El óxido de grafeno en un compuesto con nanopartículas de plata reduce la citotoxicidad de fibroblastos y células endoteliales de una nanoplataforma antibacteriana

Materiales y métodos

Preparación y caracterización de nanoplataformas recubiertas con nanomateriales

Se prepararon nanoplataformas hechas de láminas de poliuretano recubiertas de nanopartículas como se describió anteriormente [25]. Se cubrieron láminas de poliuretano de forma cuadrada (15 × 15 mm, 0,05 mm de espesor) con suspensiones de AgNP (HydroSilver1000, Amepox, Łódź, Polonia) sintetizadas por reacción de reducción química en presencia de alcohol polivinílico desarrollado por Amepox y / o GO sintetizado por método de Hummers modificado. Se mezclaron diez gramos de polvo de grafito con 230 ml de ácido sulfúrico concentrado (98%) (Sigma-Aldrich Co., St. Louis, MO, EE.UU.) a una temperatura por debajo de 10ºC. Posteriormente, se añadieron 4,7 g de nitrato de sodio (Sigma-Aldrich) y 30 g de permanganato de potasio (Sigma-Aldrich) a la mezcla de grafito, manteniendo la temperatura por debajo de 10 ° C. Luego, la mezcla se calentó a 30ºC y se agitó durante 2 h. Posteriormente, se añadieron 100 ml de agua y la mezcla se trató con 10 ml de peróxido de hidrógeno. El GO se purificó por filtración y se lavó con agua desionizada hasta que el pH del filtrado alcanzó 6,5. Se prepararon suspensiones de GO, AgNP y el compuesto de AgNP y GO (Ag-GO) en agua desionizada. Durante el recubrimiento, las concentraciones de nanomateriales fueron las siguientes:GO, 200 mg / l; AgNP, 100 mg / l; Ag-GO, 200 mg / l; y AgNP, 100 mg / l. El recubrimiento de nanopartículas se realizó utilizando un cuerno ultrasónico (cuerno de Ti, Ø13 mm, 60% de eficiencia, 20 kHz; Sonics &Materials, Inc., Newtown, CT, EE. UU.) A una temperatura de 30 ± 1 ° C. Las muestras cubiertas se lavaron con agua desionizada y se secaron en condiciones estériles. La caracterización de nanoplataforma con un microscopio electrónico de barrido (SEM), un microscopio de fuerza atómica (AFM) y un microscopio de fuerza lateral (LFM) se informó anteriormente, mostrando nanoplataformas cubiertas casi en su totalidad con nanomateriales [25].

Se tomaron imágenes de los nanomateriales utilizados para obtener las nanoplataformas utilizando un microscopio electrónico de transmisión (TEM). Las imágenes TEM se adquirieron utilizando un microscopio JEM-1220 (JEOL, Tokio, Japón) a 80 kV con una cámara Morada de 11 megapíxeles (Olympus Corporation, Tokio, Japón). Las muestras se prepararon colocando gotas de hidrocoloides en rejillas de cobre recubiertas de formvar (Agar Scientific, Stansted, Reino Unido), que se dejaron secar al aire antes de las observaciones.

Los espectros Raman se recogieron usando un espectrómetro Renishaw inVia con una fuente láser de 532 nm (Wotton-under-Edge, Reino Unido). Para evitar el calentamiento de la muestra, la potencia del láser se mantuvo baja (0,3 mW, calibrada en la muestra). Se utilizó el modo de mapeo Raman con un área de escaneo de aproximadamente 10 × 10 μm, que contiene 25 espectros). Cada espectro consta de dos bandas principales, una banda G (~ 1578 cm ^{- 1} ) y banda D (~ 1347 cm ^{- 1} ), se ajustó utilizando la forma de línea de Lorentz. Las mediciones de FT-IR se realizaron usando un espectrómetro Nicolet iS10 (Thermo Fisher Scientific, Waltham, EE. UU.) En modo de reflectancia total atenuada en un cristal de diamante. La suspensión de óxido de grafeno se secó sobre la superficie de polietileno a temperatura ambiente para crear una lámina fina GO. El espectro se recopiló en el rango de 400 a 4000 cm ^{- 1} .

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.