은 나노 입자가 포함된 복합 재료의 산화 그래핀은 항균 나노 플랫폼의 섬유모세포 및 내피 세포 세포 독성을 감소시킵니다.

자료 및 방법

나노물질로 코팅된 나노플랫폼의 준비 및 특성화

나노입자로 코팅된 폴리우레탄 호일로 만든 나노플랫폼은 이전에 설명한 대로 준비되었습니다[25]. 사각형 모양의 폴리우레탄 호일(15 × 15 mm, 0.05 mm 두께)은 Amepox 및/또는 GO 합성에 의해 개발된 폴리비닐 알코올의 존재 하에 화학적 환원 반응에 의해 합성된 AgNP(HydroSilver1000, Amepox, Łódź, Poland)의 현탁액으로 덮였습니다. Hummers의 방법을 수정했습니다. 흑연 분말 10g을 10°C 미만의 온도에서 진한 황산(98%)(Sigma-Aldrich Co., St. Louis, MO, USA) 230㎖와 혼합하였다. 이어서, 4.7g의 질산나트륨(Sigma-Aldrich) 및 30 g의 과망간산칼륨(Sigma-Aldrich)을 온도를 10°C 미만으로 유지하면서 흑연 혼합물에 첨가하였다. 그 다음, 혼합물을 30℃로 가열하고 2시간 동안 교반하였다. 이어서, 물 100ml를 첨가하고 혼합물을 과산화수소 10ml로 처리하였다. GO를 여과에 의해 정제하고 여과액의 pH가 6.5에 도달할 때까지 탈이온수로 세척하였다. GO, AgNPs 및 AgNPs와 GO의 합성물(Ag-GO) 현탁액은 탈이온수에서 준비되었습니다. 코팅하는 동안 나노물질의 농도는 다음과 같았다:GO, 200 mg/l; AgNPs, 100 mg/l; Ag-GO, 200 mg/l; 및 AgNPs, 100 mg/l. 30 ± 1 °C의 온도에서 초음파 혼(Ti 혼, Ø13 mm, 60% 효율, 20 kHz; Sonics &Materials, Inc., Newtown, CT, USA)을 사용하여 나노입자 코팅을 수행했습니다. 덮인 샘플을 탈이온수로 세척하고 멸균 조건에서 건조했습니다. 주사전자현미경(SEM), 원자간력현미경(AFM) 및 측면간력현미경(LFM)을 사용한 나노플랫폼 특성화는 이전에 보고되었으며, 나노플랫폼이 거의 전체가 나노물질로 덮여 있음을 보여줍니다[25].

나노플랫폼을 얻기 위해 사용된 나노물질은 투과전자현미경(TEM)을 사용하여 이미지화되었다. TEM 이미지는 Morada 11 메가픽셀 카메라(Olympus Corporation, Tokyo, Japan)와 함께 80 kV에서 JEM-1220 현미경(JEOL, Tokyo, Japan)을 사용하여 획득했습니다. 샘플은 하이드로콜로이드 방울을 formvar로 코팅된 구리 그리드(Agar Scientific, Stansted, UK) 위에 놓고 관찰 전에 공기 건조되도록 하여 준비했습니다.

라만 스펙트럼은 532nm 레이저 소스(Wotton-under-Edge, UK)가 있는 Renishaw inVia 분광계를 사용하여 수집되었습니다. 샘플의 가열을 피하기 위해 레이저 출력을 낮게 유지했습니다(샘플에서 보정된 0.3 mW). 라만 매핑 모드는 25개의 스펙트럼을 포함하는 약 10 × 10 μm의 스캔 영역에서 사용되었습니다. 두 개의 주요 밴드로 구성된 각 스펙트럼은 G 밴드(~ 1578 cm ^{− 1} ) 및 D 밴드(~ 1347 cm ^{− 1} ), Lorentzian 선 모양을 사용하여 적합했습니다. FT-IR 측정은 다이아몬드 결정에서 감쇠된 전체 반사율 모드에서 Nicolet iS10 분광계(Thermo Fisher Scientific, Waltham, USA)를 사용하여 수행되었습니다. 그래핀 옥사이드 현탁액은 GO 얇은 포일을 만들기 위해 실온에서 폴리에틸렌 표면에서 건조되었습니다. 스펙트럼은 400–4000 cm ^{− 1} 범위에서 수집되었습니다. .

Zeta potential measurements of GO (20 mg/l), AgNPs (10 mg/l) and Ag-GO (GO 20 mg/l and AgNPs 10 mg/l) were carried out with a Nano-ZS90 Zetasizer (Malvern Instruments, Malvern, UK) at 25 °C, using the Smoluchowski approximation. Nanomaterials were sonicated for 30 min and zeta potential was immediately measured. Subsequently, nanomaterials were left for 24 h at room temperature and the zeta potential was measured again. Each measurement was repeated at least seven times after 60 s of stabilisation at 25 °C.

The hydrodynamic diameter of nanoparticles in water and their size distribution were measured with dynamic light scattering (DLS) using a Nano-ZS90 Zetasizer (Malvern). Similar to for the zeta potential analysis, GO (20 mg/l), AgNPs (10 mg/l) and Ag-GO (GO 20 mg/l and AgNPs 10 mg/l) were sonicated for 30 min and left for 24 h at room temperature. Each sample was measured at least seven times at 25 °C.

Bacterial Cultivation

Salmonella enteritidis subspecies enterica serovar Enteritidis (ATCC 13076) was obtained from LGC Standards (Łomianki, Poland). The bacteria were grown on tryptic soy agar (Merck Millipore, Darmstadt, Germany). The bacteria, grown on agar plates, were harvested by gently washing the plates with sterile distilled saline solution. To calculate the number of bacteria in the cell suspension, the optical density of the suspensions at 600 nm (OD600) was measured using a spectrophotometer (Helios Epsilon, Unicam, Milwaukee, WI, USA). A calibration curve was prepared by performing serial tenfold dilutions of bacterial suspensions of a known optical density, up to 10 ^− 5 . After 24 h of incubation at 37 °C, the number of formed colonies was enumerated and the number of colony-forming units (CFU) of the original bacterial suspension was calculated.

Bacteria Viability Assay

Viability was evaluated using a PrestoBlue Cell Viability Assay (Thermo Fisher Scientific). Bacteria were cultured onto foils coated with GO, AgNPs and Ag-GO, located on inserts inserted into six-well plates (200 μl MH broth with 5 × 10 ³  CFU per foil) and incubated for 24 h. Subsequently, 90 μl of each sample was transferred to 96-well plates and 10 μl of PrestoBlue reagent was added to each well and incubated for an additional 2 h at 37 °C. The optical density of each well was recorded at 570 nm using a microplate reader (Infinite M200, Tecan, Durham, NC, USA). Bacteria viability was expressed as the relative value after substitution of the absorbance from the blank samples. Experiments were repeated three times.

Scanning Electron Microscopy Analysis

Bacteria were incubated on foils with Ag-GO and a sterile cover glass. Bacteria cultures (100 μl, 10 ⁶  CFU/ml) were incubated on foils and a cover glass for 24 h at 37 °C. All samples were dried and covered with gold. Cells were fixed with 2.5% glutaraldehyde in phosphate-buffered saline (PBS, pH 7.2) and contrasted with 1% osmium tetroxide (Sigma-Aldrich) and 1% carbohydrazide (Sigma-Aldrich). Subsequently, cells were dehydrated in increasing concentrations of hexylene glycol (Sigma-Aldrich). Drying was performed using a Polaron CPD 7501 critical point dryer (Quorum Technologies, Laughton, UK). Finally, the samples were imaged with a SEM (FEI Quanta 200, Tokyo, Japan) at an acceleration voltage of 15 kV.

Human Cell Lines

Human umbilical vein endothelial cells (HUVECs; catalogue number:C0035C) and human fibroblasts (catalogue number:C0135C) were obtained from Thermo Fisher Scientific. HUVECs were maintained on low-serum Medium 200 basal media supplemented with Large Vessel Endothelial Supplement (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific), whereas fibroblasts were cultured in low-serum Medium 106 (Thermo Fisher Scientific) supplemented with Low Serum Growth Supplement (Thermo Fisher Scientific) and 1× penicillin/streptomycin (Thermo Fisher Scientific). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂ /95% air.

To analyse biological interactions, the nanoplatforms were put into six-well plates. After detachment from the cell culture flask, HUVECs or fibroblasts were placed directly on the nanoplatform with 100 μl of growth media. To avoid the media drying during incubations, plates were kept in humidity chambers.

Analysis of Nanoplatform Toxicity to HUVECs and Fibroblasts

To analyse HUVEC and fibroblast viability on the nanoplatforms, cells were cultured in the droplet directly on the nanoplatforms or uncoated foil (1 × 10 ⁴ cells in 100 μl growth media). After 24 h of incubation, cell viability was analysed using a PrestoBlue assay (Thermo Fisher Scientific). PrestoBlue reagent was incubated with assessed cells for 2 h in a cell culture incubator. Subsequently, 50 μl of growth media with PrestoBlue reagent was transferred to a 96-well plate where fluorescence (excitation λ  = 560 nm, emission λ  = 590 nm) was analysed using a Tecan Infinite 200 microplate reader (Tecan, Durham, USA). Cell viability was expressed as the relative value after substitution of the fluorescence from blank samples. Experiments were repeated three times.

Fibroblast morphology was observed using an inverted optical microscope (Olympus Corporation) using phase contrast. Fibroblasts were seeded in 35-mm diameter Petri dishes directly on the nanoplatforms (1 × 10 ⁴ cells in 100 μl growth media). Images were taken after 24 h of incubation.

Chorioallantoic Membrane Assay

Fertilised eggs from Ross 308 hens were obtained from a certified hatchery and kept for 4 days at 12 °C. The eggs were cleaned, sterilised with UVC light and divided into four groups (4 × 20 eggs). Embryos were incubated at standard conditions (temperature 37 °C, humidity 60% and turned once per hour). At 8 days of embryonic development, small holes (1 cm ² ) were made in the shell above the air space, the inner membrane was gently striped off and the nanoplatforms were placed on the chicken embryo chorioallantoic membrane. Subsequently, chicken embryos were incubated for the next 48 h, when nanoplatforms were cut out with the chorioallantoic membrane that was directly below the nanoplatform. The chorioallantoic membrane on the nanoplatforms was imaged using a stereoscopic microscope (SZX10, Olympus Corporation).

Antibody Array Analysis

An analysis of inflammation cytokines in fibroblast growth medium was performed using an antibody array (Abcam, Cambridge, UK; catalogue number ab134003). Fibroblast cells (1 × 10 ⁴ ) were incubated on nanoplatforms coated with AgNPs, GO, Ag-GO and uncoated foil with 100 μl of media. After 24 h, 80 μl of growth medium was collected. For each experimental group, the growth medium from six foils was used for analysis. Pooled growth medium from the six experiments was centrifuged (1600 rpm for 5 min), and 500 μl of growth media was diluted in 500 μl of PBS. Therefore, 1 ml of diluted growth media was used per each analysed membrane. The assay was performed in accordance with the manufacturer’s instructions. Diluted growth media was incubated with the membranes for 24 h at 4 °C. Subsequently, antibodies conjugated with biotins were added and incubated for the next 24 h at 4 °C. In the next step, the membranes were incubated with streptavidin conjugated with horseradish peroxidase for 2 h at room temperature. Membranes were visualised after the addition of the provided horseradish peroxidase substrate using a ChemiDoc imaging system (Bio-Rad, Hercules, USA).

Statistical Analysis

Data were analysed using one-way analysis of variance with GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Differences between groups were tested with Tukey’s HSD post hoc tests. Results are shown as means with standard deviations. Differences at P  < 0.05 were considered significant.