Antibacterial dressing compounded with extracellular trap and preparation method of antibacterial dressing

Abstract

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

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to an antibacterial dressing of composite extracellular traps and a preparation method thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Bacterial infection is one of the main reasons that affect wound healing. Mild infection delays wound healing, while severe infection can lead to complications such as sepsis. Although simply treating the wound with ordinary disinfection methods 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 make bacteria resistant, leading to an increase in the use of antibiotics.

[0004] Medical dressings refer to temporary protective coverings that are applied to wounds. When the skin is injured, choosing a suitable dressing to cover the wound can help control bleeding, prevent infection, and absorb secretions, thereby promoting rapid wound healing. Although commonly used dressings in clinical practice, such as chitosan-based dressings, collagen dressings, and silver ion dressings, have certain anti-inflammatory and antibacterial effects, they have defects such as non-waterproofness, tissue adhesion, drug resistance, and heavy metal poisoning. The antibacterial components on the dressings are mostly inorganic ions, organic matter, or antibiotics, which are easily unable to meet the needs of wound antibacterial, anti-inflammatory, and healing due to bacterial resistance, poor biocompatibility, and inherent cytotoxicity. Summary of the invention

[0005] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide an antibacterial dressing with a composite extracellular trap 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 object, the present invention is implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides an antimicrobial dressing with composite extracellular traps, comprising a dressing and extracellular traps loaded on the dressing.

[0008] Preferably, the extracellular traps are obtained by treating immune cells with mechanical stimulation using nano-piezoelectric particles.

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

[0010] Further preferably, the nano piezoelectric particles are prepared by depositing Au on BaTiO 3 Surface obtained.

[0011] Preferably, the dressing is a hydrogel. The hydrogel is formed by a water-soluble or hydrophilic polymer through certain chemical crosslinking or physical crosslinking, such as polysaccharides (cellulose, chitosan, alginic acid, 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] Further preferably, the hydrogel is formed by cross-linking alginate and a soluble calcium salt, 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 the antibacterial dressing of the composite extracellular trap as described in the first aspect, comprising the following steps:

[0014] The soluble calcium salt solution containing the extracellular traps is added dropwise into the alginate solution to obtain a reaction.

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

[0016] The macrophages are incubated with nano-piezoelectric particles, and after incubation, the unphagocytosed nano-piezoelectric particles are washed away, and the lysate is collected by ultrasonication, washing, freezing and thawing, and the whole cells and debris are removed.

[0017] Preferably, the concentration of nano-piezoelectric particles is 1-5 mg / ml; the ultrasonic power is 0.05-0.15 W / cm 2 10s, ultrasonic 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 soluble calcium salt solution containing extracellular traps is 18.5-37 mg / mL, and the concentration of the extracellular traps is 1×10 7 -2×10 7 mL -1 .

[0020] Preferably, the dropping and reaction are carried out on a shaker at 70-90 rpm, and the reaction time is 4-6 min.

[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 has biological activity, can actively capture and efficiently kill bacteria, and has good antibacterial properties.

[0024] 3. The antibacterial dressing of the present invention uses hydrogel as a matrix, has good water absorption capacity, can keep the wound moist, and can better absorb wound exudate, which is beneficial to the healing of the wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 This is a comparison chart of the results of in vitro antibacterial experiments of METs-SA of Example 1 and BMDM-SA of Comparative Example 1;

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

[0028] Figure 3 It is a comparison chart of the antibacterial properties of the SA hydrogel of Comparative Example 2, the BMDM-SA hydrogel of Comparative Example 1, the METs-SA hydrogel of Example 1, the freeze-dried BMDM-SA hydrogel of Comparative Example 1, and the freeze-dried METs-SA hydrogel of Example 1. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0030] Example 1

[0031] BT&Au preparation: 20 mg BaTiO 3 The nanoparticles were dispersed in 20 mL of anhydrous ethanol, and then 200 μL of MTS was added. After mixing, they were ultrasonically treated for 30 min, centrifuged at 6000 rpm for 10 min, washed with anhydrous ethanol three times, 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 HauCl was added. 4 solution and 10mL of 20% methanol solution, with 0.5MK 2 CO 3The solution was adjusted to pH 9.7 and subjected to ultrasonic treatment (40 kHz, 80 W) at 4° C. for 1 h. The product was centrifuged at 12,000 rpm for 10 min, washed three times with deionized water, and dried at 70° C. to obtain BT&Au.

[0032] Preparation of extracellular traps (METs) solution: Macrophages (BMDM) were seeded in CellAttach six-well plates, with 5×10 6 Cells were cultured normally until they adhered to the wall. 875 μl of 2 mg / ml BT&Au was added to each well. After incubation for 1 hour, the unphagocytosed BT&Au was washed away and ultrasound was applied: P = 0.1 W / cm 2 10s, incubate for 4h. Rinse with PBS, add 200μl PBS to each well, freeze and thaw repeatedly at -80℃ and 37℃ for 10 times, collect lysate, centrifuge at 4℃×300g for 5min, and remove whole cells and debris.

[0033] 3%wt% sodium alginate: 1.5g sodium alginate was added to 48.5ml deionized water in several portions, stirred at room temperature for 4h, stirred in a 50℃ hot water bath for 30min, and allowed to stand for 24h.

[0034] 3% wt% calcium gluconate-METs: 150 mg calcium gluconate monohydrate was added to 4.85 ml METs solution (about 5 × 10 7 METs), and stirred at room temperature for 1 h.

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

[0036] Comparative Example 1

[0037] Preparation of BMDM solution: BMDM were seeded in a CellAttach six-well plate, with 5×10 cells per well. 6 Cells were cultured normally until adhered to the wall for 4 hours. PBS was rinsed, 200 μl PBS was added to each well, and the cells were repeatedly frozen and thawed at -80℃ and 37℃ for 10 times. The lysate was collected and centrifuged at 4℃×300g for 5 minutes to remove the whole cells and debris.

[0038] 3%wt% sodium alginate: 1.5g sodium alginate was added to 48.5ml deionized water in several times, stirred at room temperature for 4h, stirred in 50℃ hot water for 30min, and allowed to stand for 24h.

[0039] 3% wt% calcium gluconate-BMDM: 150 mg calcium gluconate monohydrate was added to 4.85 ml BMDM solution (approximately 5 × 10 7 BMDM), and stirred at room temperature for 1 h.

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

[0041] Comparative Example 2

[0042] Preparation of SA hydrogel: 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. SA hydrogel was obtained after 5 min.

[0043] Example 2

[0044] S. aureus bacterial suspension was added to the SA hydrogel of Comparative Example 2, the BMDM-SA of Comparative Example 1, and the METs-SA hydrogel of Example 1, and cultured at 37°C for 12 hours. The suspension was collected and plated for counting. The number of bacteria and the number of macrophages were approximately 50:1. Figure 1 As shown, compared with Comparative Examples 1 and 2, the dressing METs-SA hydrogel prepared in Example 1 exhibited significant antibacterial effect.

[0045] Construction of wound infection model: 6-week-old C57 mice (weight 20 g, male) were shaved on the back and anesthetized with isoflurane inhalation. A hole was punched on the back of the mouse with an 8 mm hole puncher. 50 μl of 1×10 8 CFU / ml MRSA (ATCC33591) was dripped into the wound surface, and the 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 puncher, and cover the wound with 8×8mm BMDM-SA and METs-SA hydrogels. After 24 hours, observe the infected wound.

[0047] from Figure 2 It can be seen that compared with Comparative Example 1, the dressing METs-SA hydrogel 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, the freeze-dried BMDM-SA hydrogel, and the freeze-dried METs-SA hydrogel were placed in 1 ml 1×108 CFU / ml S.aureus, incubate at 37℃ for 1h, collect the bacterial solution, and count the number of CFU / ml S.aureus at the original concentration, 100-fold dilution, and 10,000-fold dilution.

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

[0051] Example 4

[0052] BT&Au preparation: 20 mg BaTiO 3 The nanoparticles were dispersed in 20 mL of anhydrous ethanol, and then 200 μL of MTS was added. After mixing, they were ultrasonically treated for 30 min, centrifuged at 6000 rpm for 10 min, washed with anhydrous ethanol three times, 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 HauCl was added. 4 solution and 10mL of 20% methanol solution, with 0.5MK 2 CO 3 The solution was adjusted to pH 9.7 and subjected to ultrasonic treatment (40 kHz, 80 W) at 4° C. for 1 h. The product was centrifuged at 12,000 rpm for 10 min, washed three times with deionized water, and dried at 70° C. to obtain BT&Au.

[0053] Preparation of extracellular traps (METs) solution: Macrophages (BMDM) were seeded in CellAttach six-well plates, with 5×10 6 Cells were cultured normally until they adhered to the wall. 875 μl of 2 mg / ml BT&Au was added to each well. After incubation for 1 hour, the unphagocytosed BT&Au was washed away and ultrasound was applied: P = 0.1 W / cm 2 10s, incubate for 4h. Rinse with PBS, add 200μl PBS to each well, freeze and thaw repeatedly at -80℃ and 37℃ for 10 times, collect lysate, centrifuge at 4℃×300g for 5min, and remove whole cells and debris.

[0054] 2%wt% sodium alginate: add 49ml deionized water to 1g sodium alginate in several times, stir at room temperature for 4h, stir in a 50℃ hot water bath for 30min, and let stand for 24h.

[0055] 2% wt% calcium gluconate-METs: 100 mg calcium gluconate monohydrate was added to 4.9 ml METs solution (about 5 × 10 7 METs), and stirred at room temperature for 1 h.

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

[0057] Example 5

[0058] BT&Au preparation: 20 mg BaTiO 3 The nanoparticles were dispersed in 20 mL of anhydrous ethanol, and then 200 μL of MTS was added. After mixing, they were ultrasonically treated for 30 min, centrifuged at 6000 rpm for 10 min, washed with anhydrous ethanol three times, 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 HauCl was added. 4 solution and 10mL of 20% methanol solution, with 0.5MK 2 CO 3 The solution was adjusted to pH 9.7 and subjected to ultrasonic treatment (40 kHz, 80 W) at 4° C. for 1 h. The product was centrifuged at 12,000 rpm for 10 min, washed three times with deionized water, and dried at 70° C. to obtain BT&Au.

[0059] Preparation of extracellular traps (METs) solution: Macrophages (BMDM) were seeded in CellAttach six-well plates, with 5×10 6 Cells were cultured normally until they adhered to the wall. 875 μl of 2 mg / ml BT&Au was added to each well. After incubation for 1 hour, the unphagocytosed BT&Au was washed away and ultrasound was applied: P = 0.1 W / cm 2 10s, incubate for 4h. Rinse with PBS, add 200μl PBS to each well, freeze and thaw repeatedly at -80℃ and 37℃ for 10 times, collect lysate, centrifuge at 4℃×300g for 5min, and remove whole cells and debris.

[0060] 4%wt% sodium alginate: 2g sodium alginate was added to 48ml deionized water in several portions, stirred at room temperature for 4h, stirred in a 50℃ hot water bath for 30min, and allowed to stand for 24h.

[0061] 4% wt% calcium gluconate-METs: 200 mg calcium gluconate monohydrate was added to 4.8 ml METs solution (about 5 × 10 7 METs), and stirred at room temperature for 1 h.

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

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antibacterial dressing with composite extracellular traps, It is characterized in that The invention comprises a dressing and extracellular traps loaded in the dressing.

2. The antibacterial dressing according to claim 1, It is characterized in that The extracellular traps are obtained by physically stimulating immune cells with nanomaterials.

3. The antibacterial dressing according to claim 2, It is characterized in that The immune cells include macrophages, the physical stimulation includes mechanical stimulation, preferably ultrasound; the nanomaterial is preferably a nano piezoelectric material.

4. The antibacterial dressing according to claim 1, It is characterized in that The dressing is a hydrogel; Preferably, the hydrogel is formed by cross-linking alginate and a soluble calcium salt, the alginate includes sodium alginate, and the soluble calcium salt includes calcium gluconate.

5. A method for preparing the antibacterial dressing of the composite extracellular traps as claimed in claims 1-4, It is characterized in that The following steps are involved: The soluble calcium salt solution containing the extracellular traps is added dropwise to the alginate solution to react, and the extracellular trap cells are derived from macrophages.

6. The preparation method according to claim 5, It is characterized in that Also included is a method for preparing extracellular traps: The macrophages are incubated with nano-piezoelectric particles, and after incubation, the unphagocytosed nano-piezoelectric particles are washed away, and the lysate is collected by ultrasonication, washing, freezing and thawing, and the whole cells and debris are removed.

7. The preparation method according to claim 6, It is characterized in that The concentration of nanopiezoelectric particles is 1-5 mg / ml; the ultrasonic power is 0.05-0.15 W / cm 2 10s, ultrasonic time is 3-5h.

8. The preparation method according to claim 5, It is characterized in that The concentration of the alginate solution was 18.5-37 mg / mL.

9. The preparation method according to claim 5, It is characterized in that The concentration of soluble calcium salt in the soluble calcium salt solution containing extracellular traps is 18.5-37 mg / mL, and the concentration of extracellular traps is 1×10 7 -2×10 7 mL -1 .

10. The preparation method according to claim 5, It is characterized in that Place the mixture on a shaker at 70-90 rpm for dropwise addition and reaction, with a reaction time of 4-6 min.

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