the dressing can degrade rapidly on its own, avoiding secondary damage to newly formed tissue when changing dressings, thus achieving "targeted degradation".

[0136] SEM images of microstructure Instrument: Scanning Electron Microscope (SEM).

[0137] Sample preparation: Take a small piece of dry sample from Example 1, carefully adhere it to the conductive adhesive, and perform gold sputtering to increase the conductivity of the sample surface.

[0138] Observation conditions: Observe the surface and cross-sectional morphology of the sample under an accelerating voltage of 5-10 kV.

[0139] Figure 1 This is a SEM image of the porous material surface in Example 1.

[0140] Depend on Figure 1It is evident that the material exhibits a highly porous and interconnected three-dimensional network structure with uniformly distributed pores. The pore size is mainly distributed between 50-200 μm, belonging to the micron-scale porous structure. The pore walls are relatively thin, and abundant wrinkles and lamellar structures are visible on the surface, which is determined by the arrangement of polysaccharide molecular chains under the action of ice crystal templates.

[0141] Figure 2 SEM image of the cross-section of the porous material in Example 1: Figure 2 (a) and (a') are SEM images of the sample from Example 1 at different magnifications.

[0142] Results Analysis: SEM results visually confirm the successful construction of an ideal, interconnected porous microstructure via freeze-drying. This structure provides ample space for rapid capillary penetration and large-scale liquid storage, which is corroborated by the test results of ultra-high liquid absorption rate.

[0143] The introduction of the hydrophilic modifier (carboxymethyl chitosan) not only enhanced hydrophilicity but also appeared to improve the stability and uniformity of the polysaccharide network, resulting in a more uniform ice crystal template formed during freezing and thus a superior pore structure. This indicates a synergistic effect between hydrophilic modification and microporous structure construction.

[0144] The organic combination of antibacterial testing, degradation behavior research, and microstructure characterization, together with liquid absorption performance testing, constitutes a complete chain of evidence, fully demonstrating that the functionalized porous materials prepared in this invention have great application potential and industrial value in biomedical fields such as advanced wound care.

[0145] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A biopolysaccharide composite material, characterized in that, It has a porous structure, including cross-linked biopolysaccharides and active factors loaded on the biopolysaccharides; The biopolysaccharide includes one or more of the following: hydrophilic-modified trehalose, hydrophilic-modified alginate, and hydrophilic-modified carrageenan, along with hydrophilic-modified chitosan.

2. The biopolysaccharide composite material according to claim 1, characterized in that, The hydrophilic agent includes carboxymethylated chitosan.

3. The biopolysaccharide composite material according to claim 1 or 2, characterized in that, The active factors include antimicrobial peptides and / or anti-inflammatory factors.

4. The method for preparing the biopolysaccharide composite material according to any one of claims 1 to 3, characterized in that, Includes the following steps: A solution of biopolysaccharide raw material is modified by mixing it with a hydrophilic agent to obtain a solution of biopolysaccharide; the biopolysaccharide raw material includes one or more of trehalose, alginate and carrageenan, and chitosan; The solution of the biopolysaccharide, the solution of the pH-sensitive crosslinking agent, and the pH adjuster are mixed to carry out the first crosslinking reaction to generate the crosslinked biopolysaccharide, thus obtaining the crosslinking reaction system; The cross-linking reaction system is mixed with a solution of active factors for loading to generate biopolysaccharides loaded with active factors. The resulting loaded system is then subjected to pre-freezing and freeze-drying to obtain the biopolysaccharide composite material.

5. The preparation method according to claim 4, characterized in that, The mass of the hydrophilic agent is 1-5% of the mass of the solution of the biopolysaccharide raw material; The modification was performed at room temperature for 1 hour, and the modification was carried out under stirring conditions at a speed of 300-600 rpm.

6. The preparation method according to claim 4, characterized in that, The mass ratio of trehalose to solvent volume in the solution of the biopolysaccharide raw materials is 1~5g:100mL, the mass ratio of chitosan to solvent volume is 0.5~2g:100mL, the mass ratio of alginate to solvent volume is 0.5~2g:100mL, and the mass ratio of carrageenan to solvent volume is 0.5~2g:100mL. The pH-sensitive crosslinking agent contains an aldehyde group; the pH-sensitive crosslinking agent includes citral; The mass ratio of the pH-sensitive crosslinking agent to the volume of the solvent in the solution is 5~10g:100mL; The ratio of the amount of aldehyde group in the pH-sensitive crosslinking agent to the amount of amino group in chitosan is 0.2~0.8:1; The pH adjuster includes a sodium hydroxide solution, and the concentration of the pH adjuster is 1M; The pH value of the system obtained by mixing the solution of the biopolysaccharide, the solution of the pH-sensitive cross-linking agent, and the pH adjuster is 5-6.

7. The preparation method according to claim 4, characterized in that, The mass ratio of the active factor to the volume of the solvent in the solution of the active factor is 0.05~1.5g:100mL; when the active factor includes antimicrobial peptides and / or anti-inflammatory factors, the concentration of the antimicrobial peptides in the solution of the active factor is 0.1~1g:100mL, and the mass ratio of the anti-inflammatory factors to the volume of the solvent is 0.05~0.5g:100mL. The loading was carried out under light-proof and stirring conditions, with the temperature of the loading being <4℃ and the time being 30min.

8. The preparation method according to claim 4, characterized in that, The pre-freezing temperature is -20~-40℃, and the time is 6~12h; The freeze-drying temperature is below -50°C, the vacuum degree is below 0.1 mbar, and the time is 24~48 h.

9. The preparation method according to claim 4, characterized in that, When the biopolysaccharide raw material includes alginate and / or carrageenan, the loading process further includes: immersing the loaded system in a calcium chloride solution to carry out a second crosslinking reaction.

10. The application of the biopolysaccharide composite material according to any one of claims 1 to 3 or the biopolysaccharide composite material prepared by the preparation method according to any one of claims 4 to 9 as a dressing.

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