Preparation method and application of fluorine-containing modified chitosan antibacterial hydrogel

Abstract

The invention relates to a preparation method and application of fluorine-containing modified chitosan antibacterial hydrogel, and the preparation method comprises the following steps: 1) dissolving chitosan powder in an acetic acid solution to obtain a chitosan solution; 2) adding potassium persulfate into the chitosan solution, and pre-reacting under nitrogen to obtain an activating solution; (3) dropwise adding hexafluorobutyl methacrylate into the activating solution, and carrying out free radical polymerization reaction under the condition of continuously introducing nitrogen; (4) after the reaction is finished, cooling reaction liquid to room temperature, transferring the reaction liquid into a dialysis bag, dialyzing the reaction liquid with deionized water for 72 hours, and freeze-drying the reaction liquid to obtain hexafluorobutyl methacrylate HFMA grafted chitosan which is marked as CS-g-HFMA; 5, CS-g-HFMA is dissolved in a phosphate buffer solution, genipin is added for a reaction, and the three-dimensional network hydrogel is obtained.According to the method, free radical grafting is completed in one step, complex aftertreatment is not needed, the method is suitable for large-scale production, and the prepared hydrogel product is long in antibacterial time and good in antibacterial effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to grafting hexafluorobutyl methacrylate (HFMA) onto the chitosan molecular chain through free radical polymerization to prepare a fluorine-modified chitosan hydrogel with enhanced antibacterial properties, which is applicable to biomedical fields such as wound dressings. Background Art

[0002] Chitosan is a natural cationic polysaccharide and is widely used in wound dressings due to its biocompatibility, degradability, and mild antibacterial properties. However, its inhibitory effect on Gram-positive bacteria (such as Staphylococcus aureus) is weak, mainly due to its single antibacterial mechanism (relying on the interaction between positive charges and bacterial membranes). In the prior art, antibacterial properties can be improved through chemical modification (such as quaternization, metal ion loading), but it is often accompanied by an increase in cytotoxicity or insufficient long-term effectiveness. Fluorine-containing compounds can disrupt the integrity of bacterial membranes due to their strong hydrophobicity and chemical stability, but how to efficiently introduce them into the chitosan backbone while maintaining the hydrogel properties remains a technical difficulty. Summary of the Invention

[0003] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a method for modifying chitosan by grafting a fluorine-containing monomer (HFMA) through free radicals to prepare a hydrogel with both high antibacterial properties, good mechanical properties, and biocompatibility. The specific scheme is as follows: I. Dissolution and Activation of Chitosan 1) Dissolve chitosan powder with a deacetylation degree ≥ 90% in a 2% acetic acid solution to prepare a 3% (w / v) chitosan solution, and stir magnetically until transparent.

[0004] 2) Add potassium persulfate (KPS) with a concentration of 0.08 - 0.12 mol / L as an initiator, preferably 0.1 mol / L, and pre-react at 60 °C for 30 minutes under nitrogen protection to activate the free radical sites on the chitosan molecular chain.

[0005] Selecting potassium persulfate as the initiator can not only fully activate chitosan hydroxyl groups but also avoid cytotoxicity caused by initiator residues.

[0006] II. Grafting Reaction of Hexafluorobutyl Methacrylate (HFMA) 1) Drop hexafluorobutyl methacrylate into the above activation solution, and carry out free radical polymerization reaction under the condition of continuous nitrogen flow; 2) After the reaction is completed, cool the reaction solution to room temperature, transfer it to a dialysis bag with a cut-off molecular weight of 8 - 14 kDa, and dialyze with deionized water for 72 hours to remove unreacted monomers and initiators, and then freeze-dry to obtain hexafluorobutyl methacrylate HFMA-grafted chitosan, denoted as CS-g-HFMA.

[0007] Preferably, the molar ratio of chitosan to hexafluorobutyl methacrylate is controlled at 1:6 - 10, preferably 1:8. At this ratio, the grafting rate can reach 28% - 35%, and the fluorine element content (EDS analysis) is increased to 12 - 15 wt%, significantly enhancing the hydrophobicity and antibacterial activity.

[0008] The temperature of the free radical polymerization reaction is 65 - 75 °C, and the reaction time is 5 - 8 hours. Preferably, the reaction is carried out at 70 °C for 6 hours. At this time, the grafting efficiency is the highest (>30%), and there is no obvious degradation of the chitosan main chain.

[0009] Using hexafluorobutyl acrylate (C4F6) instead of short-chain fluorinated monomers (such as trifluoroethyl acrylate), its long-chain structure is more likely to insert into the bacterial membrane phospholipid layer. The diameter of the inhibition zone against Staphylococcus aureus reaches 16 - 20 mm, which is more than twice that of unmodified chitosan (6 - 8 mm). Through Zeta potential testing, the surface potential of the modified hydrogel drops from +25 mV to +15 mV, indicating that the fluorinated hydrophobic effect and the positive charge of chitosan cooperate to damage the bacterial membrane (fluorescent staining shows that 90% of the bacterial membranes are ruptured).

[0010] III. Preparation of hydrogel 1) Dissolve CS-g-HFMA in phosphate buffer (PBS, pH = 6.5), and add 0.5 - 1.2% (w / v) genipin as a cross-linking agent for reaction to form a three-dimensional network hydrogel.

[0011] At this concentration, the compressive modulus (25 - 30 kPa) and swelling ratio (180% - 220%) of the hydrogel reach equilibrium, meeting the mechanical requirements of wound dressings (ASTM F2900 standard).

[0012] Preferably, the temperature of the cross-linking reaction is controlled at 35 - 45 °C, and the cross-linking reaction time is 3 - 5 hours. The best condition is cross-linking at 40 °C for 4 hours. At this time, the cross-linking density (calculated by the swelling method) is 0.12 - 0.15 mol / m³, and the network structure is uniform (SEM shows that the pore size distribution is 10 - 50 μm).

[0013] The fluorinated modified chitosan antibacterial hydrogel prepared by the above preparation method, and the application of the hydrogel as a wound dressing.

[0014] Specifically, cut the hydrogel into a standard shape (such as a disc or film), soak it in PBS for equilibrium swelling, and then use it.

[0015] The beneficial effects of the present invention are as follows: 1) The fluorinated side chain of HFMA destroys the bacterial membrane structure through hydrophobic interaction, and the antibacterial rate against Gram-positive bacteria is >99%, and the long-term effect is better than that of quaternization modification; 2) In the slightly acidic wound environment (pH 5.5 - 6.5), the main chain of chitosan accelerates hydrolysis, releasing fluorinated side chains for continuous antibacterial effect, and the degradation rate is > 80% within 14 days (measured by the weight loss method); 3) The microphase separation structure and cross - linked network synergistically enhance the mechanical strength to meet the flexibility requirements of wound dressings; 4) The free - radical grafting is completed in one step without complex post - treatment, which is suitable for large - scale production. Description of the Drawings

[0016] Figure 1 It is a schematic diagram of the synthesis route and chemical structure of CS - g - HFMA. Detailed Embodiments

[0017] The present invention will be further described below in conjunction with the drawings of the specification to better understand the technical solution. Example 1

[0018] The present invention provides a fluorine - modified hydrogel. The amounts of raw materials for the grafting reaction are as follows: 1 mol of chitosan, 6 mol of hexafluorobutyl methacrylate (HFMA), potassium persulfate (KPS), and 100 ml of deionized water.

[0019] The preparation method of the above - mentioned fluorine - modified chitosan antibacterial hydrogel is as follows: Dissolve chitosan in 2% acetic acid solution (3% w / v), add 0.1 mol / L KPS, and pre - react at 60 °C for 30 minutes under nitrogen protection. Gradually add 6 mol of HFMA dropwise to the activated solution and react at 70 °C for 6 hours. After the reaction is completed, cool the mixture to room temperature, transfer it to a dialysis bag with a molecular weight cut - off of 8 - 14 kDa, and dialyze with deionized water for 72 hours to remove unreacted monomers and initiators, and then freeze - dry to obtain HFMA - grafted chitosan. Dissolve CS - g - HFMA in phosphate - buffered saline, add 0.5% genipin as a cross - linker, and cross - link at 40 °C for 4 hours to form a three - dimensional network hydrogel. Example 2

[0020] The present invention provides a fluorine - modified hydrogel. The amounts of raw materials for the grafting reaction are as follows: 1 mol of chitosan, 8 mol of hexafluorobutyl methacrylate (HFMA), potassium persulfate (KPS), and 100 ml of deionized water.

[0021] The preparation method of the above-mentioned fluorinated modified chitosan antibacterial hydrogel is as follows: Dissolve chitosan in 2% acetic acid solution (3% w / v), add 0.1 mol / L KPS, and pre-react at 60 °C for 30 minutes under nitrogen protection. Dropwise add 8 mol HFMA to the activation solution and react at 70 °C for 6 hours. After the reaction is completed, cool the mixture to room temperature, transfer it to a dialysis bag with a molecular weight cut-off of 8 - 14 kDa, and dialyze with deionized water for 72 hours to remove unreacted monomers and initiators, and then freeze-dry to obtain HFMA-grafted chitosan. Dissolve CS-g-HFMA in phosphate buffer, add 0.5% genipin as a cross-linking agent, and cross-link at 40 °C for 4 hours to form a three-dimensional network hydrogel. Example 3

[0022] The present invention provides a fluorinated modified hydrogel, and the usage amounts of the raw materials for the grafting reaction are as follows: 1 mol of chitosan, 10 mol of hexafluorobutyl methacrylate (HFMA), potassium persulfate (KPS), and 100 ml of deionized water.

[0023] The preparation method of the above-mentioned fluorinated modified chitosan antibacterial hydrogel is as follows: Dissolve chitosan in 2% acetic acid solution (3% w / v), add 0.1 mol / L KPS, and pre-react at 60 °C for 30 minutes under nitrogen protection. Dropwise add 8 mol HFMA to the activation solution and react at 70 °C for 6 hours. After the reaction is completed, cool the mixture to room temperature, transfer it to a dialysis bag with a molecular weight cut-off of 8 - 14 kDa, and dialyze with deionized water for 72 hours to remove unreacted monomers and initiators, and then freeze-dry to obtain HFMA-grafted chitosan. Dissolve CS-g-HFMA in phosphate buffer, add 0.5% genipin as a cross-linking agent, and cross-link at 40 °C for 4 hours to form a three-dimensional network hydrogel.

[0024] The synthetic route and chemical structure schematic diagram of the above CS-g-HFMA are as Figure 1 shown.

[0025] The performance test results of the hydrogels prepared in the above Examples 1 - 3 are shown in Table 1.

[0026] Table 1 Test Items Example 1 Example 2 Example 3 Grafting Ratio (%) 18 32 30 Inhibition Zone (mm) 10±2 18±1 16±1 Compression Modulus (kPa) 15 25 35 Swelling Ratio (%) 250 210 180 Cell Survival Rate (%) 98 96 82 As can be seen from Table 1: For the hydrogel prepared in Example 2 with a molar ratio of chitosan:HFMA of 1:8, its grafting rate and fluorinated side chain distribution are optimal, and the antibacterial performance (inhibition zone of 18 mm) and mechanical properties (compression modulus of 25 kPa) reach an equilibrium, which is the preferred condition. For the hydrogel prepared in Example 1 with a molar ratio of chitosan:HFMA of 1:6, the lack of HFMA leads to the failure of antibacterial performance and is only applicable to low-infection-risk scenarios. For the hydrogel prepared in Example 3 with a molar ratio of chitosan:HFMA of 1:10, the excessive HFMA leads to a decrease in biocompatibility.

[0027] Through precise regulation of the molar ratio of chitosan to HFMA, the present invention realizes the ternary synergistic optimization of "high antibacterial property - mechanical strength - biocompatibility", providing a reliable parameter window for the industrialization of fluorine-containing modified chitosan hydrogels.

Claims

1. A preparation method of a fluorine-modified chitosan antibacterial hydrogel, characterized in that It includes the following steps: 1) Dissolve chitosan powder in 2% acetic acid solution to prepare a 3% (w / v) chitosan solution, and magnetically stir until transparent to obtain the dissolved chitosan solution; 2) Add potassium persulfate with a concentration of 0.08 - 0.12 mol / L as an initiator to the chitosan solution dissolved in step 1), and pre-react under nitrogen protection to activate the free radical sites on the chitosan molecular chain to obtain an activation solution; 3) Drop hexafluorobutyl methacrylate into the activation solution in step 2), and carry out a free radical polymerization reaction under the condition of continuous nitrogen flow; 4) After the reaction is completed, cool the reaction solution to room temperature, transfer it to a dialysis bag with a molecular weight cut-off of 8 - 14 kDa, and dialyze with deionized water for 72 hours to remove unreacted monomers and initiators, and then freeze-dry to obtain hexafluorobutyl methacrylate HFMA-grafted chitosan, denoted as CS-g-HFMA; 5) Dissolve the CS-g-HFMA prepared in step 4) in phosphate buffer solution, add 0.5 - 1.2% (w / v) genipin as a cross-linking agent to carry out the reaction to form a three-dimensional network hydrogel.

2. The preparation method of a fluorine-modified chitosan antibacterial hydrogel as described in claim 1, characterized in that In step 1), the deacetylation degree of chitosan is ≥90%.

3. The preparation method of a fluorine-modified chitosan antibacterial hydrogel as claimed in claim 1, wherein In step 2), the pre-reaction temperature is 60 °C and the pre-reaction time is 30 minutes.

4. The preparation method of a fluorine-modified chitosan antibacterial hydrogel according to claim 1, characterized in that In step 3), the molar ratio of chitosan to hexafluorobutyl methacrylate is controlled at 1:6 - 10, the temperature of the free radical polymerization reaction is 65 - 75 °C, and the reaction time is 5 - 8 hours.

5. The preparation method of a fluorine-modified chitosan antibacterial hydrogel according to claim 1, characterized in that In step 5), the temperature of the cross-linking reaction is controlled at 35 - 45 °C, and the cross-linking reaction time is 3 - 5 hours.

6. The fluorine-modified chitosan antibacterial hydrogel prepared by the preparation method according to any one of claims 1 - 5.

7. The application of the fluorine-modified chitosan antibacterial hydrogel according to claim 6 as a wound dressing.

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