Silver nanowires as prospective carriers for drug delivery in cancer treatment: an in vitro biocompatibility study on lung adenocarcinoma cells and fibroblasts

Introduction

In spite of considerable advancements in the medical and oncological fields, lung cancer is still a devastating disease (1) and one of the most prominent causes of cancer-related mortality in Europe and USA (2, 3). The main reason for the short survival of lung cancer patients is the poor efficacy of currently available chemotherapeutic agents (4). In order to increase the success rate of the medical treatments, the pharmaceutical industry has recently developed targeted chemotherapeutic agents that are designed to be effective against specific subtypes of lung cancer defined by precise gene mutations (5, 6). Unfortunately, these mutations define mechanisms of drug sensitivity and/or resistance that are not shared by most patients or by other lung cancer subtypes (5, 7). For example, the patients with epidermal growth factor receptor (EGFR) mutations show great response to tyrosine kinase inhibitors (e.g., gefitinib and erlotinib), but patients with mutations to the KRAS gene are resistant to such therapy (6, 8). As a consequence, novel and more efficient anti-cancer treatments are needed against lung cancer.

Nanotechnology has tremendous potential to improve the precision of lung cancer therapy, and has become a key technology in the development of more sensitive diagnostic tools and more powerful pharmaceutical treatments (frequently offered as two-in-one (theranostic) systems) against cancer (6, 9–11). Nanomedicine products can, for example, be loaded with chemotherapeutic agents and specifically targeted to the tumor site, thus decreasing the side effects and increasing the therapeutic effect of the drugs (12). In particular, silver nanomaterials can find many remarkable applications in clinical practise, since they possess optimal chemical reactivity for the functionalisation of their surfaces with biologically active moieties (such as targeting and therapeutic molecules) (13), and are characterised by localised surface plasmon modes (14) that enable the development of tunable contrast agents, thermal ablation therapies and thermal-induced drug release (15). The working hypothesis behind this study was that silver nanowires (AgNWs) can be potentially implemented as theranostic, nano-enabled drug delivery platforms for lung cancer treatment once their biocompatibility has been proven.

The use of silver for biomedical purposes is quite widespread. In colloidal form, it is used for various purposes such as preparation of ointments (e.g., silver sulfadiazine remains the main topical product used in treating burn) (16) and treatment of bacterial infections in open wounds. In nano-form (17, 18), advanced technology has enabled scientists to increasingly exploit silver nanoparticles (AgNPs) (also known as nanosilver) as an antimicrobial agent (17, 19) (e.g., in wound dressings (namely, Acticoat-7, Actisorb Silver 220, Aquacel-Ag hydrofiber and Silverlon (14)) catheters (20) and various household products), and for the treatment of retinal neurovascularisation (21) and of acquired immunodeficiency syndrome (22). Nanosilver has also been shown to improve the anti-thrombogenic surface properties of nanocomposite polymers for bypass grafts (23), providing a synergistic approach where the release of nitric oxide (24) (responsible of maintaining the haemostasis between pro-thrombotic and anti-thrombogenic states of the endothelium lining the vessels (25)) is associated with the release of Ag⁺ ions, thus reducing the onset of cardiovascular implant-associated infection (26). In addition, silver nanowires (AgNWs) have very high electric conductivity (27), meaning that they can transfer electricity efficiently when incorporated in elastic conductors (28), and therefore, similarly to gold nanowires (29), they might find application in the production of cardiac patches. Preclinical studies have also been carried out to evaluate the use of silver nanomaterials for cancer treatment (30–32) and/or diagnosis (33).

Despite decades of use, the toxicity of silver is still not clear. In small concentrations colloidal silver is considered non-toxic to human cells (34), even if topical and systemic side effects (such as argyria (35) and liver injury (36), respectively) have been associated with long-term exposure to silver-containing products. In parallel, results concerning the toxicity of silver nanomaterials are often inconclusive (37). Recent studies show that, in vitro, nanosilver leads to oxidative stress, lipid peroxidation, inhibition of mitochondrial activity, DNA damage and apoptotic cell death (38–48). Nanosilver has also been reported to cause hepatotoxicity (49–51), while the cytotoxicity of AgNWs is dependent on cell type, nanowire lengths, doses and incubation times (52). The proposed mechanism of action is that silver nanomaterials release Ag⁺ ions (53), which interact with the thiol groups of proteins in the cell cytoplasm and the inner mitochondrial membrane, interrupting and/or perturbing the normal cells activity (54, 55). However, a number of recent studies have indicated that the transformation of silver nanoparticles to (Ag₂S)NPs reduces the adverse effects of these materials (56–60). Researchers have also provided evidence that in lung epithelial cells the Ag⁺ ions released from AgNWs precipitate in the form of insoluble metallic Ag₂S, thus limiting the toxic impact of AgNWs. In addition, it is known that fibre length is an important factor in determining the cytotoxic (52) and inflammatory responses (53, 61) to AgNWs, and to HARNs more in general (62). Recent studies envisage in fact that a higher inflammatory state is associated with increasing AgNWs lengths (53, 61): this is known as the “fibre pathogenicity paradigm” (43). In vivo studies have reported frustrated macrophage uptake of AgNWs that are longer than 10 μm (61, 63); this phenomenon has been identified as an important factor in the post-exposure inflammatory response and associated asbestos-like lung pathogenicity of such nanomaterial (53, 62, 63).

In light of this diverse evidence, this study aimed to investigate the in vitro internalisation, cytotoxicity and the influence on the cell cycle of commercially-available AgNWs longer than 10 μm which were incubated with lung adenocarcinoma (A549) cells and normal lung fibroblasts (MRC-5 cells). The A549 human lung carcinoma cell line is considered one of the closest cell models mimicking alveolar epithelial type II cells (e.g., membrane-bound inclusions resemble lamellar bodies of type II cells) (64), and it has proven to be a robust cell line/alveolar model for several previous nanomaterial-based studies (52, 65–68). In addition, A549 cells are a physiologically relevant in vitro model of non-small cell lung cancer (NSCLC) (69), the most prevalent form of lung cancer originating from epithelial cells. Therefore, this cell line was chosen as an appropriate epithelial alveolar model (70) for testing AgNWs internalisation and cytotoxicity in lung cancer tissue. Conversely, the MRC-5 human fibroblast cell line was selected as a model of normal/non-cancerous lung cell type, in order to investigate the cytotoxicity of AgNWs when coming into contact with the healthy cells surrounding the malignantly transformed tissue areas in the lung.

In both cell lines tested, our data clearly demonstrated that AgNWs could penetrate and localise within the cells without causing any acute and/or detectable cytotoxicity (up to 72 h exposure) or any significant change in the cell cycle. From these observations, we conclude that AgNWs could be used as suitable candidates for subsequent functionalisation, drug delivery and diagnostic purposes (such as lung cancer treatment) with several advantages over conventional therapies.

Materials and methods

Chemicals and solvents were purchased from commercial sources [Sigma-Aldrich (Ireland), Fisher Scientific (Ireland), Invitrogen Bioscience (Ireland) and Calbiochem (USA)] and used as supplied unless differently stated in the text.

Results

Cellular internalisation of AgNWs

Confocal microscopy was used to identify whether AgNWs were internalised into A549 (Figure 1A–C) and MRC-5 cells (Figure 1D–F). After 24 h exposure, it resulted evident that AgNWs were internalised into both cell culture models at all concentrations tested.

Figure 1

Representative confocal images of (A–C) A549 and (D–F) MRC-5 cells exposed to AgNWs (1, 5 and 10 μg/mL) for 24 h. Cells were stained for F-actin (in red) and nuclei (in blue). AgNWs were imaged in confocal reflectance mode and are pseudo-colored in green. Arrows highlight the internalised AgNWs. Scale bars: 10 μm (63× oil-immersion objective lens).

Cytotoxicity of AgNWs

Trypan Blue exclusion assay

Trypan Blue exclusion assay was used to quantify A549 and MRC-5 cell viability changes (Figure 2A and B, respectively) to three concentrations of AgNWs (1, 5 and 10 μg/mL) at two time points 24 and 72 h. No significant change in cell viability was evidenced after 24 or 72 h exposure in all cell models tested and, as the concentration of AgNWs increased, the percentage of live cells did not show any unusual decrease.

Figure 2

Percentage (%) of live cells in (A) A549 and (B) MRC-5 cells untreated (negative control), exposed to 70% methanol for 30 min (positive control) and to AgNWs (1, 5 and 10 μg/mL) for 24 or 72 h, as calculated by Trypan Blue exclusion assay. The symbol (***) indicates significant changes (p<0.001) as compared to the negative controls.

Live/Dead viability assay

Live/Dead cytotoxicity assay was performed in order to confirm the data obtained by the Trypan Blue exclusion assay. Epifluorescence microscopy (Figure 3A) and quantitative (Figure 3B–C) analysis confirmed that AgNWs did not cause any cytotoxic response in A549 cells or MRC-5 cells after 24 and 72 h exposure.

Figure 3

(A) Representative epifluorescence images of A549 cells untreated (negative control), treated with 70% methanol for 30 min (positive control), and exposed to AgNWs for 24 h. Cells were stained with Calcein AM (live cells, in green) and EthD-1 (dead cells, in red). Scale bars: 20 μm (10× objective lens). (B-C) Percentage (%) of live cells as quantified by Live/Dead cytotoxicity assay after exposing (B) A549 and (C) MRC-5 cells to AgNWs. The symbols (**) and (***) indicate significant changes (p<0.01 and p<0.001, respectively) as compared to the negative controls.

Influence of AgNWs on the cell cycle

Flow cytometry was used to evaluate the AgNWs interactions with the cell cycle in A549 and MRC-5cells exposed to such nanomaterial. Exposure of A549 cells to AgNWs for 24 h caused an increase in the cell population in G0/G1 phase at all the concentrations tested (Figure 4A). This was correlated with a decreased number of cells in G2/M phase. A similar trend was found for MRC-5 cells exposed to AgNWs for 24 h (Figure 4C). In addition, after 72 h the increase in A549 cell population in G0/G1 phase was associated with a moderate but statistically significant increased number of cells entering G2/M phase (Figure 4B). In contrast, MRC-5 cells demonstrated a rise in the G0/G1 population only after 72 h (Figure 4D).

Figure 4

Effects of AgNWs on the cell cycle (A–B) A549 and (C–D) MRC-5 cells after (A, C) 24 or (B, D) 72 h exposure. The symbols (*), (**) and (***) indicate significant changes (p<0.05, p<0.01 and p<0.001, respectively) compared to the negative controls.

Discussion

Analysis of the biological interactions of AgNWs with lung epithelial (A549) cells was carried out with the aim to assess (1) the capability of AgNWs as future drug delivery platforms to be internalised into lung cancer cells, and (2) to evaluate the biocompatibility of AgNWs in the lungs. Cell cultures of lung fibroblasts (MRC-5 cells) were also tested, in order to investigate the cytotoxicity of AgNWs coming into contact with non-cancerous tissue.

Confocal reflectance microscopy imaging clearly demonstrated that AgNWs internalisation was effectively achieved after 24 h exposure at all three concentrations tested (Figure 1). It has been observed that the cell type is one of the key factors that influence the mechanism of cellular entry of silver nanomaterials (37). In this study, it was not clear whether the cellular entry of AgNWs occurred through phagocytosis or some other mechanism (such as autophagy (52) in human phagocytic cells) and whether AgNWs entered into the various cell types through different mechanisms. Further studies are therefore needed to elucidate the mechanism of cellular internalisation of AgNWs in the various cell types and to identify the precise localisation of the internalised AgNWs in specific cellular compartments. Similarly to previously reported data on A549 cells’ responses to AgNWs (52), Live/Dead viability assay showed that AgNWs did not cause any detectable cytotoxicity when incubated with A549 and MRC-5 cells for up to 72 h (Figure 3). Likewise, the Trypan Blue exclusion assay (Figure 2) suggested that AgNWs did not cause any detectable cytotoxicity, thus supporting the idea that such nanomaterial could be used as a safe drug delivery platform following ad hoc engineered functionalisation. Our investigation focused on the cells response after 24 and 72 h due to the fact that AgNWs identical to those tested in this study have been reported to be subject to dissolution over longer periods (53). In particular, the authors would like to highlight that the AgNWs used in this study are reported not be bio-persistent beyond a few days (53); this partly reduces the relevance of the “fibre pathogenicity paradigm” for the nanomaterial tested. Bio-persistence is in fact one of three key factors in determining the pathogenic effects of long nanofibres (71) and HARNs (62), which are known to evoke chronic inflammatory responses and cancer development when resistant to dissolution and/or breakage in the biological environment. In addition, it has been recently reported that the length threshold for fibre pathogenic effects is a higher value in the lung than at the mesothelial surface, the cut-off length for AgNWs inducing acute pulmonary inflammation being 14 μm (63). This, together with our results, supports the concept that the AgNWs tested in this study could be translated in carriers for functionalisation and drug delivery purposes. Additional research is however needed to evaluate if, similarly to nanosilver (51), AgNWs could cause hepatotoxicity following inhalation or injection. A further issue is the formation of different silver species over time via AgNWs dissolution. The AgNWs used in this study do in fact slowly dissolve (53) and, depending on the biological environment, the resulting Ag⁺ ions may form soluble complexes such as AgCl₂^-, AgCl₃^2-, [Ag(NH₃)₂]⁺, covalent adducts with thiol group containing compounds (e.g., glutathione, cysteine, proteins) or insoluble salts (AgCl, Ag₂S) (72). Furthermore, Ag⁺ could be reduced to Ag⁰ (e.g., by glucose), thus leading to the de novo formation of Ag⁰ nanoparticulates. It becomes therefore crucial to fully characterise the physico-chemical properties of AgNWs and the biological effects of their products of conversion at the various timepoints as we provided in this work.

Finally, while AgNWs did not have strong influence on the cell cycle of MRC-5 cells following 72 h exposure (Figure 4D), significant changes in the percentage of cell population in the various phases were detected when A549 cells were exposed to AgNWs for 24 h and 72 h (Figure 4A–C). This indicates that AgNWs had very diverse effects on cells with different proliferation rates, with higher effect on the fast-growing, carcinogenic epithelial lineage. On the other hand, it is known that cells with damaged DNA accumulate in the G1 phase, the DNA synthesis (S) phase or the G2/M phase (73). Therefore, the increased A549 cell population in G1 and G2/M phases after 24 h or 72 h, respectively, indicated a DNA damage triggered by AgNWs exposure, which might ultimately cause genotoxicity in the chronic phase similarly to what has been demonstrated for nanosilver in various in vitro and in vivo studies (73). Further investigation is needed to better elucidate the cell line-dependent differences and understand whether the effects detected in A549 cells could translate into genotoxicity over longer exposure times.

In conclusion, our data showed that AgNWs could penetrate and localise within A549 and MRC-5 cells, without causing any detectable change in cell viability. Further investigations into the systemic side effects and bio-distribution of this nanomaterial are required, as well as to demonstrate that, by selective functionalisation with biologically active moieties, AgNWs could be used as platforms for drug delivery against lung cancer.