In 2008 elemental copper and its alloys have been listed as antimicrobial materials and officially recognised as the first metallic antimicrobial agents by US Environmental Protection . Later this statement has been amended to include also the residual antiviral properties . The enhanced antimicrobial activity of Copper oxides nanoparticles towards pathogenic microorganisms and viruses has been already displayed in several studies [29 – 32]. This characteristic makes copper oxides commercially applicable in paints, fabrics, agriculture and in production of medical devices to prevent potential infections . Such wide use and reactivity pose many concerns for human and environmental health and necessary precautions should be taken to reduce the risk related to occupational or environmental exposure.
Different mechanisms of action have been suggested to explain the antimicrobial activity of CuO NPs depending on the original oxidative state of copper (I and II) in the nanomaterial and his tendency to be released in the medium, and the same properties could be partially responsible for the observed cytotoxicity in mammalian cells. Finally, a recent work performed by  was able to show in detail the mechanism of action of Cu/Cu5Zn5Al1SnASW through High resolution microscopical studies, against bacteria.
In the current study, we investigated the toxicity mechanisms of sonochemically synthesized CuO NPs in comparison with commercial ones, on A549 cells for very short time (1h and 3h). The aim was to 1) identify specific NP physico-chemical features responsible for the precocious toxicity (e. g. crystalline defects, surface reactivity); 2) understand the role of particle internalization and the Lysosomal Enhanced Trojan Horse mechanism at early exposure stages in inducing cell death; 3) define suitable and precocious marker for the NP-related oxidative effects which could be considered as key events in the view of an AOP-oriented testing strategy. These aspects are crucial to be considered for designing safer or more active biocidal agents.
3.1 Role of P-chem properties in the precocious effects of CuO NPs
The physical and chemical properties of metal-based nanoparticles drastically influence their toxic effects.
The particles used in this study were fully characterised by combining several techniques (HRTEM, DLS, ICP-OES), while information about ESR, XRD and DSC analyses could be found in previous works [2, 21].
The two CuO NPs have quite similar primary size respectively of 34,4±0,8 nm (nCuO) and 24,0±0,3 nm (sCuO) but present a different crystalline structure, as confirmed by HRTEM, and ROS generation potential. cCuO nanoparticles appeared spherical in shape, with a smooth surface and a coherent diffraction volume of tens nanometers. Electron diffraction patterns suggest that their crystallite size is bigger than the one of sCuO, which shows indeed more structural defects as already demonstrated in a previous work by DSC analyses . As previously discussed, surface defects are one of the potential triggers for ROS production, making those particles potentially more hazardous when interacting with biological structures compared to cCuO.
The presence of corners, edges, or defects (increased abrasiveness) were found positively correlated with the increase of particle toxicity, potentially because (i) the increased area helps in adsorption and binding of active compounds and (ii) the increased surface defects also increases the surface area-to-volume ratio which has a direct effect on ROS generation .
A surface with defects and corrugation it’s characterised by high energy due to the presence of dangling bonds that are finally responsible of the local electron density and reactivity .
In addition,  calculated conduction bands for several metal oxide nanoparticles and they found that the value estimated for CuO lies in the range of biological redox potentials, suggesting that the cytotoxic effects observed for this material might depend on its ability to act as a catalyst; this material is potentially capable of accepting electrons from the oxidation of biological matter and subsequently transferring them to molecular acceptors forming reactive compounds.
The first parameter we tested to compare the two compounds was the cell viability. Although both CuO NPs were able to induce a significant and concentration dependent cytotoxicity on A549 within the first hour of exposure, sCuO resulted to have a greater cytotoxicity compared to cCuO after 3h and the differences became even more visible after 6h [see Additional file 2]. Together with the surface defects also the size and aggregation could have played an important role. In a recent paper , CuO NPs with the same primary size as sCuO have been shown inducing higher cytotoxicity in human epithelial cells than NPs with the same composition but smaller in size (4nm), normally considered as more toxic. The results were partially attributed to the nanoparticle size itself, which probably facilitated and promoted the internalization and then the intracellular accumulation of NPs.
Besides the size, solubilisation of toxic ions is also recognized as a leading mechanism of metal-based NPs effect.
After 3h of incubation in complete culture medium (CCM) in a cell-free experiment, the estimated extracellular ion release from sCuO was around 50% and most of it occurred within the first hour, whether only 10% was determined when cCuO (50 mg /ml) was incubated in the same conditions.
Although we found that sCuO is partially soluble in CCM already during the first hours of incubation, the toxicity was practically independent from the extracellular solubility of CuO for both the NPs. Indeed, after exposure to copper ions at the maximum theoretical concentration released by 100 mg/ml CuO NPs (corresponding to 80 mg/ml of soluble copper) we didn’t observe any reduction in cell viability and these findings agree with previous studies in which the cell death induced by Cu++ exposure was significantly lower than that produced by NPs . In other papers, it was even reported that the presence of small concentrations of copper could stimulate growth, as found in human glioma, astrocytoma, and neuroblastoma cells .
Since sCuO was more soluble than cCuO in culture medium and, in parallel, the observed toxicity induced by sonochemical particles themselves was higher, we can assume that the intrinsic toxic potential related to the structure reactivity of the sonochemical CuO itself is very high.
3.2 Role of NPs internalization and the Lysosomal Enhanced Trojan Horse mechanism at early exposure time
In our previous study, where A549 cells were exposed to cCuO NPs for 24h, we attributed the responsibility for the lysosomal destabilization and for cell autophagy processes to the oxidative burst induced by the massive intracellular copper release from CuO NPs taken up through the endo-lysosomal pathway .
This phenomenon, known as “Lysosome-Enhanced Trojan Horse effect” (LETH), has been suggested as a general mechanism to explain the cytotoxicity of different metal containing nanoparticles (such as metallic, metal oxide, and semiconductor NPs) . In that work, authors pointed up on the role of the endocytosis as the starting point for CuO NP toxicity and identified copper ions as the main mediators. Moreover, they found that particles entering cells following the endocytic process resulted in a very strong cytotoxic effect, while particles internalised directly in the cytosol by a non-energy dependent mechanism were non-toxic.
In the present work, the attention was focused on short-term events and precocious oxidative phenomena that characterize the cytotoxic mechanism induced by CuO NPs exposure. It has been demonstrated that, at 1h and 3h post-exposure, the intracellular ion dissolution, involving the lysosomal compartment, was not responsible for the observed cytotoxicity since we did not detect any intracellular release (or limited) of copper ions through the rhodanine staining, in parallel to cell viability decrease. After short-term exposure we can hypothesize that for cCuO endocytosis works as temporary and protective mechanism to reduce particle toxicity. Cells try to modulate the stress induced by the interactions with particles through the endocytic process but, in this way, they internalise big amount of NPs, later able to release ions in the intracellular environment and to induce the cell death through LETH at later stages (24h) .
In sCuO-exposed A549 cells we suggest that the precocious oxidative damages observed were independent from LETH mechanism, supporting once again the idea that sCuO NPs are able to act very quickly depending on their own surface reactivity.
The first biological barrier encountered by particles is the plasma membrane, but that interaction could only partially explain the precocious cytotoxic effect induced by CuO NPs. Apparently, NP internalization is mandatory to start the cytotoxic process as confirmed by viability results obtained by co-incubating cells with NPs and cytocalasin D (CytD). This molecule is known to inhibit energy dependent mechanisms of internalisation even if the exact endocytic route seems to be cell-type specific [40, 41]. In our study, we observed a significant reduction in cell mortality only after co-incubation of sCuO (25, 50 mg/ml) with 4 mM CytD. This evidence confirms that the endocytosis of particles is playing a pivotal role even in promoting the very early toxicity of sCuO.
Furthermore, blocking the proton pumps responsible for the lysosomal acidification (by pre-incubating cells with Bafilomycin A1) we did not observe any recovery in term of cell viability after exposure to both CuO NPs at the short exposure times used for this study. Thus, sCuO does not necessarily need to follow the lysosomal pathway or being dissolved by the surrounding acidic environment to exert its toxicity. In fact, as shown by TEM analyses, the cell ultrastructure looked affected by the presence of sCuO free aggregates (Fig. 9d), which in turn can be considered responsible of the increased level of oxidized proteins (Fig. 4).
Likely, these particles are more prone to generate free radicals because of their size and surface defects leading to the oxidization of macromolecules (DNA, lipids and proteins) and resulting in significant oxidative stress and cell death, as previously suggested for CuO NPs by .
In particular,  observed that CuO NPs clearly localised in both mitochondria and cell nucleus after being taken up by A549 cells through endocytosis. There they stimulate ROS via impaired electron transport chain inducing structural damage, activation of NADPH-like enzyme system, and depolarization of the mitochondrial membrane  which then resulted in apoptosis .
In our study around 70% of the cell population turned out to be positive to annexin V after 3h exposure to sCuO (by flow cytometry analysis), confirming apoptosis as main mechanism driving to cell death. Usually, cancer cells tend to avoid apoptosis and continue to propagate  but it has been demonstrated that increased ROS level in cancer cells alters the mitochondrial functions and plays a key role in the apoptosis induction [46-48]. Moreover,  found apoptosis as main mechanism of cell death in cancer cells exposed to different sizes of CuO NPs. The involvement of oxidative events in the process driving to cell death in our model was testified by the viability recovery observed in presence of NAC, which works as ROS scavenger.
Co-incubation with N-acetyl cysteine was able to significantly prevent cytotoxicity after exposure to sCuO (25, 50 mg/ml) even if the oxidative insult induced by the highest concentration of NPs was probably too strong to be totally inhibited thus, we cannot exclude the possibility of a direct damage provoked by the particles themselves .
Further investigations therefore are needed to understand the potential of sCuO nanoparticles to target specifically cancer cells, preserving the normal ones, in order to evaluate the possibility of applying this material as potential and effective agent in anticancer therapies, of course in combination with proper surface functionalization. The use of more realistic ad complex models like 3D co-culture systems and organoids, could additionally help in the assessing of this targeting ability.
Although the uptake of particles was observed also in cCuO- exposed cells, NP internalization seems to have a negligible contribution to the precocious cytotoxicity induced by this compound. As testified by the experiments performed co-incubating cells with particle and cytocalasin D, no significant recovery in cell viability was observed.
Only after 6h of exposure, there was evidence of activation of LETH that could be responsible for later effects, here not investigated. In fact, intralysosomal dissolution of copper was detected in 6h-exposed cells, as testified by the presence of rhodanine-positive precipitate in the cytoplasm [see Additional file 3].
We know from TEM and DLS analyses that the single particle size and the hydrodynamic diameter of the commercial CuO are bigger than those of the sonochemical CuO. These findings suggest a potential high deposition rate on the top of the cell layer, as also evident by our SEM analyses, with a consequent increase of interaction between particles and cellular proteins or lipids as suggested by .
3.3 Precocious markers for CuO-induced oxidative stress
As reported in , in living cells ROS as H2O2 and O2-- can be converted into the more reactive hydroxyl radical, OH-, which can cause DNA-strand breaks, damage membrane lipids or attack proteins.
Proteins are particularly sensitive to the presence of ROS/RNS. The interaction between amino-acids residues with this chemical species or with different intermediates generated by the oxidation of other cellular compounds (as lipids and carbohydrates) could have repercussions on their activity, unfolding, degradation, and, ultimately, on cell functioning [53 – 56].
The increase in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) plays an important role in copper-induced organic dysfunction . Moreover, we already hypothesized that the precocious events involved in the early cytotoxicity of CuO could depend on CuO NP specific action against thiol groups as already suggested in .
For these reasons, we investigated also thiolation and carbonylation as potential markers for reversible and irreversible protein modification respectively.
Here, we have the evidence that showed how nCuO is powerful in inducing thiol group oxidation and protein carbonylation in A549 cells after short exposure time at high concentration (100 mg/ml), with sCuO retaining such effect also at lower concentrations, which testifies that the sonochemically synthesized nCuO is a stronger oxidating agent compared to the commercial form and that thiolation could be consider a good marker for nCuO precocious biological reactivity and toxicity. Decline in the levels of NPSH (non-protein thiol groups) and PSH (protein-thiol groups) were also observed in the liver and kidney as reported in .
In particular for sCuO we observed precocious irreversible oxidative damages to biomolecules, as confirmed by the immunochemistry of protein carbonyls, since after 3h-exposure a significant protein carbonylation increase was detected in A549 cells treated with 50 mg/ml sCuO. Under these exposure conditions, size reduction and morphological changes on cells were detected by SEM. Thus, our results suggest that sCuO NPs activate a cell death pathway through an oxidative stress that involves at first proteins and then lipids and is reasonably independent from extracellular and intracellular release of copper ions, but likely depends on the defects of the crystalline structure of the particles, which ultimately dictates the ability to locally generate ROS.