3.4. Biological Evaluation of Nanosilver-Based Dressings

The in vitro biocompatibility was evaluated on L929 fibroblast cell cultures, by employing the tetrazolium-based viability assay (MTT) to quantitatively assess the cell proliferation, whereas the morphology was observed using the optical microscopy technique for living cells. Fluorescence staining using Phalloidin, which specifically binds to filamentous actin was performed to verify the cytoskeleton integrity of the treated cells.

L929 cell line is highly recommended for cytotoxicity studies, due to its high sensibility. The cells were cultured in MEM Earle’s (MEM) (Biochrom, Merck Milipore, Berlin, Germany), supplemented with 10% fetal bovine serum (Biochrom, Merck Milipore), 1% l-glutamine (Biochrom, Merck Milipore) and 1% antibiotics (penicillin and streptomycin) (Biochrom, Merck Milipore).

For the MTT assay, 5000 cells/well were seeded in 96-well plates and cultured for 24 h in standard conditions (37 ± 2 °C, 5% ± 1% CO₂, more than 90% humidity). Meanwhile, solutions of nanoparticles in different concentrations (10, 5, and 1 µg/mL) were prepared by ultrasound dispersion in complete culture medium and added in each of the sample wells, while in control wells (untreated cells) complete culture medium was added; blank samples were also prepared (wells with no cells) in order to eliminate possible interferences. The viability measurements were done at 24, 48, and 72 h after the treatment: the nanoparticles were gently removed from the wells and 10 µL of MTT solution was added over 90 µL complete culture medium (supplemented with 5% Fetal Bovine Serum) in each well. The plates were incubated for 2 h in standard conditions. After this time, 100 µL of acid isopropyl alcohol solubilization solution was added in each well and plates were subjected tovigorous shaking for several minutes. The absorbance was read at 570 nm using Mitras LB 940 (Berthold Technologies, Calmbacher, Germany).

For the morphology evaluation using optical microscopy, the images were recorded using an Olympus CKX31CF microscope, from the samples prepared for the viability assay, prior to the addition of the MTT solution.

For the qualitative evaluation of the cytotoxic effects, we evaluated the morphology and cytoskeleton integrity of treated cells using Texas Red^®-X Phalloidin (ThermoFisher Scientific) fluorescence staining. For this purpose, 5000 cells were seeded on each 10 mm diameter glass slides placed in 24-well plates and cultured for 24 h in standard conditions. The treatment was done similarly to the cytotoxicity assay. At 24 h after the treatment, the nanoparticles were removed from the wells and the cells were gently washed with Phosphate Buffer Saline (PBS) for 3 times; then, 3.7% Paraformaldehyde solution was added for fixing, for 5 min, the cells were permeabilized with 1% Triton X for 10 min and colored with Texas Red-Phalloidin for 40 min (at dark). The cells were gently washed with PBS after each step. The visualization of the as-prepared samples was performed using an Olympus LX71 fluorescence microscope and the image recording was performedusing an ixon+ image recorder (Andor Technology).

Values were presented as means ± standard error of the mean. Data werestatistically analyzed using a two-tailed Student’s test, with p ≤ 0.05 accepted as statistically significant.

The antibacterial potential of the experimentally modified wound dressings was assessed in vitro against two clinically relevant bacterial strains that are often incriminated in nosocomial infections, namely the Gram-positive species Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC27853. The strains are maintained as glycerol stocks in the culture collection of Microbiology Immunology Department of Faculty of Biology, University of Bucharest. The antibacterial efficiency was assessed by considering the bacterial development and colonization inthe presence of regular polyester-nylon dressings (positive control) and nanosilver-coated wound dressings. Thus, bare and AgNPs-based wound dressing sections were placed in 6-wellplates, followed by the inoculation of 2 mL of microbial suspension of 0.5 McFarland standard density (1.5 × 10⁸ CFU/mL) from each bacterial strain obtained directly into sterile broth medium. Subsequently, the inoculated plates were incubated for 24 h at 37 °C. Thereafter, the culture medium was removed and the specimens were washed with sterile phosphate buffered saline (PBS). The wound dressing sections (both uncoated and nano-modified) were afterwards placed in fresh medium and incubated at 37 °C for 24 and 48 h. After incubation, the wound dressing samples were gently washed with sterile phosphate buffered saline and further placed in 1.5 mL centrifuge tubes containing 750 μL of PBS. The as-obtained specimens were centrifuged for 30 s and subsequently subjected to ultrasounds for 10 s. Serial ten-fold dilutions were performed and distributed on Petri dishes containing Luria agar, for viable cell counts assay. All the experiments were performed in triplicate and repeated in three separate occasions.

The experimental protocol was applied according with the European Council Directive No. 86/609 (24 November 1986), the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (2 December 2005), and the Romanian Parliament Law No. 43 (11 April 2014) on the protection of animals used for scientific purposes. The study was approved by the Ethics Committee of the University of Medicine and Pharmacy of Craiova, Romania (Approval Report No. 118/27.05.2015).

Three weeks old BALB/c mice were aseptically injected with 100-μL of 1 mg/mL dispersion of nanostructures, obtained in saline and previously sterilized by UV irradiation for 30 min. Intravenous administration was carried out slowly, under general anesthesia (Ketamine/Xylazine mixture), into the left jugular vein, using a catheter. Reference mice were injected with 100-μL of saline. The mice were kept in standard conditions before the organs removal. At 2 days and 10 days after the beginning of the experiment, the animals were euthanized, under general anesthesia, for the sampling of internal organs (brain, liver, myocardium, pancreas, lung, kidney and spleen).

Directly after the sampling, the biological material was washed in PBS to remove blood. Then, the internal organs were fixed in 10% buffered neutral formalin, for 72 h, at room temperature, and processed for routinely histological paraffin-embedding technique.

For the histological analysis of nanostructures, 4-μm thick serial sections were cut on a MICROM HM355s rotary microtome (MICROM International GmbH, Walldorf, Germany) equipped with a waterfall-based section transfer system (STS, MICROM). The cross-sections were placed on histological blades treated with poly-l-Lysine (Sigma-Aldrich, Munich, Germany). After Hematoxylin–Eosin (HE) classical staining, cross-sections were evaluated and photographed using a Nikon Eclipse 55i light microscope equipped with a Nikon DS–Fi1 CCD high definition video camera (Nikon Instruments, Apidrag, Romania). Images were captured, stored and analyzed using Image ProPlus 7 AMS software (Media Cybernetics Inc., Marlow, Buckinghamshire, UK) [36,37,38].