Active silver nanoparticles for wound healing

Abstract

In this preliminary study, the silver nanoparticle (Ag NP)-based dressing, Acticoat™ Flex 3, has been applied to a 3D fibroblast cell culture in vitro and to a real partial thickness burn patient. The in vitro results show that Ag NPs greatly reduce mitochondrial activity, while cellular staining techniques show that nuclear integrity is maintained, with no signs of cell death. For the first time, transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS) analyses were carried out on skin biopsies taken from a single patient during treatment. The results show that Ag NPs are released as aggregates and are localized in the cytoplasm of fibroblasts. No signs of cell death were observed, and the nanoparticles had different distributions within the cells of the upper and lower dermis. Depth profiles of the Ag concentrations were determined along the skin biopsies. In the healed sample, most of the silver remained in the surface layers, whereas in the unhealed sample, the silver penetrated more deeply. The Ag concentrations in the cell cultures were also determined. Clinical observations and experimental data collected here are consistent with previously published articles and support the safety of Ag NP-based dressing in wound treatment.

Figure 1

Mitochondrial activity in silver nanoparticle (Ag NP)-treated 3D fibroblast cultures, (one sample n = 2 readings ± SD versus time). The mitochondrial activity in the treated samples is expressed as a percentage of the activity of the untreated samples. For each time point, MTT values were obtained from duplicate readings of a single sample.

Figure 2

Dermal-like tissue reconstructed in vitro. Cells, visible thanks to the Hoechst blue staining of the nuclei, can be seen inside the collagen-based scaffold and appear to be organized in layers. (a) Un-treated control after three days from the beginning of the experiments; (b) Untreated control at nine days; (c) Enlargement of selected area; (d) Ag NP-treated fibroblast at three days; (e) Ag NPs fibroblast at nine days; (f) Enlargement of selected area.

Figure 3

Progression of cell growth in time in a 3D dermal-like tissue after Ag NPs treatment (dark grey) and in the control sample (light grey); mean value ± SD (n = 2) samples versus time. The count of the live cells in the sample is obtained as the sum of the live cells at various depths at each position.

Figure 4

Optical microscopy (OM) Images of the skin samples: burnt (a), healed after seven days (b and c), unhealed after seven days (d and e) and after complete re-epithelialization (f) after 10 more days of treatment. Healed and unhealed skin sections are shown with H/E and toluidine blue staining. Scale bar: 100 μm.

Figure 5

TEM images of the healed skin sample. (a) epidermis and dermis; (b) detail of a fibroblast surrounded by Ag NPs, in the upper part of the dermis; (c) endocytic vesicle containing Ag NP agglomerates; (d) magnification of endocytic vesicle containing Ag NP agglomerates; (e) Ag NPs have been released into the cytoplasm of a fibroblast and are located near the mitochondria; (f) a fibroblast in the lower part of the dermis—Ag NP aggregates are near the nuclear membrane; (g) a healthy undamaged mitochondrion. Key: DE, dermis; EP, epidermis; EV, endocytic vesicle; IS, intercellular space; M, mitochondrion; N, nucleolus; NM, nuclear membrane. Arrows indicate Ag NP agglomerates. Scale bars: (a) 500 nm; (b) 1 μm; (c) 100 nm; (d) 20 nm; (e) 200 nm; (f) 1 μm; (g) 100 nm.

Figure 6

TEM images of the unhealed skin sample, DM (dark matter), contained in vesicle. Scale bar: 1 μm.

Figure 7

SEM images of the unhealed sample (a) and energy-dispersive X-ray spectroscopy (EDS) spectrum (b) of the particle indicated by the arrow. Scale bar: 50 μm.

Figure 8

Depth profiles of Ag concentration (ng mg⁻¹) after seven days of treatment. In this pilot study, the biopsies were obtained from the same patient. One biopsy for silver analysis was taken from the healed part of the wound (top) and one from the unhealed (bottom) tissue. Panoramic images of the other duplicate sample were obtained by optical microscopy, and zoomed in areas of representative portions are also shown for comparison.

Figure 9

Flow chart of the experimental plan adopted for the in vitro study. The cycle of applications is repeated to carry out the chemical, toxicological and mitochondrial functionality determinations for 3, 6 and 9 days of cumulative duration of the treatment.

Figure 10

Flow chart of the experimental plan adopted for the in vivo study.