An antibacterial polymer f127acbr with terminal modification of acetyl bromide, a preparation method and application thereof

Abstract

This application belongs to the field of biomaterials, specifically relating to an antibacterial polymeric derivative of F127 with terminal modification of acetyl bromide, its preparation method, and its application. This application obtains F127AcBr by terminal modification of polymeric F127 with acetyl bromide via an acylation reaction. Its good antibacterial effect and biocompatibility were verified in vitro using plate antibacterial assays against Staphylococcus aureus and Escherichia coli, as well as live / dead staining assays of L929 cells and HUVEC cells. This application optimized the concentration of F127AcBr and loaded it into an MS hydrogel system. Excellent biocompatibility was confirmed in a diabetic mouse wound model using HE staining of mouse organs. Immunofluorescence staining against Staphylococcus aureus further confirmed the good antibacterial activity of F127AcBr.

Description

Technical Field

[0001] This application belongs to the field of biomaterials, specifically relating to an antibacterial polymer F127AcBr with terminal modified acetyl bromide, its preparation method and application. Background Technology

[0002] In diabetic foot wounds, infection is the leading cause of poor healing, deterioration, and even amputation. The harsh microenvironment of high glucose, hypoxia, and necrotic tissue accumulation greatly promotes bacterial proliferation, causing the wound to remain in the inflammatory phase for a prolonged period and hindering granulation cell growth. Studies have shown that once diabetic foot becomes infected, the risk of amputation increases significantly, and treatment costs multiply. Using novel antibacterial agents to treat diabetic wound infections can effectively alleviate the inhibitory effect of persistent inflammation on tissue repair. Unlike the mechanism of action of traditional antibiotics, novel antibacterial agents can effectively avoid the development of drug-resistant bacteria and are widely used in antibacterial therapy research. Among these bactericides, quaternary ammonium salts have been widely used in bacterial infection treatment research, but they have problems such as environmental pollution and the development of drug-resistant bacteria. While photosensitizers can further improve the bactericidal effect, they require light exposure and are difficult to use for treating deep tissue infections.

[0003] Polymer F127 (Planick F127) is an amphiphilic block copolymer composed of polyoxyethylene (PEO) and polypropylene (PPO) in a specific ratio. It has high biocompatibility and is often used in the biomedical field to construct thermosensitive hydrogel drug delivery systems. As a drug carrier raw material, it does not have antibacterial effects itself. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this application provides a polymer F127 with terminally modified acetyl bromide, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution of this application is as follows: a polymer F127 with terminal modified acetyl bromide, which is synthesized by acylation reaction of F127 with bromoacetyl bromide.

[0006] Preferably, the number-average molecular weight of F127 is not less than 10,000.

[0007] The preparation method of the end-modified acetyl bromide polymer F127 as described above includes the following steps: dissolving F127 in a mixed solution containing dichloromethane and triethylamine, adding dichloromethane containing dissolved acetyl bromide dropwise under an ice bath, and then reacting at 20-30°C for 20-28 hours.

[0008] Preferably, after the reaction is complete, the organic phase is washed with saturated sodium chloride solution, dried, filtered to remove the desiccant, concentrated, precipitated with cold petroleum ether and vacuum dried to obtain a crude product, dissolved in pure water, dialyzed, and the terminal-modified acetyl bromide polymer F127 is obtained.

[0009] Preferably, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa.

[0010] The application of the terminally modified acetyl bromide polymer F127 as described above in the preparation of antibacterial agents.

[0011] An antibacterial hydrogel system comprising the end-modified acetyl bromide polymer F127 as described above and a hydrogel carrier.

[0012] Preferably, the hydrogel carrier is a hydrogel system formed by click chemistry of maleimide-modified gelatin and thiol-modified F127.

[0013] Preferably, the terminally modified acetyl bromide polymer F127 has a loading rate of 0-1% in the system.

[0014] Preferably, the terminally modified acetyl bromide polymer F127 is loaded at a rate of 0.5% in the system.

[0015] The antibacterial hydrogel system described above is used in the preparation of drugs for treating bacterial infections of wounds.

[0016] The beneficial effects of this application are as follows: By terminally modifying the biocompatible polymer F127 with acetyl bromide (F127AcBr), a novel material with good antibacterial properties and biodegradability is synthesized. This material can effectively inhibit the growth of Staphylococcus aureus and Escherichia coli while exhibiting good biocompatibility. In a diabetic mouse wound model, loading F127AcBr into an MS hydrogel system (a hydrogel system formed by click chemistry of maleimide-modified gelatin and thiol-modified F127) showed good biocompatibility in HE staining of mouse heart, liver, spleen, lungs, and kidneys, and fluorescent staining with anti-Staphylococcus aureus antibody showed good antibacterial effect. Therefore, this novel antibacterial agent F127AcBr has good application potential. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0018] Figure 1 The synthetic route for F127AcBr is shown below. Figure 2 The 1H NMR spectrum of F127 and F127 modified with acetyl bromide (F127AcBr); Figure 3 To demonstrate the antibacterial effect of different concentrations of F127AcBr on Escherichia coli in a plate experiment; Figure 4 A quantitative graph showing the antibacterial effect of F127AcBr; Figure 5 Fluorescence imaging of live / dead staining after L929 cells were co-cultured with different concentrations of F127AcBr in a composite hydrogel system via Transwell was performed. Figure 6 F127AcBr was loaded into a composite hydrogel system, and fluorescence imaging of live / dead staining was performed after co-culturing with L929 cells and HUVEC cells via Transwell. Figure 7 The antibacterial effect of F127AcBr loaded on the composite hydrogel system against Escherichia coli and Staphylococcus aureus was demonstrated in a plate experiment. Figure 8 In a diabetic mouse wound model, paraffin sections of various organs of mice were stained with hematoxylin and eosin (HE) after different groups of hydrogel treatment. Figure 9 These are representative images of immunofluorescence staining of anti-Staphylococcus aureus antibodies on frozen skin sections after different groups of hydrogel treatment in a diabetic mouse wound model. Detailed Implementation

[0019] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application. The terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0021] Example 1: Preparation and Characterization of F127AcBr 10 g F127 (M n= 12.6 kDa) was dissolved in a mixed solution containing dichloromethane (80 mL) and triethylamine (3.3 mL), and preheated in an ice bath for 30 min. Under ice bath conditions, 10 mL of dichloromethane containing 2.052 mL of bromoacetyl bromide was added dropwise to the mixed solution. The reaction system was then reacted in an oil bath at 25 °C for 24 h. After the reaction was completed, the mixture was washed three times with saturated sodium chloride solution (90 mL). The organic phase was collected and dried with anhydrous magnesium sulfate for 30 min. After removing the magnesium sulfate by filtration, the resulting organic phase was rotary evaporated. The product was then precipitated with cold petroleum ether and dried under vacuum to obtain the crude product. The product was then dissolved in 300 mL of pure water at 4 °C and dialyzed for 3-4 days (MW cutoff 3.5 kDa), with the water changed 2-3 times a day. Finally, the dialysate was freeze-dried to obtain F127-AcBr, which was stored at -20 °C.

[0022] Synthetic routes such as Figure 1 As shown.

[0023] The results of the 1H NMR spectrum test are shown below. Figure 2 Acetyl bromide was successfully modified, and the modification rate of acetyl bromide was calculated to be 72%.

[0024] Example 2: Exploration of the application concentration of F127AcBr (1) Antibacterial effect and concentration exploration: The in vitro antibacterial activity of F127AcBr was evaluated using the plate coating method with Escherichia coli. Different concentrations of F127AcBr (0, 0.01%, 0.05%, 0.1%, 0.5%, 1%) were mixed with bacterial suspension (≈1×10⁻⁶). 5 CFU mL -1 The culture was incubated for 4 hours. Then, the bacterial suspension was diluted with PBS, and 200 μL of the diluted solution was evenly spread onto a solid LB agar plate using a spreader. The plate was then incubated upside down at 37 °C for 24 hours. The resulting colonies were photographed and counted. The antibacterial efficacy of 0.5% was 95%. (See attached table). Figure 3 .

[0025] Quantifying the results yields Figure 4 Bacterial survival rates at different concentrations. It was found that with only 0.01% F127AcBr added, the bacterial survival rate reached nearly 60%, showing a significant dose-dependent decrease with increasing concentration. When the concentration increased to 0.5%, the survival rate dropped to ≤5%, achieving a significant antibacterial effect. Further increasing to 1%, the bacterial survival rate essentially approached 0%, indicating that the material possesses complete bactericidal ability at high concentrations.

[0026] Example 3: Preparation of F127AcBr composite hydrogel: Prepare a 20% (w / v) Gelatin-MAL (M) solution using PBS solution, heat at 37 ℃ to dissolve evenly, and denote this as solution M; F127-AcBr (F) solutions of different concentrations were prepared using PBS solution and dissolved evenly in an ice bath at 4 ℃. These solutions were denoted as F solutions. Prepare an 8% (w / v) F127-SH (S) solution using PBS solution, dissolve it evenly in an ice bath at 4 ℃, and denote it as solution S; Mix M solution and S solution according to a 1:1 ratio, stir well, and the mixed solution will quickly form a gel in a short time to obtain MS sterile hydrogel; Mix the MF solution according to the 1:1 ratio, then mix the mixed solution with the S solution and stir until homogeneous. The mixed solution will quickly form a gel in a short time to obtain MSF antibacterial hydrogel (F127-AcBr final concentration is 0.1%, 0.5%, 1%).

[0027] Example 4: Biocompatibility of F127AcBr Composite Hydrogel (1) L929 cells were fed at a rate of 2 × 10⁻⁶ 5 Cells were seeded at a density of 10 cells / well into 24-well plates, with 3 replicates per group to improve experimental reliability. After 24 h, once cells adhered, the existing culture medium was discarded, replaced with basal medium, and the cells were placed in Tanswell chambers containing pre-prepared MSF sterile hydrogels containing different concentrations of F127AcBr (0, 0.1%, 0.5%, 1%) for another 24 h of incubation. After incubation, the chambers and culture medium were discarded, and the cells were gently rinsed once with PBS. Following the kit instructions, appropriate concentrations of Calcein-AM and PI working solutions were prepared, and 0.25 mL of diluted working solution was added to each well, ensuring uniform dye coverage of the cells. The cells were incubated at 37°C for 30 min in the dark. After incubation, the cells were observed under a fluorescence microscope. Results are shown below. Figure 5 The 0.1% and 0.5% F12AcBr loadings showed good biocompatibility, but at high concentrations (1%), cytotoxicity increased significantly.

[0028] (2) L929 cells and HUVEC cells were respectively fed with 2×10 5Cells were seeded at a density of 10 cells / well into 24-well plates, with 3 replicates per group to improve experimental reliability. After 24 h, once cells adhered, the existing culture medium was discarded, replaced with basal medium, and the cells were placed in pre-prepared sterile hydrogels (MS, MSF) in Tanswell chambers for another 24 h of incubation. After incubation, the chambers and culture medium were discarded, and the cells were gently rinsed once with PBS. Following the kit instructions, appropriate concentrations of Calcein-AM and PI working solutions were prepared, and 0.25 mL of diluted working solution was added to each well, ensuring uniform dye coverage. Cells were incubated at 37°C for 30 min in the dark. After incubation, the cells were observed under a fluorescence microscope. Results are shown below. Figure 6 All groups exhibited good biocompatibility.

[0029] Example 5: In vitro antibacterial properties of F127AcBr composite hydrogel The in vitro antibacterial activity of MSF hydrogel loaded with 0.5% F127AcBr was evaluated using the plate coating method against *Escherichia coli* and *Staphylococcus aureus*. MS and MSF sterile hydrogels were coated with 1 mL of bacterial suspension (≈1×10⁻⁶). 5 CFU mL -1 Co-incubate. Then dilute the bacterial suspension with PBS, and spread 200 μL of the dilution evenly onto solid LB agar plates using a plate-spreading glass bead. Incubate upside down in a 37 ℃ bacterial incubator for 24 h, then photograph and count the resulting colonies. Results are shown below. Figure 7 MSF hydrogel has good antibacterial effects against Escherichia coli and Staphylococcus aureus.

[0030] Example 6: Evaluation of the biocompatibility and antibacterial effect of F127AcBr in an MS hydrogel system in vivo. Forty-five male Balb / c mice, weighing 20-22 grams, were used in this study. After one week of acclimatization in an SPF-grade animal facility, animal experiments began. A diabetic mouse model was established by intraperitoneal injection of 50 mg / kg streptozotocin (STZ). After successful modeling, a 0.8 cm diameter wound was created on the skin surface. The wound was treated according to the following groups: control group, MS hydrogel group, and MSF hydrogel group. On days 3, 7, and 14 postoperatively, the mice were euthanized by intraperitoneal injection of an excessive amount of chloroform. Square full-thickness skin, subcutaneous tissue, and the heart, liver, spleen, lungs, and kidneys of the mice were harvested, cleaned with physiological saline, and fixed with 4% paraformaldehyde for 48 hours.

[0031] Fixed mouse organs were dehydrated using a gradient of ethanol (30%, 50%, 70%, 80%, 95%, and 100%). The dehydrated organs were then immersed in xylene solution for 30 min and paraffin for 2 h. The dehydrated tissue embedding cassettes were embedded with tissue, and then cooled. Before sectioning skin tissue, paraffin blocks were frozen at -20°C for 30 min, then fixed on a microtome, with the water bath set to 40°C. Tissue sections were lifted from the microtome with a brush. The excised skin tissue was spread in water and placed on a glass slide. The slides were dewaxed in a 70°C oven for 30 min and then in an 85°C oven for 15 min. Cover slips were then dewaxed in xylene solution for 5 min, repeated twice. Soak the coverslip sequentially in anhydrous ethanol, 95% ethanol, and 75% ethanol for 2 min each, then in tap water and deionized water for 2 min each. Stain the coverslip with hematoxylin solution for 5-10 min, differentiate with hydrochloric acid-alcohol solution for 15 s, rinse with tap water, stain with eosin solution for 5-10 min, immerse the coverslip in saturated lithium carbonate for differentiation for 5 s, rinse repeatedly with anhydrous ethanol, and then immerse the coverslip in xylene solution. Gently wipe away any residual reagents around the tissue on the coverslip with a paper towel. Apply a drop of neutral resin glue to the slide and cover with the coverslip. Air dry in a ventilated area for 72 h, then observe and photograph the slide using a slide scanner. Results are shown below. Figure 8 The organs and tissues of the mice in each group were intact without abnormal proliferation or necrosis, and there were no significant differences between the groups, further demonstrating that it has good biocompatibility.

[0032] After fixation, the skin was dried with paper towels, dehydrated with 15% sucrose for 1 day, then with 30% sucrose for 1 day, and stored at 4°C. After dehydration, the skin was thoroughly dried with paper towels, embedded with OCT, taking care to prevent the tissue from sinking to the bottom and to avoid air bubbles, and incubated at -140°C overnight. Then, sections were prepared using a cryostat and stored at -80°C. For staining, remove frozen sections stored at -80℃, air-dry at room temperature for 15 minutes, then soak in PBS for 10 minutes to remove OCT. Use a histochemical pen to circle the tissue to be stained, fix with 4% paraformaldehyde for 30 minutes, wash three times with PBS for 5 minutes each time, then incubate with 0.5 mL of 0.1% Triton X-100 for 10 minutes, followed by three washes with PBS for 5 minutes each time. Block the sections with PBS containing 10% normal goat serum at room temperature for 1 hour, then discard the liquid. Add diluted anti-Staphylococcus aureus antibody to the sections, then incubate overnight at 4℃ in a humidified chamber. The next day, wash the sections three times with PBS for 5 minutes each time. Add the corresponding secondary antibody to the tissue, incubate in the dark at room temperature for 2 hours or at 37℃ for 1 hour. After incubation, wash three times with PBS for 5 minutes each time, then stain the cell nuclei with DAPI-containing mounting medium and mount the slide. Be careful to avoid air bubbles during mounting; a layer of nail polish can be applied around the coverslip for reinforcement. After drying, observe and photograph the samples under a confocal or fluorescence microscope. Results are shown below. Figure 9 The MSF group showed the weakest fluorescence intensity and the fewest bacteria compared to the control group and the MS group, indicating that F127AcBr also has a good antibacterial effect in vivo.

[0033] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A polymer F127 with terminally modified acetyl bromide, characterized in that, It was synthesized by the acylation reaction of F127 with bromoacetyl bromide.

2. The method for preparing the polymer F127 with terminal-modified acetyl bromide as described in claim 1, characterized in that, Includes the following steps: F127 was dissolved in a mixed solution containing dichloromethane and triethylamine. Dichloromethane containing bromoacetyl bromide was added dropwise under ice bath conditions, and the mixture was then reacted at 20-30°C for 20-28 hours.

3. The method for preparing the terminally modified acetyl bromide polymer F127 according to claim 2, characterized in that, After the reaction was completed, the organic phase was washed with saturated sodium chloride solution, collected, dried, filtered to remove the desiccant, concentrated, precipitated with cold petroleum ether and dried under vacuum to obtain the crude product, dissolved in pure water and dialyzed to obtain the end-modified acetyl bromide polymer F127.

4. The method for preparing the terminally modified acetyl bromide polymer F127 according to claim 3, characterized in that, The dialysis uses dialysis bags with a molecular weight cutoff of 3.5 kDa, among others.

5. The application of the terminal-modified acetyl bromide polymer F127 as described in claim 1 in the preparation of antibacterial agents.

6. An antibacterial hydrogel system, characterized in that, It includes the end-modified acetyl bromide polymer F127 and hydrogel carrier as described in claim 1.

7. The antibacterial hydrogel system according to claim 6, characterized in that, The hydrogel carrier is a hydrogel system formed by click chemistry of maleimide-modified gelatin and thiol-modified F127.

8. The antibacterial hydrogel system according to claim 7, characterized in that, The terminally modified acetyl bromide polymer F127 as described in claim 1 has a loading rate of 0-1% in the system.

9. The antibacterial hydrogel system according to claim 7, characterized in that, The terminally modified acetyl bromide polymer F127 as described in claim 1 was loaded at a rate of 0.5% in the system.

10. The use of the antibacterial hydrogel system according to any one of claims 5-9 in the preparation of a medicament for treating bacterial infections of wounds.

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