3.3.1. Cytotoxicity
The new materials developed for biomedical applications require the simultaneous fulfillment of three major conditions: biocompatibility, low toxicity, and biodegradability. Various nanostructures based on iron and silicon have been reported to be highly biocompatible [
65,
66,
67], and easily metabolized by the body as orthosilicic acid Si(OH)
4 or iron ions. The retention of NPs in the body primarily can negatively affect organs, including liver, kidneys, stomach, and intestines.
To assess the biocompatibility of hybrid Fe-Si NPs coated with
l-DOPA and CMC-Na, doses ranging from 0–200 µg/mL were exposed to Caco2 cells for 24 and 72 h. Untreated cells and cells treated with the individual components of NPs were used as controls. The results indicated no significant change in Caco2 cell viability exposed to concentrations between 0–100 μg/mL
l-DOPA-coated Fe-Si NPs and 0–50 μg/mL CMC-Na-coated Fe-Si hybrid NPs (
Figure 6). At the highest dose of 200 μg/mL, NP cell viability decreased after 72 h exposure by 29%, 43%, and 11% in the presence of Fe-Si2_
l-DOPA NPs, Fe-Si3_
l-DOPA NPs, and, respectively, Fe-Si7_
l-DOPA NPs. By comparison, Fe-Si7_CMC-Na NPs (200 μg/mL) were the most toxic, causing a decrease by 73% of cell viability after 72 h of exposure, compared to untreated control. By comparison, the decrease of viability in the Caco2 cells exposed to uncoated NPs was lower, the maximum decrease was induced by 200 μg/mL Fe-Si NPs after 72 h. Surprisingly, the most toxic component for Caco2 cells was
l-DOPA which has an IC
50 value of 81.2 μg/mL. However, the IC
50 values for the
l-DOPA stabilized NPs were higher compared with those stabilized with CMC-Na (
Table 4).
By now,
l-DOPA and CMC-Na have been used to stabilize various NPs, including iron oxide [
68], magnetic Fe-MWCNT [
69], Fe@C [
70], zinc oxide (ZnO) [
71], silica (SiO
2), and titania (TiO
2) ones [
72], as well as anticancer drug carriers [
73] etc., but the interactions with cells and tissues were not entirely described and understood.
l-DOPA is an amino acid, precursor of dopamine, biosynthesized naturally by a number of plants and animals. In humans, it is obtained through the metabolic pathway of catecholamines, being biosynthesized directly from
l-tyrosine. Some in vitro studies have reported toxic effects of
l-DOPA [
74,
75], whereas others have demonstrated a protective action, similar to the beneficial effects of some dopamine receptor agonists. Furthermore, the results of many in vivo experiments, as well as of clinical trials, did not demonstrate
l-DOPA toxicity or remained inconclusive [
76]. On the other hand CMC-Na “generally recognized as safe” (GRAS) by the US Food and Drug Administration is widely used in diverse industries including medical applications. The in vitro and in vivo biocompatibility CMC has been demonstrated [
22,
77] as well as limited biodegradation by glucose residues releasing [
78].
In our case, as resulted from
Figure 6, NPs coated with CMC-Na presented a higher toxicity compared with those coated with
l-DOPA at high doses in Caco2 cells.
The release of lactate dehydrogenase (LDH) into the surrounding culture medium was assessed as a marker for membrane integrity and necrotic events. Upon incubation with all tested suspensions, high activity of LDH was found in the medium of Caco2 cells exposed to stabilized NPs and to
l-DOPA (
Figure 7). A lower increase of LDH level was also noticed after incubation of Caco2 cells with Fe-Si2, Fe-Si3, and Fe-Si7 NPs compared to untreated control, whereas no release of LDH was observed after cell exposure to Fe-Si7_CMC-Na NPs and CMC-Na alone. These data were in accordance with the results of MTT test and suggest that stabilization of hybrid Fe-Si NPs with
l-DOPA and CMC-Na could influence the interaction with Caco2 cells and increases their toxicity at higher doses.
3.3.2. Cell Morphology and Dispersion of NPs in Culture
To study the morphology of treated Caco2 cells, two doses of 25 and 50 µg/mL of Fe-Si NPs were chosen based on cytotoxicity test outcomes. We inspected the filamentous actin cytoskeleton of Fe-Si NP-treated Caco2 cells as a measure of preservation of the overall cellular architecture. Untreated cells showed a regular structure made up of aligned and tightly compacted F-actin or actin bundles (
Figure 8a). After incubation of Caco2 cells with each type of NP, no obvious changes in the cortical F-actin system were caused by doses of 25 μg/mL (data not shown). The corresponding morphologic structures remained detectable underneath the plasma membranes of most cells. However, some alterations were noticed in cells exposed for 72 h to 30 µg/mL
l-DOPA and 50 µg/mL Fe-Si3, Fe-Si3_CMC-Na, and Fe-Si7_CMC-Na (
Figure 8b). Formation of some F-actin aggregates and/or inclusion bodies and a great proportion of actin bundles accumulated on cell periphery were noticed (
Figure 8a). By contrast, F-actin cytoskeleton was largely reserved after incubation with Fe-Si7 and Fe-Si7_
l-DOPA samples. As stated above, we show that cell viability was not compromised by the presence of the F-actin alterations.
The degree of internalization was different between Fe-Si NPs samples. Unstabilized Fe-Si NPs presented a low dispersion in the aqueous suspension, as well as in the culture medium, which hindered the internalization on cellular level. The large aggregates of NPs are impossible to penetrate the cellular membrane, and so the interaction with the cells remained, for the most part, limited to the outer membrane. All stabilized suspensions of NPs were initially well-dispersed solutions, but after their addition in the culture medium,
l-DOPA -coated NPs have formed large aggregates, especially the Fe-Si7_
l-DOPA sample (
Figure 9), probably due to the presence of ionized carboxyl and amino groups that could interact electrochemically between individual NPs and between NPs and amino acids and proteins from the culture media.
Figure 9 shows the dispersion of hybrid Fe-Si NPs in the cell culture medium, and their internalization in the Caco2 cell cytoplasm. The Fe-Si NPs stabilized with CMC-Na presented a better dispersion in medium compared with those stabilized with
l-DOPA. As it can be seen, no internalization was observed for Fe-Si7_
l-DOPA sample.
In a culture medium environment, rich in serum proteins, NPs are covered by the so-called protein corona, which is influenced by the surface properties of the coated NPs in terms of type and amount of absorbed proteins. This protein corona plays an important role in their interaction with the cells and tissues, and thus in biological responses, therapeutic efficiency, and toxicity of NPs [
79].
l-DOPA and CMC-Na are chemically different. In the culture medium (pH 7.4),
l-DOPA has a neutral charge. According to several protein corona studies, neutral surfaces are prone to adsorb high amount of serum proteins, which can result in higher blood circulation time in vivo [
80,
81]. Carboxylic acid groups can act as anchor points for addition of secondary molecules by covalent bonding with amine groups, e.g., via carbodiimide chemistry [
80].
On the contrary, CMC sodium salt is an anionic derivative of cellulose, negatively charged at neutral pH (pKa of 4.3), in which the hydroxyl groups are partially or fully substituted by carboxymethyl groups (–CH2–COOH). Due to the ionic nature of CMC-Na, it can interact with proteins to form soluble and stable complexes. Also, the polar groups of CMC-Na (–OH, –COOH) can react with metal ions (Fe2+, Fe3+, Ca2+, Mg2+) by electrostatic forces.
The formation of aggregates in cell culture medium favors the absorption of more serum protein onto the NP surface, so the interaction of NPs with cell membrane is quite limited.
In the case of Fe-Si7_
l-DOPA sample, the interaction occurred most likely at the cell membrane surface, thus explaining the increased level of LDH, starting with 24 h of incubation with 100 and 200 µg/mL doses. Also, the NP uptake was low. On the contrary, CMC-Na NPs (Fe-Si7_CMC-Na sample) presented a high dispersion in the culture medium and a high internalization in Caco2 cellular cytoplasm occurred (
Figure 9). Similar changes were registered for the other CMC-Na coated NP samples. This could indicate that mechanism of toxicity is influenced by the type of coating and degree of dispersion in the culture medium.
3.3.3. Oxidative Stress
The generation of reactive oxygen species (ROS) in Caco2 cells was analyzed up to 4 h of exposure to 25 and 50 µg/mL hybrid Fe-Si NPs. As shown in
Table 5, the production of ROS was time-dependent. The unstabilized Fe-Si NPs generated the highest amount of ROS. All samples CMC-Na-coated Fe-Si NPs induced ROS post-exposure at both doses of 25 and 50 µg/mL. In the case of
l-DOPA-coated Fe-Si NPs, higher ROS levels were obtained for 25 µg/mL dose. No significant increase of ROS levels was registered in Caco2 cells exposed to Fe-Si7_
l-DOPA NPs. Furthermore, a similarity between the results obtained for a dose of 25 µg/mL Fe-Si 2_
l-DOPA and Fe-Si2_CMC-Na samples, as well as of Fe-Si3_L_DOPA and Fe-Si3_CMC-Na ones, was observed, which suggests that ROS generation at a low dose of NPs is not influenced by the nature of stabilizer. However, it is clear that a 50 µg/mL dose of
l-DOPA-coated Fe-Si NPs induced less ROS compared to CMC-Na-coated Fe-Si NPs. Moreover, we showed that stabilization of Fe-Si NPs with
l-DOPA and CMC-Na reduced considerable the ROS production.
According to Zhou et al., studies [
82], CMC-Na could play a major role as protective coating by decreasing the toxicity against microorganisms and oxidizing capacity of nanoscale zerovalent iron after suppressing the available oxidants from the surrounding media.
In cancer cells, ROS levels are higher in comparison to normal cells, due to mitochondrial dysfunction, peroxisome activity, increased cellular receptor signaling, increased activity of oxidases, cyclooxygenases, lipoxygenases and thymidine phosphorylase [
83]. The high rate of ROS production is counterbalanced by an equally high rate of antioxidant activity in cancer cells to maintain redox balance in order to ensure the cell survival.
The concentration of reduced glutathione (GSH), a major component of cellular non-enzymatic antioxidant defense system, increased post-exposure in a time dependent manner. In the first 24 h, no significant variation of the GSH concentration was observed in Caco2 cells exposed to stabilized Fe-Si NPs, but a significant increase was detected in cells exposed to non-stabilized Fe-Si2 and Fe-Si3 NPs, and CMC-Na. After 72 h, its level rose significantly for all types of stabilized NPs, except Fe-Si7_
l-DOPA (
Figure 10). The highest level of GSH was found in Caco2 cells incubated with 25 µg/mL Fe-Si7_CMC-Na and Fe-Si2_
l-DOPA, the percentage increases being 67% and 56% respectively. This suggests the activation of protecting mechanisms by elevating the stock supply of GSH, in order to face the increased ROS production, and thus, preventing the damage of lipids, proteins, and DNA caused by oxidative stress.
GSH is a major player in oxidative adaptation of cancer cells which is why it is exploited by researchers as a valid target for cancer targeted therapy [
84]. The glutathione level is elevated in cancer cells in order to maintain the redox state and to protect cells from damage induced by free radicals, peroxides, and toxins. Glutathione is a powerful reducing compound, able to react with cellular toxic agents directly or via the reactions catalyzed by the glutathione
S-transferase family of enzymes. Studies on NPs toxicity showed that GSH content could vary depending on NP dose. Increased GSH contents were reported by Saddick et al. [
85] in brain tissue exposed to 500 μg/L Zn NP, while a significant decrease in GSH content was registered after exposure to 2000 μg/L. Similarly, Hao and Chen (2012) [
86] found that GSH increased in the liver, gills, and brain of carp exposed to 0.5 and 5 mg/L of ZnO NPs, and decreased in all tissues of fish exposed to 50 mg/L ZnO NPs. Manke et al. [
87] studied oxidative stress as an underlying mechanism for NP toxicity, and postulate that overexpression of antioxidant enzymes is indicative of mild oxidative stress, whereas mitochondrial apoptosis is induced during conditions of severe oxidative stress. According to the abovementioned, we can conclude that a mild oxidative stress was induced by 25 and 50 μg/mL stabilized Fe-Si NPs in Caco2 cells, which results in GSH level increase, and no cell death. During conditions of mild oxidative stress, transcriptional activation of phase II antioxidant enzymes occurs via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) induction, another important mechanism by which cancer cells ensure their antioxidant protection. This transcription factor binds to antioxidant response element (ARE), and activates defensive gene expression, thus increasing the antioxidant proteins levels and protecting against oxidative damage [
88]. Normally, Nrf2 interacts with Kelch-like ECH-associated protein 1 (KEAP1), thus leading it to proteasomal degradation. Elevated ROS levels oxidize redox sensitive cysteine residues on KEAP1, which cause the dissociation of KEAP1 from Nrf2, resulting in the increase of active Nrf2 level in the cytoplasm.
The Nrf2 protein expression was assessed in Caco2 cells after exposure for 24 and 72 h to 25 and 50 µg/mL of non-stabilized and stabilized hybrid Fe-Si NPs (
Figure 11). In Caco2 cells, the activation of Nrf2 was time-dependent. After 24 h incubation, an increase of Nrf2 level in cells exposed to 25 µg/mL Fe-Si2_
l-DOPA, Fe-Si3_
l-DOPA, Fe-Si3_CMC-Na, Fe-Si7_CMC-Na samples, and to 50 µg/mL Fe-Si2_CMC-Na, Fe-Si3_
l-DOPA, Fe-Si7_CMC-Na samples, compared with untreated control, was noticed. No change was registered for Fe-Si7 and Fe-Si7_
l-DOPA samples. These results were in correlation with ROS and GSH levels. After 72 h, the increase of Nrf2 protein expression was significant in cells treated with both doses of all types of NPs. Furthermore, these results were in accordance with the increase of GSH content. By comparison, in cells exposed to the non-stabilized Fe-Si NPs and both stabilizers, the Nrf2 expression was slightly increased in accordance with the low GSH levels.
In this study, we showed that the coating of Fe-Si NPs with l-DOPA or CMC-Na influenced the in vitro biological response in Caco2 cells. The effects of the individual components of the NPs were also investigated in tandem, and were different from those of stabilized Fe-Si NPs, most likely due to their low dispersion in cell culture medium and low internalization into Caco2 cells. According to the cell viability test results, the coating of Fe-Si NPs with l-DOPA increased the biocompatibility of NPs compared to individual components, whereas the capacity of Fe-Si NPs to induce cell death was not changed after coating with CMC-Na. However, instead, this combination reduced significantly the LDH leakage. The coating of hybrid Fe-Si NPs had a great influence on NP dispersion in the cell culture medium and on cellular internalization. The CMC-Na-coated NPs were far better dispersed compared with those stabilized with l-DOPA, which could explain their higher toxicity. Changes of F-actin cytoskeleton organization were highlighted also in the cells treated with CMC-Na-coated NPs, as well as with uncoated NPs and l-DOPA. After treatment with 25 and 50 µg/mL stabilized Fe-Si NPs, ROS were produced in Caco2 cells and a mild oxidative stress was most likely induced. Consequently, the GSH level increased in order to counteract the oxidative stress, which was in accordance with the increased Nrf2 protein expression. Biological data indicated also that Caco2 cells are able to deal with the effects induced by Fe-Si NPs at a dose below 50 µg/mL by activating the scavenging mechanisms against ROS production. Thus, we showed that the Caco2 biological response to Fe-Si NPs displays hormesis, which is an adaptive response of cells to a moderate stress. By comparison, in Caco2 cells treated with unstabilized Fe-Si NPs, a higher amount of ROS was generated, but a lower antioxidant response was induced.
Studying of NP toxicity is a significant challenge. Numerous studies have appeared in the literature to differentiate between the toxicity of uncoated and coated NPs, which is extremely difficult, due to the diversity of factors influencing NP toxicity, such as size, shape, surface chemistry, synthesis techniques, coating agents, types of tissues/cells, etc. Similarly with our study, several papers found a higher toxicity of uncoated NPs than coated NPs [
89], but some research studies have noticed the toxicity of uncoated NPs to be less than of the coated ones, which was associated with the NP cellular interaction and uptake [
90]. Other researchers showed that the coating of iron NPs could decrease the leaching of iron ions and the lysosomal degradation of iron ions, thus reducing the oxidative stress and alterations in iron homeostasis [
91,
92].
A potential advantage of these new Fe-Si nanoparticle aggregates came from their dual composition: the iron-base phases gives them the sensitivity to magnetic fields which open the path for magnetic-guided/trapped drug delivery or variable-magnetic field-induced hyperthermia and/or heat-controlled drug release, whereas the luminescent nanosized silicon particles allow them to be simultaneously used for bio-labeling. Moreover, the silicon quantum dot toxicity is known to be much lower when compared with classical quantum dots, such as CdSe, CdTe, CdS, ZnS, or ZnSe [
93], as proved for PEGylated micelle-encapsulated Si quantum dots synthesized also by laser pyrolysis, allowing them to be used for multiple in vivo applications, such as tumor vasculature targeting, sentinel lymph node, and multicolor near-infrared (NIR) imaging map** [
94]. The possibility of coupling Fe-based magnetic and Si fluorescent nanoparticles in a biocompatible probe was already proved in [
95] by co-encapsulation of hydrophobized Fe
2O
3 superparamagnetic NPs and alkyl-capped Si quantum dots in PEGylated phospholipid micelles, yet in this case, those two functions can be uncoupled once the micelle is degraded. In our case, there are some aggregates in which the Fe-based and Si nanoparticles are strongly linked, and more difficult to break apart, which keeps connected, the magnetism and luminescence properties in biological media. However, the problem of luminescence loss in the aqueous-based biological fluids, due to the advanced oxidation of Si nanoparticles, still need to be countered by future studies for the founding of proper cap** agents, and also by testing in this direction the CMC-Na and
l-DOPA-coated NP aggregates reported in this work.
We think that the knowledge on toxicity of NPs is still limited, and much attention is required on development of new strategies and methods to explore the toxicity of all kind of NPs, in order to keep up with the rapid progress in the synthesis of novel nanomaterials for clinical applications.