Effects of silver nanoparticles on primary cell cultures of fibroblasts and keratinocytes in a wound-healing model

Preparation and characterization of AgNPs

AgNPs were obtained from NanoTrade Company as commercial samples, and were prepared by dissolving AgNO₃ in distilled water. After adding NaBH₄ under magnetic stirring, the AgNPs were formed very quickly. They were characterized by ultraviolet-visible (UV-VIS) spectrum spectroscopy (from 200 nm to 800 nm) and transmission electron microscopy (TEM). The analysis was performed using a JEOL JEM 2011 transmission electron microscope at an accelerating voltage of 100 kV. Photographs were taken with a Morada or Keen View II digital camera, using the iTEM program (SIS, Olympus). Zeta Plus analyzer (Brookhaven) was used for the detection of the zeta potential.

Cell cultivation and maintenance

NHDFs and NHEKs were isolated from plastic surgery skin sections with approval from the ethics committee of the University Hospital Olomouc and the patients’ consent. The study was performed in accordance with the Code of Ethics of the World Medical Association. The morphology and origin of the cells were authenticated in the Histology Department of the University Hospital Olomouc.

NHDFs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin under standard culture conditions (5% CO₂, 37°C). Cells were used between the second and third passages. For the experiments, the cells were seeded in 6-well plates at a final concentration of 0.5 × 10⁶ cells/well.

NHEKs were cultured in keratinocyte basal medium-2 (KBM-2) supplemented with KGM-2 SingleQuots and 2% FBS for 3 days (17). After the incubation period, KBM-2 was substituted with EpiLife growth medium supplemented with the Human Keratinocyte Growth Supplement Kit, 100 mg/mL penicillin, 100 mg/L streptomycin and 250 µg/mL ampicillin, and incubated at 37°C in a 5% CO₂ atmosphere. The medium was changed every other day until the keratinocytes reached 50%-60% confluence. Cells were used at the third passage. For the experiments, the cells were seeded in 6-well plates at a final concentration of 3 × 10⁴ cells/well.

Scratch wound assay

Cells were seeded in 6-well plates at 0.5 × 10⁶ cells/well for NHDF and 3 × 10⁴ cells/well for NHEK, and grown to 100% confluence. After 1 day of culture, the scratch wound assay was performed using a 10-cm portion of a plastic pipette, making 1 scratch across each well. The cells were washed with phosphate-buffered saline, and AgNPs at different concentrations were added (0.25, 2.5 and 25 parts per million [ppm]), diluted in serum-free cultivation medium. The concentrations were chosen based on our previous experiments (data not shown). As a positive control, we used scratched cells without AgNPs.

The medium was collected for the enzyme-linked immunosorbent assay (ELISA) and Bio-Plex analyses, and the cells were lysed with RIPA buffer supplemented with protease inhibitors at specified times (24 and 48 hours) for the Western blot analysis.

Bio-Plex analysis

The healing mechanism is a complex system that is regulated by cytokines and growth factors. The levels of TNF-α, IL-6, IL-8, IL-10, IL-12, basic FGF, VEGF and granulocyte macrophage colony-stimulating factor (GM-CSF) were measured using a Bio-Plex cytokine assay (Human Group I 8-Plex Panel; Bio-Rad) according to the manufacturer’s instructions. The detection range was 0.18 to 37,000 pg/mL. All of the Bio-Plex results are expressed as the ratio between the concentrations of markers (pg/mL) in treated cells vs. the concentrations of markers (pg/mL) in the positive control.

ELISA

MMP-1, MMP-2, MMP-3 and MMP-9 in the cell supernatant were examined using the Human Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) at 24 and 48 hours according to the manufacturer’s instructions.

Western blot

Western blot was used for the detection of COX-2. Cell lysates were briefly ultrasonicated in a cleaning sonicator bath before being centrifuged for 10 minutes at 10,000 g at 4°C. The protein concentration was quantified by the Bradford method, and 50 μg of protein was loaded in each well with 10% sodium dodecyl sulfate–polyacrylamide gel for the analysis of COX-2 and β-actin. Gels were wet-transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% low-fat milk in Tris-buffered saline (TBS) with 0.1% Tween 20 for 2 hours at room temperature and washed with 0.1% Tween 20 TBS before incubation with primary antibodies overnight at 4°C. The dilutions of the antibodies were 1:500. The membranes were washed in TBS, and secondary antibody (goat antirabbit or rabbit antigoat) was added for 2 hours and imaged (18). A chemiluminescence system was applied to each membrane for 2 minutes. Excess substrate was removed, and the film was exposed to the membrane for 1-10 minutes. The films were then developed with a film processor.

Statistical analysis

All of the experiments were repeated at least 3 times using different donors in duplicate. The data are expressed as the means and standard deviation. Student’s t-test was used for the statistical analysis. Values of p<0.05 were considered statistically significant.

Characterization of silver nanoparticles

Silver nanoparticles (1 mL) were diluted in 50 mL of water for the UV-VIS characterization (Fig. 1A). The silver colloid was characterized by strong absorption in the visible region (called the surface plasmon resonance band) at 400 nm. The position of the maximum and width of an absorption band provide information about the form, average size and size distribution of nanoparticles (NPs). The average size of AgNPs was 10 ± 5 nm (>50% of the NPs), confirmed by TEM (Fig. 1B). The pH of the AgNPs was 7.1, with a zeta potential of-22 mV.

Bio-Plex

Cytokines play an important role in the wound-healing process. We tested whether AgNPs are able to change the production of proinflammatory cytokines, antiinflammatory cytokines and growth factors after 24 and 48 hours. The secretion of these cytokines was detected by the Bio-Plex multiple-bead system. Only the levels of cytokines that were altered by AgNPs compared with the positive control cells are discussed. The production of TNF-α and VEGF by NHDFs and NHEKs decreased after 24 and 48 hours at all of the tested concentrations (Fig. 2A-D). AgNPs decreased the secretion of IL-12 by NHDFs at all of the tested concentrations after 24 and 48 hours compared with untreated cells. AgNPs also decreased the secretion of IL-12 by NHEKs after 48 hours at all of the tested concentrations (Fig. 3A, B). AgNPs (2.5 and 0.25 ppm) slightly increased the production of IL-12 by NHEK after 24 hours (Fig. 3A).

ELISA

Extracellular matrix remodeling is mainly influenced by MMPs through the removal of damaged tissue and decrease in the overproduction of extracellular matrix proteins. We evaluated the effects of AgNPs on the remodeling of wound healing in cell supernatants and detected the volume of MMP-1, MMP-2, MMP-3 and MMP-9. The production of MMP-2 by NHDFs was unchanged, and MMP-9 production was below the limit of detection (data not shown). MMP-3 levels in NHEKs decreased compared with positive control after 48 hours (Fig. 4B), whereas AgNPs increased the production of MMP-1 after 24 and 48 hours (Fig. 4A).

Western blot

COX-2 is one of the most important proinflammatory mediators and is involved in acute inflammation. In the present study, we determined whether AgNPs influence the expression of COX-2 in NHEKs and NHDFs after AgNP application. The expression of COX-2 in NHEKs was monitored by Western blot. It decreased after the application of AgNPs after 24 hours at concentrations of 2.5 and 25 ppm (Fig. 5A). AgNPs at a concentration of 25 ppm decreased the expression of COX-2 in NHDFs after 24 hours (Fig. 5C).