Oxidative stress (over production of reactive oxygen species, i.e., ROS) induced by NPs could damage the cellular components and lead to cell death via apoptosis (Fu et al. 2013). Therefore, studies reporting on cytotoxicity and oxidative stress were summarized in this section.
Cytotoxicity associated with oxidative stress
Duan et al. (2013a) showed that S-SiNPs (62 nm) induced time- (6, 12, and 24 h) and dose-dependent (25–100 µg/ml) reduction in cell viability (assessed by 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide, i.e., MTT), loss of membrane integrity (lactate dehydrogenase (LDH) release) and apoptosis (Annexin V/PI staining) in human umbilical vein endothelial cells (HUVECs). Apoptosis was also induced in lung (A549) and skin epithelial cells (A431) treated with Pr-SiNPs (15 nm). A dose-dependent increase (25–200 µg/ml for 72 h) in cytotoxicity (MTT and LDH), ROS production (assessed by dichlorodihydrofluorescein assay, i.e., DCFH-DA), lipid peroxidation (measurement of malondialdehyde, i.e., MDA), and apoptosis (caspase 3 and 9 activity) was observed in both cell lines. The lung cells showed, in general, a slightly higher toxic response compared to skin cells (Ahamed 2013).
SiNPs (20 and 80 nm) induced P53-mediated apoptosis in human fetal lung fibroblasts (HFL-1). At the dose of 500 µg/ml, 20 nm SiNPs induced a threefold increase in DCF fluorescence compared to 80 nm. In addition, increased expression of P53, upregulation of cytochrome C (CytC) and caspase 9, and downregulation of anti-apoptotic protein B-cell lymphoma 2 (bcl2) was observed in cells treated with 1000 µg/ml for 48 h (Xu et al. 2012). Another study with lung fibroblasts also showed that SiNPs (20 nm) could reduce cell viability (MTT) by inducing apoptotic cell death (fluorescence microscopy) in a dose-dependent manner (250–1000 µg/ml for 48 h) (Zhang et al. 2011). Athinarayanan et al. (2014) isolated SiNPs (10–50 nm) from commercial food products processed with food additive silica (E551) and exposed human lung fibroblasts (WI-38 cell line) with increasing doses (25–400 µg/ml). After 24 h, they observed cytotoxicity (MTT) in a dose-dependent manner and ROS production (DCFH-DA) at 50 µg/ml.
Cytotoxicity not associated with oxidative stress
Py-SiNPs (12 and 40 nm) induced a significant size and dose- (31.3, 93.8, and 156.3 µg/cm2 culture well) dependent cytotoxicity (LDH, Sulphorodhamine B assay (SRB) and water-soluble tetrazolium-1(WST-1)) in human colon carcinoma cell line (HT29), while no induction of ROS (DCFH-DA) was observed (Gehrke et al. 2013). In the study by Napierska et al. (2012a), 50 μg/ml (24 h) of 16 nm iron-doped S-SiNPs and pure S-SiNPs induced strong cytotoxicity (MTT and LDH) in a human endothelial cell line (EA.hy926), but a significant increase in oxidative stress markers [GSH depletion, malondialdehyde (MDA formation), induction of heme oxygenase-1, glutathione reductase, and NADPH oxidase-1] was observed only for iron-doped SiNPs.
Cytotoxicity of SiNPs was investigated using different cell lines and incubation times, making the comparison between studies difficult. However, from Table 3, it is clear that all types of SiNPs induced cytotoxicity. Significant (compared to untreated cells) cytotoxic effects were observed only at or above the concentration of 25 µg/ml. Furthermore, it can be clearly seen that SiNPs induced oxidative stress and mediated apoptosis mainly via the intrinsic or mitochondrial pathway (caspase-dependent pathway) in a size- and dose-dependent manner. ROS-mediated toxicity is believed to be an important mechanism of NP toxicity including SiNPs (Manke et al. 2013). Nevertheless, Py- and S-SiNPs caused cytotoxicity without measurable levels of ROS production. It was demonstrated that the disturbance of membrane integrity due to direct cell-membrane interaction might be another possible mechanism of NP cytotoxicity (Fröhlich et al. 2009; Thomassen et al. 2011). However, neither of these studies did substantiate these observations and, therefore, SiNPs cytotoxic effects in the absence of oxidative stress remain poorly understood.
Furthermore, some authors used very high concentrations that may cause “overloading” of cells and modify the nature of NP–cell interactions (Wittmaack 2011). In these cases, it is difficult to evaluate whether the observed effects are physiologically relevant. Although it is challenging, we consider a dose of 384 µg/cm2 or higher as irrelevant to human inhalation exposure for amorphous silica, based on the estimation that can be derived from the occupational exposure levels (OELs) (Fig. 2).
In this section, we presented studies on genotoxic effects of SiNPs as it is used as another major endpoint to characterize hazard of NMs. Direct interaction with DNA, oxidative DNA damage, depletion of anti-oxidants, cell cycle arrest, and abnormal expression of genes have been identified as potential mechanisms of NP mediated (geno)toxicity (Donaldson et al. 2010).
DNA damage associated with oxidative stress
Exposure to SiNPs (15, 30, and 100 nm) resulted in a size- and dose- (2.5–10 µg/ml for 24 h) dependent increase in 8-hydroxy-2′-deoxyguanosine levels (8-OH-dG), phosphorylation of histone on serine-139 (ɣH2AX), and DNA strand breaks (comet) in human keratinocytes (HaCaT) (Gong et al. 2012). Nabeshi et al. (2011a) also demostrated that exposure to SiNPs (70 nm; 10–90 µg/ml for 24 h) resulted in the increase of oxidative DNA damage (8-OH-dG levels) in HaCaT cells. SiNPs were taken up via actin-mediated endocytosis. Micron-sized particles used in these studies showed no or little effects.
The viability of human Caucasian colon adenocarcinoma (Caco-2) cells dropped to 40% when exposed to 15 nm C-SiNPs (64 µg/ml for 24 h), and, at this same concentration, nearly a threefold increase in micronuclei formation, fivefold increase in histone phosphorylation (ɣH2AX), and a significant increase in DCF fluorescence were observed. The particles were localized within lysosomes and endocytic compartments, but not in the nucleus. 55 nm C-SiNPs did not induce any of these effects at the same concentration (Tarantini et al. 2015b).
A non-significant increase in % tail DNA (comet assay) and no chromosomal aberrations were induced by 17 nm SiNPs in human peripheral lymphocytes treated with 100 µg/ml, while a dose-dependent (50–100 µg/ml for 24 h) ROS production (DCFH-DA) and GSH depletion were observed (Rajiv et al. 2015).
Cell cycle arrest associated with oxidative stress
S-SiNPs (62 nm) induced increase in DCF fluorescence and decrease in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in HUVECs in a dose-dependent manner (25–100 µg/ml for 24 h). Oxidative stress was linked to cell cycle arrest at G2/M checkpoint (upregulation of chk 1 and downregulation of Cdc25c, Cyclin B1, and Cdc2) and increase in apoptosis (Duan et al. 2013a). In the study by Li et al. (2011), a size-dependent (19, 43, and 68 nm) increase in oxidative stress (DCF fluorescence) and cell cycle arrest in S and G2/M was observed in HepG2 cells exposed to 100 µg/ml of C-SiNPs. Cell cycle arrest in G2/M phase along with the increase in ROS was also noticed in human hepatic cell line (LC-02) treated with S-SiNPs (50 nm) in a dose-dependent manner (50–200 µg/ml for 24 h) (Wang et al. 2013).
DNA damage not associated with oxidative stress
Genotoxicity of Py-SNP (20 and 25–70 nm), Pr-SNP (20 nm), and C-SNP (15 and 40–80 nm) SiNPs were studied in Chinese hamster lung fibroblasts. Py-SiNPs (20 nm) induced a significant increase in DNA strand breaks at 66 µg/ml (24 h), while C-SiNPs (15 nm) showed a similar effect only at 252 µg/ml. Neither of these SiNPs did induce ROS. SiNPs in the size range 25–80 nm exerted no or little genotoxicity (Guichard et al. 2016).
Genotoxicity reports without the assessment of oxidative stress
M-SiNPs (100 nm) induced a significant increase in phosphorylated-ɣH2AX-foci in HT-29 cells treated with a dose of 10 µg/ml for 24 h (Sergent et al. 2012). In the human embryonic kidney cell line (HEK293), 579 genes were upregulated and 1263 genes were downregulated after 24 h of exposure (100 µg/ml) to 100 nm M-SiNPs (Zhang et al. 2015). In another study, 15-nm C-SiNPs induced a significant increase in DNA strand breaks (comet assay) in chinese hamster cells (V79) and A549 cells at 100 µg/ml (24 h), but, for 55 nm, this effect was observed only in A549 cells (Maser et al. 2015). A significant increase in DNA tail length (comet assay) was observed in HaCaT cells treated with 30 µg/ml (24 h) of 70 nm SiNPs (Nabeshi et al. 2011b).
Exposure to C-SiNPs (~7 nm) resulted in positive genotoxic effects (Lymphoma assay) in mouse lymphoma cells treated with 100 and 150 µg/ml for 4 h (Demir and Castranova 2016). SiNPs (12 nm) induced DNA strand breaks in RAW 264.7 at 200 and 400 µg/ml, but the induction of micronuclei was noticed only at 400 µg/ml. The particles were internalized in vesicles and in the nucleus (Hashimoto and Imazato 2015).
At any tested concentrations (1–100 µg/ml for 72 h), SiNPs (10–25, 5–30, 35, 15, 80, and 90 nm) neither induced cytotoxicity nor micronuclei in immortalized Balb/3T3 fibroblasts (Uboldi et al. 2012). In another study, Pr-SiNPs (NM-200 and NM-201) and Py-SiNPs (NM-202 and NM-203) with primary size between 14.5 and 16 nm did not induce any micronuclei (cytokinesis block micronucleus assay) in human peripheral lymphocytes exposed to different concentrations (200–1250 µg/ml) over 24 h (Tavares et al. 2014). It is also worthy to note that, in the latter study, the positive control used did not differ from control conditions.
C-SiNPs and S-SiNPs induced genotoxicity in human tumor cell lines (lung, kidney, skin, and gastro-intestinal systems) and the amplitude of the effect negatively correlated with the size of the NPs. DNA strand breaks were observed at low concentrations (2.5–10 µg/ml), particularly in skin-derived cell lines. The genotoxic effects of C- and S-SiNPs were mainly associated with the induction of oxidative stress, while such information is very limited for other types (Py- and Pr-SiNPs). One study indicated that Py-SiNPs induce DNA damage without the generation of ROS, suggesting that other mechanisms such as direct DNA damage might be involved (Magdolenova et al. 2014). However, it is very difficult to judge whether such genotoxic effect is direct or indirect, since the cellular uptake and subcellular localization of SiNPs are not often reported. Furthermore, several factors such as SiNP properties, cell type, and exposure scenarios (such as concentrations, assays, and endpoints) may influence the outcomes (Magdolenova et al. 2014), making the comparison difficult between studies and indicating an urgent need for the standardization of genotoxicity studies.
NPs entering the body will most probably interact with immune cells, as they are the first line of defence in human body. In this section, we presented the immune responses induced by SiNPs in different cell lines.
Immunotoxicity associated with oxidative stress
Hara et al. (2014) exposed THP-1-derived macrophages to 100 µg/ml of SiNPs (30 nm) for 6 h and found a significant increase in interleukin-1-beta (IL-1β), ROS production, and SiNP uptake via phagocytosis. In the study of Choi et al. (2010), larger sized SiNPs (150–200 nm) were effectively phagocytosed by primary rat microglial cells after 24 h of exposure to different concentrations (0.0728–7.28 µg/ml). A significant increase in ROS, reactive nitrogen species (RNS) and IL-1β was detected at all concentrations.
Immunotoxicity not associated with oxidative stress
At 10 and 20 µg/ml, Di Cristo et al. (2016) found that Py-SiNPs (~14 nm) induced a stronger increase of tumor necrosis factor-alpha (TNF-α), interleukin(IL)-6, and IL-1β in RAW.264.7 macrophages compared to similar sized Pr-SiNPs; Notably, no SiNPs induced ROS in RAW.264.7 macrophages.
Immunotoxicity reports without the assessment of oxidative stress
A significant increase in TNF-α, IL-6, and IL-1α, mitogen activated protein kinases (MAPKs), and nuclear factor (NF)-κB were observed only for C-SiNPs (100 nm) in J774A.1 macrophages exposed to 100 µg/ml of same sized (100 nm) C-SiNPs or M-SiNPs (Lee et al. 2011).
Uemura et al. (2016) showed that SiNPs (10 and 50 nm) caused dose-dependent (6.25–100 µg/ml) increase in the production of TNF-α and decrease of IL-6 in RAW.264.7 macrophages, while their amine surface-modified counterparts did not. Furthermore, 300 and 1000 nm micron-sized particles (both bare and amine modified) also showed a dose-dependent decrease of IL-6. Notably, the effects were stronger for 50 nm compared to other particles. The same cell line was utilized by Yu et al. (2011) to investigate phagocytosis using inductively coupled plasma mass spectroscopy (ICP-MS), and they found that S-SiNPs (25 nm) were phagocytosed at least ten times more than M-SiNPs of same size and high aspect ratio SiNPs (AR 2, 4, and 8). In the study by Napierska et al. (2012b), THP-1 cells dosed with 5 µg/cm2 S-SiNPs (2 nm) showed a significant increase of IL-8, TNF-α, and macrophage inflammatory protein (MIP)-1α, while only a non-significant increase in MIP-1α expression was observed for 16 and 104 nm S-SiNPs.
The main cells used to study immune responses to SiNPs were ‘innate’ cells such as monocytes and macrophages. Therefore, the identified in vitro studies only address a very limited part of the immune system, essentially pro-inflammatory responses and potential phagocytosis. Furthermore, the data on immune responses and oxidative stress are very limited and, therefore, no firm conclusions can be made. SiNPs, not only induced stronger pro-inflammatory responses compared to sub-micron and micron sized particles but also size-specific effects within the nano-range in immune cells are observed. Besides size, shape and porosity seem to influence the phagocytosis of SiNPs.
Recently, a growing body of evidence identified autophagy as a cellular defence mechanism against NP toxicity, since it plays a key role in removing misfolded proteins and clearing damaged organelles (Glick et al. 2010). Hence, we present here studies that show induction of autophagy upon exposure to SiNPs.
Autophagy associated with oxidative stress
The same S-SiNPs (62 nm) were used in three studies to investigate the induction of autophagy. Along with the dose-dependent (25–100 µg/ml) increase in ROS production, increase in autophagy bio-marker-microtubule-associated protein 1A/1B-light chain 3 (LC3) and monodansylcadaverine (MDC) labelled autophagic vacuoles were detected in HepG2 cells treated with 62 nm S-SiNPs. In addition, transmission electron microscopy (TEM) images revealed that autophagosomes and autolysosomes induced in the presence of SiNPs (Yu et al. 2014). The same S-SiNPs (62 nm) induced increase of LC3-II/LC3-I ratio and decrease of p-mTOR/mTOR, p-P13 K/P13 K and p-Akt/Akt in HUVEC cells in a dose-dependent (25–100 µg/ml) manner (Duan et al. 2013b, 2014a, b). The results of Guo et al. (2016) also suggest that 50 µg/ml of S-SiNPs (58 nm) could induce autophagy via MAPK/Bcl-2 and PI3K/Akt/mTOR signaling in HUVECs.
After 4 h of exposure to 200 µg/ml of S-SiNPs (50 nm), autophagosomes and ROS production was observed in HaCaT cells. The TEM images revealed that SiNPs were in the cytoplasm and lysosomes, but not in the nucleus. (Liang et al. 2014).
SiNPs (4–13 nm, 62.5 µg/ml) induced a time-dependent (24, 48, and 72 h) reduction in cell viability and increase in oxidative stress (DCF fluorescence and GSH depletion) in the lung fibroblast cell line MRC-5. Compared to control, a significant increase of autophagic vacuoles and LC-3 II/LC3-I ratio was also observed in a time-dependent manner (Voicu et al. 2015). A549 cells, when exposed to 100 and 1000 µg/ml of 20-nm SiNPs, showed threefold and fivefold increase in MDC fluorescence, respectively. In addition, autophagy genes such as ATG-12 and BECN were significantly upregulated (30- and 50-fold, respectively) along with increased production of ROS in cells dosed with 1000 µg/ml (Nowak et al. 2014).
SiNPs, particularly S-SiNPs induced autophagy mainly via oxidative stress-mediated upregulation of autophagy-related genes and differential regulation of Akt/mTOR signaling. Similar to cytotoxicity, 25 µg/ml appeared to be the lowest exposure concentration at which SiNPs exhibited significant effects. Furthermore, induced autophagy is correlated to cytotoxicity, suggesting that exposure to SiNPs caused irreversible (serious) cellular damage and resulted in autophagic cell death. Besides autophagy induction, lysosomal and autophagy dysfunction could be a potential mechanism of NPs toxicity (Stern et al. 2012), which has, however, not been investigated for SiNPs.
Toxic effects on blood cells and endothelial dysfunction
Several studies suggest that NPs, when inhaled or ingested, can translocate across barriers (such as air–blood) of the body, enter the circulation, and interact with the cardiovascular system. In this section, we summarized the studies that report the effects of SiNPs on blood and endothelial cells.
Toxic effects on blood cells
Nemmar et al. (2015) showed that mouse blood platelets could aggregate after 3 min of incubation with 5 or 25 µg/ml of 50-nm C-SiNPs, while in the study of Jose Corbalan et al. (2012), such aggregation was observed in 15 min (10 µg/ml of 10 nm C-SiNPs). The latter study also showed fourfold reduction of the nitric oxide (NO)/peroxynitrite (ONOO−) ratio compared to non-treated platelets.
Maurer-jones et al. (2010) investigated the role of porosity of SiNPs on blood cell toxicity. M-SiNPs (25 nm) reduced the cell viability of red blood cells (RBCs) to 50% at the concentration of 270 µg/ml, while non-porous S-SiNPs of similar size required only 20 µg/ml to reach this level of cytotoxicity. In another study, 10% hemolysis (LC10) of RBCs was observed at 36 µg/ml for S-SiNPs (115 nm), while M-SiNPs required 154 µg/ml to induce the same effects. For amine-coated counterparts, LC10 were 97 and 30 µg/ml for S- and M-SiNPs, respectively (Yu et al. 2011).
Exposure to different concentrations (12.5–100 µg/ml) of S-SiNPs (58 nm) resulted in a dose-dependent increase in inflammatory mediators such as IL-1β, IL-8, and TNFα, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) in HUVEC cells (Guo et al. 2015). In the study of Corbalan et al. (2011), 10 µg/ml of 10-nm C-SiNPs induced extremely low NO/NOO− ratio (~0.1) in HUVEC cells. Furthermore, free radical production, pro-inflammatory cytokines (IL-6 and IL-8), and NF-κB-binding activity were significantly increased in treated cells. In these two studies, increase in ROS was observed at all tested concentrations.
S-SiNPs (62 nm) induced an imbalance in the ratio NO/nitric oxide synthase (NOS) enzyme in HUVEC cells and such imbalance resulted in a significant increase of pro-inflammatory response (c-reactive protein CRP, IL-1β, IL-6, and TNFα) in a dose- (50–100 µg/ml) dependent manner (Duan et al. 2014b).
Conclusion: Toxic effects on blood cells and endothelial dysfunction
Endothelial cells and platelets together play a key role in maintaining the vascular homeostasis (Rajendran et al. 2013). C-SiNPs induced oxidative stress and disturbed NO/NOO− ratio, which resulted in the aggregation of platelets and endothelial dysfunction. This information is not available for other types of SiNPs. Furthermore, C-SiNPs mediated endothelial dysfunction resulted in pro-inflammatory signals via the secretion of cytokines and adhesion molecules. Together, these results suggest the potential of SiNPs to cause vascular thrombosis and atherosclerosis (Radomski et al. 2005). Furthermore, SiNPs caused hemolysis of RBCs in a size-, charge-, and porosity-dependent manner.
NPs of very small size are capable of translocating across the blood–brain barrier (Hu and Gao 2010). Therefore, studies investigating effects on cells relevant for neurotoxicity are presented here.
Rat medulla tumor cells (PC12 cell line) incubated with the supernatant of 20 nm SiNPs-treated-microglial cells (250 µg/ml and 500 µg/ml for 24 h) did not show any effects compared to the control. Earlier in this study, no secretion of bio-mediators was observed in SiNPs-treated-microglial cells (Xue et al. 2012). In contrast to this study, PC12 cells exposed directly to SiNPs (25 nm; 25–200 µg/ml for 24 h) showed increased uptake and a dose-dependent increase in the induction of autophagy (increase in LC-II and Beclin 1) and inhibition of PI3 K-Akt-mTOR signaling (Xie and Wu 2016). Yang et al. (2014a) showed that exposure to SiNPs (15 nm; 10 µg/ml for 24 h) induced pathological signs of Alzheimer’s disease such as altered expression of amyloid precursor protein (APP) and neprilysin, enhanced phosphorylation of tau at Ser262 and Ser396, and activation of glycogen syntheses kinase (GSK)-3β in human SK-N-SH and mouse neuro2a neuroblastoma cells.
In vitro studies used cell lines of CNS-based cells, mainly neuron like cells. Although the data on the neurotoxic effects of SiNPs are very limited, studies above suggest that SiNPs can induce adverse effects including the markers of Alzheimer’s disease, when in direct contact with neuroblastoma cells.
Influence of cell lines on SiNPs cytotoxicity
A dose- (80–640 µg/ml) dependent decrease in the viability and increase of apoptosis were observed in HepG2 cells in presence of SiNPs (7 and 20 nm), but a significant reduction was observed in normal human liver cells (LC-02) only at the unrealistic dose level of 640 µg/ml (Lu et al. 2011). In another study, SiNPs (10–50 nm) induced a dose-dependent (100–600 µg/ml) increase in LDH release in Caco-2 cells, but a significant release of LDH was observed only at the unrealistic dose of 600 µg/ml in human gastric epithelial cells (GES-1). Furthermore, exposure to these SiNPs (200 µg/ml for 48 h) also induced cell cycle arrest in S phase for GES cells and G2/M in Caco-2 cells (Yang et al. 2014b). In a porcine kidney cell line (LLC PK1) exposed to 20-nm SiNPs, a dose- (5–50 µg/ml) dependent increase in DCF fluorescence and MDA formation was observed, whereas human kidney cells (HK-2) showed little effects at 50 µg/ml (Passagne et al. 2012).
Conclusion: miscellaneous issues
No firm conclusions can be drawn from these cases; however, the cytotoxicity of SiNPs appears to vary with species and cell line.
Physiologically relevant cultures
Lung co-culture models
Co-cultures of lung cells are usually made with epithelial cells on the apical and endothelial cells on the basal compartment of a transwell membrane, with or without monocytes on top of the epithelial cells. In a co-culture (A 549 at the apical and ISO-HAS-1 at the basolateral compartment) exposed to 100-µg/ml 30-nm C-SiNPs (coated with or without surfactant), nearly a fivefold increase of IL-8 release for both forms of SiNPs was observed in both compartments (Kasper et al. 2015). When other epithelial cells were used—H441 cells—at the apical together with ISO-HAS-1 cells at the basolateral compartment, these C-SiNPs induced IL-8 were expressed in both compartments, while SiCAM-1 (6–600 µg/ml) and IL-6 (at 6 and 60 µg/ml) were observed only in the apical part (Kasper et al. 2011). The same co-culture model was exposed to 100 µg/ml of S-SiNPs (15, 35, and 80 nm) and the authors noticed an increase of IL-8, TNF-α, and surfactant protein (SP-A1 and SP-A2) expression compared to the control. In addition, less IL-8 and surfactant protein expression, and more TNF-α were observed in the co-culture added with THP-1, notably the effect was the highest for 35 nm (Farcal et al. 2012).
Napierska et al. (2012b) tested SiNPs with primary size 2, 16, 60, and 104 nm (dosed at 10 µg/cm2) in a co-culture (A549 at the apical and EA. hy926 at the basolateral compartment) and observed increase in cytokines such as IL-6, IL-8, TNF-α, and MIP-1α only for 60 and 2 nm (except IL-8). When THP-1 were added to the co-culture, a significant increase in IL-8 and decrease in TNF-α were observed only for 2 nm. The expression of cytokines was also differentially regulated for 16 and 104 nm before and after THP-1 added, but the effects were stronger for 60 and 2 nm, particularly 60-nm NPs.
At the air–liquid interface (ALI), aerosolized and deposited 12-nm Py-SiNPs (52 µg/cm2) and 50-nm S-SiNPs (117 µg/cm2) induced significantly less biological effects (LDH leakage, IL-8 release, COX-2 expression, and p38 phosphorylation) in A549 cells compared to A549 exposed to 15.6 µg/cm2 under sub-merged conditions (Panas et al. 2014).
Conclusion: physiologically relevant cultures
Sub-merged (co) cultures and ALI systems (Lenz et al. 2013; Panas et al. 2014) have been claimed to more closely mimicking the in vivo exposure scenarios compared to monocultures. In these systems, the toxicity and pro-inflammatory responses are significantly modulated by SiNPs, which represent the complexity of in vivo systems and need for the establishment of physiologically relevant in vitro cultures. However, at this moment, it is difficult to know whether these biological responses were influenced by SiNPs physico-chemical properties.
Chronic in vitro studies
In vitro chronic Py-SiNPs (12 nm) exposure of intestinal epithelial cell line (C2BBe1) was examined in a recent study. The cells were exposed to 10-µg/cm2 SiNPs for 24 h. After 24 h, the medium was replaced (without NPs) and cells were allowed to grow for 4–6 days. At the end of incubation, cells were passaged and again exposed for 24 h and grown for 4–6 days; this cycle was repeated for 29 passages (total life span). The cells and supernatants were collected at the end of each passage for analysis. Though the particles were internalized (only in a fraction of cells), no significant induction of necrosis, apoptosis, and LDH release and decrease in cell viability was observed for any of these conditions (McCracken et al. 2013).