Gallium porphyrin complex / silk fibroin antibacterial dressing and preparation method thereof

Abstract

Traditional medical dressings used for bandaging or treating wounds in clinical practice usually contain antibiotics and cotton bases. The antibiotic resistance problem and the non-degradability of the cotton base seriously increase the pain and care costs of patients. In view of the above problems, the present invention provides a gallium porphyrin complex / silk fibroin antibacterial dressing using a lyophilized silk fibroin gel as a dressing base and adding a gallium porphyrin complex as an antibacterial component, and provides a method for cross-linking, fully freezing, and freeze-drying the gallium porphyrin complex / silk fibroin hydrogel to prepare an antibacterial dressing. The present invention has good biocompatibility and degradability, and its dressing has high antibacterial activity, antibacterial rate and antibacterial stability, which can further prevent wounds from becoming inflamed due to infection.

Description

Technical Field

[0001] The invention relates to the field of sanitary materials and preparation thereof, and in particular to an antibacterial dressing and a preparation method thereof. Background Art

[0002] Multidrug-resistant bacteria mainly refer to bacteria that are resistant to three or more types of clinically used antimicrobial drugs at the same time. The vast majority of hospital infections are caused by multidrug-resistant bacteria. These strains are widely distributed and spread quickly, posing severe challenges to clinical medical care and hospital infection control. Therefore, the demand for the development of new antimicrobial materials that are less likely to induce drug resistance is becoming increasingly strong.

[0003] Traditional medical dressings used for bandaging or treating wounds in clinical practice usually contain antibiotics and a cotton base. The antibiotic resistance problem and the non-degradability of the cotton base seriously increase the patient's pain and care costs.

[0004] Metal ion antimicrobial agents are currently the most promising antimicrobial materials, but the widely used metal ion antimicrobial agents have safety risks, which limits the development of this field. Among them, gallium ion is a new metal antimicrobial agent in recent years. Compared with other metal antimicrobial agents, the most significant advantage of gallium ion is that it has good biosafety and is effective against a variety of multi-drug resistant bacteria, but its application is still under exploration.

[0005] As a new type of metal antibacterial material, gallium porphyrin complexes are still largely unapplication in the field of medical materials. Summary of the invention

[0006] In order to solve the defects and gaps in the above-mentioned prior art, the present invention provides a gallium porphyrin complex / silk fibroin antibacterial dressing: a silk fibroin freeze-dried gel is used as a dressing base, and a gallium porphyrin complex is added as an antibacterial component; wherein the gallium porphyrin complex oxidizes the tyrosine of silk fibroin (SF) to form a stable cross-link, and the silk fibroin in the gallium porphyrin complex / silk fibroin antibacterial dressing is a porous framework structure, and the silk fibroin contains a β-folded structure.

[0007] The present invention further provides a method for preparing a gallium porphyrin complex / silk fibroin antibacterial dressing, comprising the following steps:

[0008] Step 1. Silk fibroin extraction: degumming, dissolving and dialysis of silkworm cocoons followed by reverse dialysis with polyethylene glycol, and concentrating to obtain a silk fibroin aqueous solution.

[0009] Step 2. Prepare gallium porphyrin complex with high antibacterial activity: add porphyrin small molecules and gallium chloride into anhydrous pyridine solvent, introduce nitrogen protection and heat to react, cool to room temperature after the reaction to obtain a deep red solution, remove the solvent, collect the precipitate; wash with washing solution, and dry;

[0010] Step 3. Preparation of hydrogel: uniformly dispersing the gallium porphyrin complex and silk fibroin in the solution, wherein the gallium porphyrin complex oxidizes the tyrosine of the silk fibroin to form a stable cross-link;

[0011] Step 4. Preparation of antibacterial dressing: Place the gallium porphyrin complex / silk fibroin complex in an ultra-low temperature refrigerator and freeze it thoroughly, then freeze-dry it; place the obtained gallium porphyrin complex / silk fibroin hydrogel at -80°C and freeze it thoroughly, so that the silk fibroin molecules in the system form a porous framework structure, and the solvent water solidifies into solid ice to support the stability of its framework structure. It is then freeze-dried to obtain a composite material. The gallium porphyrin complex / silk fibroin complex is freeze-dried, and during the freeze-drying process, the ice will sublimate into water vapor and be discharged outside the system, at which time a porous framework structure can be formed, and its porous structure can absorb wound exudate and accommodate cell and tissue adsorption and growth.

[0012] Step 5. Sterilize the composite material under high temperature and high pressure. Sterilize the composite material under high temperature and high pressure. Induce the silk fibroin to produce additional β-folding structure, effectively enhance its mechanical structure, and obtain the final gallium porphyrin complex / silk fibroin antibacterial dressing.

[0013] Preferably, in step 1: degumming the silkworm cocoons into a Na2CO3 solution, heating at 65°C to dissolve, placing the resulting solution in a dialysis bag and dialyzing it with deionized water. Then reverse dialyzing with a 10.0wt% polyethylene glycol (PEG) solution and allowing it to stand. Centrifugation is performed to remove undissolved residues, and the silk fibroin aqueous solution is concentrated to obtain the solution, which is then stored in a refrigerator at 4°C.

[0014] Preferably, in step 2, the porphyrin small molecule is tetra-p-methoxyphenyl porphyrin.

[0015] Preferably, in step 2, porphyrin small molecules and gallium chloride are added to anhydrous pyridine solvent and nitrogen is passed through to heat and reflux for reaction. After the reaction is completed, the solution is cooled to room temperature to obtain a deep red solution, the precipitate is collected by spin drying, and washed with a small amount of pre-cooled ethanol, the crude product is then washed with chloroform, filtered, and dried overnight in a vacuum dryer to obtain a deep purple powder.

[0016] In step 3: different proportions of gallium porphyrin complexes are added to the silk fibroin aqueous solution to obtain different hydrogel preformed solutions. The preformed solutions are cross-linked with the silk fibroin tyrosine residues via the gallium porphyrin complex under UV irradiation or left standing for a long time at room temperature to obtain covalently cross-linked hydrogels. Different contents of gallium porphyrin complexes will give the dressings different antibacterial properties.

[0017] Preferably, the amount of the gallium porphyrin complex added in step 3 is 1 g-6.5 g, and the mass volume ratio of the gallium porphyrin complex to the hydrogel solution is 3%-18%.

[0018] Preferably, the amount of the gallium porphyrin complex added in step 3 is 2.5 g to 4 g, and the mass volume ratio of the gallium porphyrin complex to the hydrogel solution is 7% to 12%.

[0019] Beneficial effects:

[0020] 1. Silk fibroin has good biocompatibility and degradability, and its degradation products are amino acids that can accelerate wound healing.

[0021] 2. The gallium porphyrin complex and silk fibroin are cross-linked with each other, which improves the antibacterial activity, antibacterial rate and antibacterial stability of the dressing, can further prevent wound inflammation due to infection, and significantly improve the limitations of existing dressings.

[0022] 3. The antibacterial dressing has good biological safety, and the preparation method is simple, rapid and low-cost, making it suitable for batch preparation for clinical use. DETAILED DESCRIPTION

[0023] Embodiment 1:

[0024] Preparation of Gallium Porphyrin Complex / Silk Fibroin Antibacterial Dressing:

[0025] 1. Silk fibroin extraction: 10g of degummed silkworm cocoons were placed in a 0.02mol / L Na2CO3 solution, and then placed in a 65°C oven for 5 hours of static dissolution. The resulting solution was placed in a dialysis bag and dialyzed with deionized water for 3 days, and the deionized water was replaced every 12 hours. Then, the solution was reverse dialyzed with a 10.0wt% polyethylene glycol (PEG) solution and allowed to stand for 12 hours. The solution was then centrifuged at 8000rpm for 40 minutes to remove the undissolved residue, and the silk fibroin aqueous solution was concentrated and stored in a 4°C refrigerator;

[0026] 2. Preparation of gallium porphyrin complex with high antibacterial activity: Weigh a certain amount of tetramethoxyphenylporphyrin (1.0mmol) and gallium chloride (0.5mmol) in a 100mL round-bottom flask, add 30mL of anhydrous pyridine solvent, pass nitrogen gas, heat and reflux for 8h. After the reaction is completed, cool to room temperature to obtain a deep red solution, collect the precipitate by spin drying, and wash it with a small amount of pre-cooled ethanol. The crude product is then washed with chloroform, filtered and placed in a vacuum dryer to dry overnight to obtain a deep purple powder.

[0027] 3. Preparation of hydrogel: 2.5 g of gallium porphyrin complex was added to silk fibroin aqueous solution to obtain 6% (w / v) hydrogel preformed solution. Under ultraviolet irradiation, the preformed solution cross-linked SF tyrosine residues via the gallium porphyrin complex to obtain a covalently cross-linked hydrogel.

[0028] 4. Preparation of antibacterial dressing: The obtained gallium porphyrin complex / silk fibroin hydrogel was fully frozen at -80°C, so that the silk fibroin molecules in the system formed a porous framework structure, and the solvent water solidified into solid ice to support the stability of its framework structure. It was then freeze-dried to obtain a composite material.

[0029] 5. The composite material is sterilized at high temperature and high pressure to induce the silk fibroin to produce additional β-folding structure, effectively enhance its mechanical structure, and obtain the final gallium porphyrin complex / silk fibroin antibacterial dressing.

[0030] Embodiment 2-4, comparative embodiment 5:

[0031] The difference from Example 1 is that the mass of the added gallium porphyrin complex is different, forming gallium porphyrin complex contents of different proportions. The added mass is shown in Table 1:

[0032]

[0033] Example 6: The difference from Example 1 is that in step 3, a pure physical cross-linked hydrogel is obtained by standing at room temperature. The relevant test data of Example 6 is not significantly different from that of Example 1.

[0034] Performance Test:

[0035] Cytocompatibility test: MTT was used to detect the effects of the antibacterial dressings of Examples 1 to 4 and Comparative Example 5 on the proliferation of MФ (RAW264.7), fibroblasts (RS-1) and vascular endothelial cells (HUVEC) cultured for 1, 3, and 7 days to determine the biocompatibility of the hydrogel. The cell survival rate calculated from the absorbance change after correction of the blank control is shown in Table 2. As the content of the gallium porphyrin complex added during the preparation of the antibacterial dressing increased, the cell compatibility of the material did not change significantly. That is, the material had no significant inhibitory effect on cell growth.

[0036]

[0037]

[0038] Blood compatibility test: The gallium porphyrin complex / silk fibroin antibacterial dressing was subjected to a hemolysis experiment to determine the blood compatibility of the dressing. Fresh rabbit blood was made into a red blood cell suspension with a concentration of 10.0% (v / v). Then, Examples 1-5 were placed in 1 mL centrifuge tubes containing 1 ml of red blood cell suspension and incubated in a constant temperature and humidity incubator at 37°C for 2 hours. Similarly, red blood cell suspensions diluted with PBS and ultrapure water were used as negative and positive controls, respectively. Finally, the red blood cell suspension after the above incubation for 2 hours was centrifuged at 3500 rpm for 10 minutes, the supernatant was collected and its absorbance at 540 nm was measured using a UV-visible spectrophotometer to determine the release of hemoglobin. The relevant data are shown in Table 3.

[0039]

[0040] The data show that both the silk fibroin dressing (Comparative Example 5) and the dressing cross-linked with the gallium porphyrin complex and silk fibroin exhibit a lower hemolysis rate, reflecting its good blood compatibility.

[0041] Antimicrobial activity testing:

[0042] The antibacterial activity of gallium porphyrin complex / silk fibroin antibacterial dressing was studied, including the inhibition of the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

[0043] 1. Inhibition zone detection: Using the filter paper method, first pick overnight bacteria and dilute and culture to the logarithmic phase. Take 100 μL of bacterial solution and evenly apply it on a non-resistant solid plate, then add the cylindrical antibacterial dressings of Examples 1-4 and Comparative Example 5 with a diameter of 1 cm, put them in a 37°C incubator for 20 hours, and measure the diameter of the area where bacterial growth is inhibited with a ruler.

[0044] 2. Antibacterial rate and antibacterial stability test: The antibacterial activity of the nanofibers of Examples 1-6 was evaluated by the oscillation method, and the antibacterial rate was calculated by the live bacteria plate count method. The samples were washed 30 times according to the washing method of standard AATCC 61-2010, and the antibacterial activity of the modified samples was evaluated by the oscillation method, and the antibacterial rate was calculated by the live bacteria plate count method.

[0045]

[0046] As the content of gallium porphyrin complex added increases, the size of the inhibition zone increases significantly. It has a good antibacterial effect on the three common hospital infection bacteria tested. Its antibacterial rate and antibacterial stability are significantly improved compared with the comparative example.

[0047] In vivo safety testing:

[0048] A mouse subcutaneous transplantation model was established to verify the safety, degradability and inflammatory response of the gallium porphyrin complex / silk fibroin antibacterial dressing in vivo: After anesthesia, the mice were implanted with the antibacterial dressings of Examples 1-5 on their backs. The mice were killed after 3, 7, 14, and 28 days, and the materials and surrounding tissues were removed, fixed with paraffin, and sliced. H&E staining was performed to observe the degradation and inflammatory response of the dressings.

[0049] No inflammatory reaction and foreign body reaction were observed in all control and test groups, and it was found that the gallium porphyrin complex / silk fibroin antibacterial dressing prepared by cross-linking treatment was biocompatible for subcutaneous transplanted tissues in mice.

[0050] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A gallium porphyrin / silk fibroin antibacterial dressing, characterized in that: The gallium porphyrin complex / silk fibroin antibacterial dressing is made of a lyophilized silk fibroin gel as a dressing base and a gallium porphyrin complex as an antibacterial component. The gallium porphyrin complex oxidizes the tyrosine of the silk fibroin to form a stable cross-link. The silk fibroin in the gallium porphyrin complex / silk fibroin antibacterial dressing is a porous framework structure, and the silk fibroin contains a β-folded structure. The method for preparing the gallium porphyrin / silk fibroin antibacterial dressing comprises the following steps: Step 1: extracting silk fibroin; Step 2: preparing a gallium porphyrin complex with high antibacterial activity; Step 3: preparing gallium porphyrin complex / silk fibroin hydrogel; Step 4: preparing an antibacterial dressing; Step 5: sterilizing the composite material at high temperature and high pressure; In the step 3, the gallium porphyrin complex and the silk fibroin are uniformly dispersed in the solution, and the gallium porphyrin oxidizes the tyrosine of the silk fibroin to form a stable cross-link; in the step 4, the gallium porphyrin complex / silk fibroin complex is placed in an ultra-low temperature refrigerator and fully frozen, and then freeze-dried.

2. A method for producing the gallium porphyrin / silk fibroin antibacterial dressing according to claim 1, characterized in that: The following steps are involved: Step 1: extracting silk fibroin; Step 2: preparing a gallium porphyrin complex with high antibacterial activity; Step 3: preparing gallium porphyrin complex / silk fibroin hydrogel; Step 4: preparing an antibacterial dressing; Step 5: sterilizing the composite material at high temperature and high pressure; In the step 3, the gallium porphyrin complex and the silk fibroin are uniformly dispersed in the solution, and the gallium porphyrin oxidizes the tyrosine of the silk fibroin to form a stable cross-link; in the step 4, the gallium porphyrin complex / silk fibroin complex is placed in an ultra-low temperature refrigerator and fully frozen, and then freeze-dried.

3. The method for preparing a gallium porphyrin / silk fibroin antibacterial dressing according to claim 2, characterized in that: The method for extracting silk fibroin in step 1 is as follows: degumming and dissolving silkworm cocoons, dialyzing them, and then reverse dialysis with polyethylene glycol to concentrate them to obtain a silk fibroin aqueous solution.

4. The method for preparing a gallium porphyrin / silk fibroin antibacterial dressing according to claim 3, characterized in that: The specific steps of silk fibroin extraction are as follows: degumming silkworm cocoons into a Na2CO3 solution, heating at 65°C to dissolve, placing the resulting solution in a dialysis bag and dialyzing it with deionized water, and then reverse dialyzing it with a 10.0wt% polyethylene glycol (PEG) solution and allowing it to stand; centrifuging to remove undissolved residues, and concentrating to obtain a silk fibroin aqueous solution.

5. The method for preparing a gallium porphyrin / silk fibroin antibacterial dressing according to claim 2, characterized in that: The preparation process in step 2 is as follows: adding porphyrin small molecules and gallium chloride into anhydrous pyridine solvent, reacting under nitrogen protection and heating conditions, cooling to room temperature after the reaction to obtain a deep red solution, removing the solvent, collecting the precipitate; washing with washing solution, and drying.

6. The method for preparing a gallium porphyrin / silk fibroin antibacterial dressing according to claim 5, characterized in that: The porphyrin small molecule is tetra-p-methoxyphenyl porphyrin.

7. The method for preparing a gallium porphyrin / silk fibroin antibacterial dressing according to claim 2, characterized in that: In step 3, the gallium porphyrin complex and the silk fibroin are cross-linked by exposing them to ultraviolet light or by standing them at room temperature.

8. The method for preparing a gallium porphyrin / silk fibroin antibacterial dressing according to claim 2, characterized in that: In step 3, the amount of the gallium porphyrin complex added is 1 g to 6.5 g, and the mass volume ratio of the gallium porphyrin complex to the silk fibroin hydrogel solution is 3% to 18%.

9. The method for preparing a gallium porphyrin / silk fibroin antibacterial dressing according to claim 7, characterized in that: In step 3, the amount of the gallium porphyrin complex added is 2.5 g to 4 g, and the mass volume ratio of the gallium porphyrin complex to the silk fibroin hydrogel solution is 7% to 12%.

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