An antibacterial dressing of composite extracellular traps and a preparation method thereof

Abstract

The present application belongs to the technical field of biomedical materials, and particularly relates to a composite extracellular trap antibacterial dressing and a preparation method thereof. The composite extracellular trap antibacterial dressing comprises a hydrogel and an extracellular trap loaded in the hydrogel. The antibacterial dressing can actively capture and efficiently kill bacteria including drug-resistant bacteria, is a novel dressing biological sterilization mechanism, has good biocompatibility, and can accelerate wound healing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to an antibacterial dressing combined with extracellular traps and a preparation method thereof. BACKGROUND

[0002] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the present application and therefore it should not be taken as an acknowledgement or any form of suggestion that it forms prior art with respect to any country.

[0003] Bacterial infection is one of the main reasons affecting wound healing. A slight infection delays the healing of a wound, and a serious infection can lead to complications such as sepsis. Although a simple disinfection of the wound can temporarily weaken the activity of bacteria, it cannot completely eliminate the infection. Although the use of antibiotics can effectively inhibit and kill most bacteria, long-term use of antibiotics can cause bacteria to develop drug resistance, resulting in an increase in the amount of antibiotics used.

[0004] Medical dressings are temporary coverings placed on wounds that have a protective effect. When the skin is damaged, a suitable dressing is selected to cover the wound, which can help control bleeding, prevent infection, and absorb secretions, thereby promoting rapid wound healing. Commonly used dressings in clinical practice include chitosan-based dressings, collagen dressings, and silver ion dressings, which have certain anti-inflammatory and antibacterial effects, but have defects such as non-waterproofness, tissue adhesion, drug resistance, and heavy metal poisoning. The antibacterial components on the dressing are mostly inorganic ions, organic substances, or antibiotics, which can lead to the inability to meet the needs of wound antibacterial, anti-inflammatory, and healing due to bacterial drug resistance, poor biocompatibility, and inherent cytotoxicity. SUMMARY

[0005] In order to solve the problems of the prior art, the present application aims to provide an antibacterial dressing combined with extracellular traps and a preparation method thereof, which can actively capture and efficiently kill drug-resistant bacteria, has good biocompatibility, and can accelerate wound healing.

[0006] In order to achieve the above-mentioned purpose, the present application is realized by the following technical solutions:

[0007] In a first aspect, the present application provides an antibacterial dressing combined with extracellular traps, comprising a dressing and extracellular traps loaded on the dressing.

[0008] Preferably, the extracellular traps are obtained by mechanically stimulating immune cells with nano piezoelectric particles.

[0009] Further preferably, the immune cells include macrophages, and the mechanical stimulation includes ultrasound.

[0010] More preferably, the nanopiezoelectric particles are obtained by depositing Au on the surface of BaTiO3.

[0011] Preferably, the dressing is a hydrogel. Hydrogels are formed from water-soluble or hydrophilic polymers through certain chemical or physical cross-linking processes. Examples include polysaccharides (cellulose, chitosan, alginate, etc.), polypeptides (poly-L-lysine, collagen, poly-L-glutamic acid, etc.), and acrylic acid or its derivatives (polyacrylamide, polymethacrylic acid, poly-N-polyacrylamide, etc.).

[0012] More preferably, the hydrogel is formed by crosslinking alginate and soluble calcium salt, wherein the alginate includes sodium alginate and the soluble calcium salt includes calcium gluconate.

[0013] In a second aspect, the present invention provides a method for preparing an antibacterial dressing with a composite extracellular trap as described in the first aspect, comprising the following steps:

[0014] The solution of soluble calcium salt containing extracellular traps is added dropwise to an alginate solution, and the reaction is completed.

[0015] Preferably, the method for preparing the extracellular trap also includes:

[0016] Macrophages were incubated with nanopiezoelectric particles. After incubation, unphagocytosed nanopiezoelectric particles were washed away. The mixture was then sonicated, rinsed, and the lysate was collected by freeze-thaw. Whole cells and debris were removed to obtain the final product.

[0017] Further preferably, the concentration of the nanopiezoelectric particles is 1-5 mg / ml; the ultrasonic power is 0.05-0.15 W / cm². 2 10s, ultrasound time is 3-5h.

[0018] Preferably, the concentration of the alginate solution is 18.5-37 mg / mL.

[0019] Preferably, the concentration of the soluble calcium salt in the solution containing the extracellular trap is 18.5-37 mg / mL, and the concentration of the extracellular trap is 1×10⁻⁶ mg / mL. 7 -2×10 7 mL -1 .

[0020] Preferably, the mixture is added dropwise and reacted on a shaker at 70-90 rpm for 4-6 minutes.

[0021] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0022] 1. The antibacterial dressing of the present invention has good biocompatibility, is non-toxic, safe, and will not cause bacterial resistance.

[0023] 2. The antibacterial dressing of the present invention is bioactive, capable of actively capturing and efficiently killing bacteria, and has good antibacterial properties.

[0024] 3. The antibacterial dressing of the present invention uses hydrogel as a matrix, which has good water absorption capacity, can keep the wound moist, and can also absorb wound exudate well, which is conducive to wound healing. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 A comparison of the results of in vitro antibacterial experiments on METs-SA of Example 1 and BMDM-SA of Comparative Example 1;

[0027] Figure 2 This is a comparison chart showing the healing results of METs-SA in Example 1 and BMDM-SA in Comparative Example 1 on infected wounds in rats.

[0028] Figure 3 This is a comparison chart showing the antibacterial properties of SA hydrogel (Comparative Example 2), BMDM-SA hydrogel (Comparative Example 1), METs-SA hydrogel (Example 1), and freeze-dried BMDM-SA hydrogel (Comparative Example 1) and METs-SA hydrogel (Example 1). Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0030] Example 1

[0031] BT&Au preparation: 20 mg BaTiO3 nanoparticles were dispersed in 20 mL of anhydrous ethanol, then 200 μL of MTS was added, mixed well, and ultrasonicated for 30 min. The mixture was then centrifuged at 6000 rpm for 10 min, washed three times with anhydrous ethanol, and dried at 70 °C to obtain MTS-BTO. 10 mg of MTS-BTO was added to 10 mL of deionized water, dispersed evenly, and then 8.35 mL of 24 mM HauCl4 solution and 10 mL of 20% methanol solution were added. The pH was adjusted to 9.7 with 0.5 M K2CO3 solution, and ultrasonicated (40 kHz, 80 W) for 1 h at 4 °C. The product was centrifuged at 12000 rpm for 10 min, washed three times with deionized water, and dried at 70 °C to obtain BT&Au.

[0032] Preparation of extracellular trap (METs) solution: Macrophages (BMDM) were seeded into CellAttach six-well plates, 5 × 10⁶ cells per well. 6 Cells were cultured normally until adherence. 875 μl of 2 mg / ml BT & Au was added to each well, and after incubation for 1 hour, unphagocytosed BT & Au was washed away, followed by sonication: P = 0.1 W / cm². 2 Incubate for 4 hours after 10 seconds. Rinse thoroughly with PBS, add 200 μl of PBS to each well, freeze and thaw at -80°C and 37°C 10 times, collect the lysate, centrifuge at 300g for 5 minutes at 4°C to remove whole cells and debris.

[0033] 3% wt% Sodium alginate: Add 1.5g sodium alginate to 48.5ml deionized water in several portions, stir at room temperature for 4 hours, stir in a 50℃ hot water bath for 30 minutes, and let stand for 24 hours.

[0034] 3% wt% Calcium gluconate-METs: 150 mg calcium gluconate monohydrate added to 4.85 ml METs solution (approximately 5 × 10⁻⁶ mg / mL). 7 METs), stir at room temperature for 1 hour.

[0035] Preparation of METs-SA hydrogel: 1 ml of 3% wt% sodium alginate was added to a six-well plate and placed on a shaker (80 rpm). 300 μl of 3% wt% calcium gluconate was added dropwise, and METs-SA hydrogel was obtained after 5 min.

[0036] Comparative Example 1

[0037] Preparation of BMDM solution: BMDM was seeded into CellAttach six-well plates, 5 × 10⁶ cells per well. 6 Cells were cultured normally until adherence, and cultured for 4 hours. After rinsing with PBS, 200 μl of PBS was added to each well. The cells were subjected to a freeze-thaw cycle of -80°C and 37°C 10 times. The lysate was collected and centrifuged at 4°C × 300g for 5 min to remove whole cells and debris.

[0038] 3% wt% Sodium alginate: Add 1.5g sodium alginate to 48.5ml deionized water in several portions, stir at room temperature for 4 hours, stir in 50℃ hot water for 30 minutes, and let stand for 24 hours.

[0039] 3% wt% Calcium Gluconate-BMDM: 150 mg calcium gluconate monohydrate is added to 4.85 ml of BMDM solution (approximately 5 × 10⁻⁶ ml). 7 BMDM), stirred at room temperature for 1 hour.

[0040] Preparation of BMDM-SA hydrogel: 1 ml of 3% wt% sodium alginate was added to a six-well plate and placed on a shaker (80 rpm). 300 μl of 3% wt% calcium gluconate-BMDM was added dropwise. After 5 min, BMDM-SA hydrogel was obtained.

[0041] Comparative Example 2

[0042] SA hydrogel preparation: 1 ml of 3% wt / wt sodium alginate was added to a six-well plate, placed on a shaker (80 rpm), and 300 μl of deionized water was added dropwise. After 5 min, SA hydrogel was obtained.

[0043] Example 2

[0044] S. aureus bacterial suspension was added to SA hydrogel (Comparative Example 2), BMDM-SA (Comparative Example 1), and METs-SA hydrogel (Example 1), and incubated at 37°C for 12 h. The suspensions were collected and plated for counting. The bacterial count to macrophage count was approximately 50:1. Figure 1 As shown, compared with Comparative Examples 1 and 2, the METs-SA hydrogel prepared in Example 1 exhibits a significant antibacterial effect.

[0045] Establishment of a wound infection model: Six-week-old male C57 mice (20g) were shaved on their backs, anesthetized with isoflurane inhalation, and punctured in their backs with an 8mm punch. 50μl of 1×10⁻⁶ mol / L wound infection solution was administered. 8 CFU / ml MRSA (ATCC33591) was infused into the wound, and a wound infection model was established after two consecutive days of infection.

[0046] Treatment of infected wounds: Trim the BMDM-SA and METs-SA hydrogels with an 8mm punch, and cover the wound with 8×8mm BMDM-SA and METs-SA hydrogels. Observe the condition of the infected wound after 24 hours.

[0047] from Figure 2 As can be seen, compared with Comparative Example 1, the METs-SA hydrogel dressing prepared in Example 1 exhibits significant anti-infection and wound healing effects.

[0048] Example 3

[0049] The BMDM-SA hydrogel of Comparative Example 1 and the METs-SA hydrogel obtained in Example 1 were freeze-dried to obtain freeze-dried BMDM-SA hydrogel and METs-SA hydrogel. The SA hydrogel of Comparative Example 2, the BMDM-SA hydrogel of Comparative Example 1, the METs-SA hydrogel of Example 1, and the freeze-dried BMDM-SA hydrogel and METs-SA hydrogel were placed in 1 ml of 1×10... 8CFU / ml S. aureus, 37℃ for 1h, collect bacterial suspension, and perform plate counting using the original concentration, 100-fold dilution, and 10000-fold dilution respectively.

[0050] from Figure 3 It can be seen that the freeze-dried METs-SA dressing still has good antibacterial effect, and the antibacterial performance of METs-SA is not affected by moisture evaporation during use.

[0051] Example 4

[0052] BT&Au preparation: 20 mg BaTiO3 nanoparticles were dispersed in 20 mL of anhydrous ethanol, then 200 μL of MTS was added, mixed well, and ultrasonicated for 30 min. The mixture was then centrifuged at 6000 rpm for 10 min, washed three times with anhydrous ethanol, and dried at 70 °C to obtain MTS-BTO. 10 mg of MTS-BTO was added to 10 mL of deionized water, dispersed evenly, and then 8.35 mL of 24 mM HauCl4 solution and 10 mL of 20% methanol solution were added. The pH was adjusted to 9.7 with 0.5 M K2CO3 solution, and ultrasonicated (40 kHz, 80 W) for 1 h at 4 °C. The product was centrifuged at 12000 rpm for 10 min, washed three times with deionized water, and dried at 70 °C to obtain BT&Au.

[0053] Preparation of extracellular trap (METs) solution: Macrophages (BMDM) were seeded into CellAttach six-well plates, 5 × 10⁶ cells per well. 6 Cells were cultured normally until adherence. 875 μl of 2 mg / ml BT & Au was added to each well, and after incubation for 1 hour, unphagocytosed BT & Au was washed away, followed by sonication: P = 0.1 W / cm². 2 Incubate for 4 hours after 10 seconds. Rinse thoroughly with PBS, add 200 μl of PBS to each well, freeze and thaw at -80°C and 37°C 10 times, collect the lysate, centrifuge at 300g for 5 minutes at 4°C to remove whole cells and debris.

[0054] 2% wt% Sodium alginate: Add 1g of sodium alginate to 49ml of deionized water in several portions, stir at room temperature for 4 hours, stir in a 50℃ hot water bath for 30 minutes, and let stand for 24 hours.

[0055] 2% wt% Calcium gluconate-METs: 100 mg calcium gluconate monohydrate added to 4.9 ml METs solution (approximately 5 × 10⁻⁶ mg / mL). 7 METs), stir at room temperature for 1 hour.

[0056] Preparation of METs-SA hydrogel: 1 ml of 2% wt% sodium alginate was added to a six-well plate and placed on a shaker (80 rpm). 300 μl of 2% wt% calcium gluconate was added dropwise, and METs-SA hydrogel was obtained after 5 min.

[0057] Example 5

[0058] BT&Au preparation: 20 mg BaTiO3 nanoparticles were dispersed in 20 mL of anhydrous ethanol, then 200 μL of MTS was added, mixed well, and ultrasonicated for 30 min. The mixture was then centrifuged at 6000 rpm for 10 min, washed three times with anhydrous ethanol, and dried at 70 °C to obtain MTS-BTO. 10 mg of MTS-BTO was added to 10 mL of deionized water, dispersed evenly, and then 8.35 mL of 24 mM HauCl4 solution and 10 mL of 20% methanol solution were added. The pH was adjusted to 9.7 with 0.5 M K2CO3 solution, and ultrasonicated (40 kHz, 80 W) for 1 h at 4 °C. The product was centrifuged at 12000 rpm for 10 min, washed three times with deionized water, and dried at 70 °C to obtain BT&Au.

[0059] Preparation of extracellular trap (METs) solution: Macrophages (BMDM) were seeded into CellAttach six-well plates, 5 × 10⁶ cells per well. 6 Cells were cultured normally until adherence. 875 μl of 2 mg / ml BT & Au was added to each well, and after incubation for 1 hour, unphagocytosed BT & Au was washed away, followed by sonication: P = 0.1 W / cm². 2 Incubate for 4 hours after 10 seconds. Rinse thoroughly with PBS, add 200 μl of PBS to each well, freeze and thaw at -80°C and 37°C 10 times, collect the lysate, centrifuge at 300g for 5 minutes at 4°C to remove whole cells and debris.

[0060] 4% wt% Sodium alginate: Add 2g of sodium alginate to 48ml of deionized water in several portions, stir at room temperature for 4 hours, stir in a 50℃ hot water bath for 30 minutes, and let stand for 24 hours.

[0061] 4% wt% Calcium gluconate-METs: 200 mg calcium gluconate monohydrate added to 4.8 ml METs solution (approximately 5 × 10⁻⁶ mg / mL). 7 METs), stir at room temperature for 1 hour.

[0062] Preparation of METs-SA hydrogel: 1 ml of 4% wt% sodium alginate was added to a six-well plate and placed on a shaker (80 rpm). 300 μl of 4% wt% calcium gluconate was added dropwise, and the METs-SA hydrogel was obtained after 5 min.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an antibacterial dressing with a composite extracellular trap, characterized in that, Includes dressings and extracellular traps loaded in the dressings; The method for preparing the antibacterial dressing with the composite extracellular trap includes the following steps: A soluble calcium salt solution containing extracellular traps is added dropwise to an alginate solution, and the reaction yields the extracellular trap cells, which are derived from macrophages. The concentration of soluble calcium salts in solutions containing extracellular traps ranged from 18.5 to 37 mg / mL, and the concentration of extracellular traps was 1 × 10⁻⁶. 7 -2×10 7 mL -1 ; The method for preparing the extracellular trap: Macrophages were incubated with nanopiezoelectric particles. After incubation, unphagocytosed nanopiezoelectric particles were washed away. The mixture was then sonicated, rinsed, and the lysate was collected by freeze-thaw. Whole cells and debris were removed to obtain the final product. The concentration of the nanopiezoelectric particles was 1-5 mg / ml; the ultrasonic power was 0.05-0.15 W / cm². 2 10s; the dressing is a hydrogel; the hydrogel is formed by cross-linking of alginate and soluble calcium salt.

2. The preparation method according to claim 1, characterized in that, The alginate includes sodium alginate, and the soluble calcium salt includes calcium gluconate.

3. The preparation method according to claim 1, characterized in that, The concentration of the alginate solution is 18.5-37 mg / mL.

4. The preparation method according to claim 1, characterized in that, The mixture was added dropwise and reacted on a shaker at 70-90 rpm for 4-6 minutes.

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