Cobalt-based nanoparticles strongly diminish CalceinAM fluorescence independently of their cytotoxic potential in human lung cell line

2.1 Chemicals and reagents

Fetal bovine serum, CalceinAM, and penicillin–streptomycin were purchased from Invitrogen Co. (Carlsbad, CA, USA). DMEM F-12, MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], NADH, pyruvic acid, perchloric acid, hydroethidine (DHE), DCFH-DA, GSH, o-phthalaldehyde (OPT), hank’s balanced salt solution (HBSS), caspase substrates, and Bradford reagent were obtained from a commercial source (Sigma–Aldrich, MO, USA). An apoptosis/necrosis triple staining kit was obtained from BD Biosciences (USA). Ultrapure water was taken from a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals used were of reagent grade.

2.2 NPs of cobalt and cobalt ferrite

NPs of Cobalt (Co), and Cobalt Ferrite (CoFe₂O₄ or simply as Co-Fe) were purchased from Sigma-Aldrich (MO, USA). All NPs were obtained and stored in powder form for later use. As per the information provided by the supplier, the average sizes of Co NPs were 28 ± 18 nm whereas that of Co-Fe was 26 ± 13 nm. Before biochemical studies of these NPs, we have characterized these NPs by conducting various characterizing techniques as described in previous publications (Ahamed et al., 2016; Akhtar et al., 2017).

2.3 NPs dissolution

NPs dissolution was determined as outlined elsewhere (Sabbioni et al., 2014). Briefly, NPs of Co and Co-Fe each at the concentration of 1 mg/mL were suspended in the aqueous media of interest and left for the incubation of 1 week with gentle shaking for 1 h/day on a shaker. After 1 week of incubation, each suspension of each NP was centrifuged at 10000 × g for 10 min. Now free Co²⁺ was measured in the supernatant collected after centrifugation in the atomic absorption spectroscopy (AAS). CoCl₂·6H₂O was used in preparing standards of Co²⁺ (Akhtar et al., 2010).

2.4 MTT and CalceinAM assays

A549 cells (ATCC, US) were cultured in DMEM media and added with 10 % fetal bovine serum (FBS) and antibiotic/antimycotic solution. Cells were passaged according to standard procedure. Cells were exposed to NPs for a 24 h exposure. After the exposure period was over, the media aspirated off and a 100 μL MTT solution prepared in HBSS was added and incubated for 2 h. The degree of MTT conversion to blue formazan was calculated in % of cell viability where 100 % cell viability was considered for control cells. Similarly, control and treated cells were labeled with CalceinAM for 1 h after exposure periods were completed. The final concentration of Calcein-AM was 5 µM in a 96-well plate. Cells labeled with CalceinAM were counterstained with a Hoechst probe for the purpose of control fluorescence as explained in the results.

2.5 Determination of lipid peroxidation and membrane integrity

Lipid peroxidation was detected by the method of Ohkawa et al that uses thiobarbituric acid as a probe for malondialdehyde (MDA) (Ohkawa et al., 1979). MDA is one of the several end products that arise as a result of LPO in polyunsaturated fatty acids constituting cell membranes. It is quantified as thiobarbituric acid reactive substances (TBARS) at 532 nm of absorbance. LPO results in damage to membranes that make cell membranes leaky for intracellular contents. One of the easily quantifiable substances is the enzyme lactate dehydrogenase which catalyzes the conversion of pyruvate to lactate in the metabolism. LDH is easily detected by a decrease in the absorbance of NADH at 340 nm as described (Welder, 1992).

2.6 Determination of O₂^•– and H₂O₂ as the primary member of ROS

ROS has a key role in cell signaling at low concentrations but can result in oxidative stress at high concentrations enough for the nullifying capacity of the cell. Moreover, cell signaling and oxidative stress are dependent on the specific ROS, their primary site of production, and the types of cells. O₂^•– which is mostly produced by mitochondria and the precursor of H₂O₂ was detected by DHE probe at 5 µM of final concentration incubated for 30 min after completion of exposures. The same cells were also co-labeled with DCFH-DA at the final concentration of 10 µM for 30 min. Cells were imaged under a fluorescence microscope using appropriate filters as described previously (Akhtar et al., 2022).

2.7 Determination of intracellular GSH content

Control and treated cells were lysed in distilled water by three cycles of freeze–thaw. Cell lysate, thus, obtained was centrifuged at 10,000 × g at 4 ℃. Supernatant was analyzed for total GSH level by a fluorescent thiol-reacting probe ortho-phthalaldehyde (OPT) as described previously (Akhtar et al., 2015). The cell lysate was also used to determine protein concentration and GSH content was presented as GSH/mg protein.

2.8 Analysis of cell death pathways

A triple staining approach was taken to assess the health status of cells as described in several publications (Akhtar et al., 2023; Atale et al., 2014). This approach involved using a universal cell-permeable dye, Hoechst 33342, to label healthy cells with blue fluorescence. Apoptotic or necrotic cells were identified by a combination of green annexin-V staining and red PI staining (Crowley et al., 2016; Sawai and Domae, 2011). In addition to direct analysis of cell health, we also indirectly estimated the activity of caspases 3 and 9 in the cells. Cells (5 × 10⁴) were grown for 24 h and got exposed to the IC50 of NPs for another 24 h. Cells were then collected, washed, and lysed to measure enzyme activities. Caspase 3 and 9 activities were measured using substrates Ac-DEVDAFC and Ac-LEHD-AFC, respectively. Enzymatic reactions were conducted by combining 20 μL of the enzyme-specific substrate, 30 μL of cell lysate from each cell, and 150 μL of enzyme buffer (50 mM Hepes, 1 mM EDTA, and 1 mM DTT, pH 7.2) for 20 min. Readings were taken at 5-minute intervals using a plate reader (Synergy HT, Bio-Tek, Winooski, Vermont, USA). The activities of caspases were expressed as a percentage of the control.

2.9 Protein estimation

The total protein content was measured by a convenient BCA Protein Assay Kit from Sigma-Aldrich as per instructions.

2.10 Statistics

ANOVA (one-way analysis of variance) followed by Dunnett's multiple comparison tests were employed for statistical analysis of results. For a particular set of experiments, a burst of images was captured at the constant exposure of time, gain, saturation, and gamma. For the calculation of corrected total cellular fluorescence (CTCF), a reasonably constant area was selected and restored in all images once opened during analysis via the 'restore selection' command present in ImageJ software (NIH, Bethesda, MD, USA).

3.1 The dissolution of Co²⁺ was higher in the suspension of Co NPs as compared to that in the suspension of Co-Fe NPs

TEM was employed to determine the morphology and size distribution of NPs (Fig. 1). The images obtained showed that both Co and Co-Fe NPs had a spherical shape with a well-defined size (Fig. 1A and 1B). The average size of Co NPs was found to be 28 ± 18 nm and the size of Co-Fe was found to be 26 ± 13 nm, which matched with the information provided by the supplier. Table 1 summarizes NPs sizes and degree of dissolutions carried out in culture media and phosphate buffer saline (PBS). There is This observation could be attributed to several factors like NPs composition, the presence of organic molecules in suspending media for the NPs may be counted as prominent ones. The presence of Fe in the Co-Fe NPs may have hindered the dissolution of Co²⁺.

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Fig. 1

NPs of cobalt (Co NPs) and cobalt ferrite (Co-Fe NPs) were captured by transmission electron microscope (TEM). TEM images are provided in (A) and (B) respectively. HR-TEM images captured at 2 nm resolution are shown in (C) and (D) depicting a matte pattern typical of crystal structure.

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3.2 Co NPs were more toxic than the Co-Fe NPs

The toxicity of Co NPs is significant, with effects starting at 25 µg/mL (as shown in Fig. 2A). Both types of NPs were found to have a concentration-dependent effect on A549 cells. However, the cytotoxicity data clearly indicates that Co NPs are more toxic than Co-Fe NPs. The IC50 for Co NPs was calculated to be 79 μg/mL after 24 h of exposure, while for Co-Fe NPs it was 163 μg/mL. Fig. 2B shows phase-contrast images of cells treated with IC50s of the two NPs, which exhibit significant deviation in cell morphology from the control cells. The treated cells appear to have similar changes in cell size and shape, with a tendency to adhere strongly to the culture vessel surface.

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Fig. 2

In A549 cells, MTT bioassay was used to evaluate cell viability due to Co NPs and Co-Fe NPs exposure. The two NPs reduced cell viability in A549 cells in a concentration-dependent manner at the concentration ranges provided in Figure (A). The online calculator (https://www.aatbio.com/tools/ic50-calculator) (accessed on 5 April 2023) was used to calculate the concentration of NPs that inhibit/reduce cell growth by 50 (i.e. IC50). Fig B is a phase contrast image of control and IC-50 treated cells. The data represent three identical experiments (n = 3) conducted in triplicate. *Statistically significant difference (p < 0.05) from control.

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3.3 CalceinAM fluorescence suppression was proportional to Co²⁺ leaching

The fluorescence of CalceinAM was found to be significantly different for the two types of NPs (NPs). This study is the first to explore the effect of cobalt NPs on the quenching of CalceinAM fluorescence, as well as the comparative mechanism of toxicity of Co NPs in bare form and Co NPs in coated form with Fe element. A time-dependent quenching potential of the two NPs was determined, and it was found that while Co NPs started quenching even at 2 h of exposure, Co-Fe did not quench CalceinAM fluorescence before 4 h of exposure. Therefore, an additional short 4-hour exposure was included for CalceinAM fluorescence measurement (Fig. 4A) in addition to 24 h of exposure (3B) carried out in each experiment.

Cell viability was measured by CalceinAM fluorescence and compared to the MTT bioassay method. Hoechst staining was used as a control of fluorescence (Fig. 3A and B), and its fluorescence (blue-colored images) was relatively uniform in each group compared to fluctuating CalceinAM fluorescence. The results showed that the CalceinAM fluorescence remaining after quenching was 41 % and 9 % after 4 h (Fig. 3C) and 24 h (Fig. 3D) of exposure to bare Co NPs, respectively. However, the same values were 86 % and 37 % in the case of Fe-containing Co (Co-Fe) NPs treatment, indicating a significant reduction in Co²⁺ leaching from Fe-containing Co-Fe NPs compared to Co NPs. In other words, the reductions in cell viability due to exposure to the two cobalt-based NPs were not as severe as the reduction in CalceinAM fluorescence.

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Fig. 3

Shows the results of an experiment where a549 cells were treated with ic50 concentrations of the two variants of cobalt-based nps. the cells were then imaged using calcein-am and hoechst co-labeling for4 h (A and B) and 24 h (C and D). The data represent three identical experiments (n = 3) conducted in triplicate. *Statistically significant difference (p < 0.05) from control. The α and β symbols represent a significant decline in CalceinAM fluorescence as compared to the cytotoxicity value obtained by MTT at IC50 concentrations of NPs of Co and Co-Fe respectively.

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Fig. 4

Presentsthe results of an experiment where A549 cells were treated with IC50 concentrations of different variants of cobalt-based NPs. The cells were then imaged using DCFH-DA and DHE co-labeling for 4 h (A) and fluorescence intensity quantified (B). C and D refer the same for a and 24 h exposure. The data represent three identical experiments (n = 3) conducted in triplicate. *Statistically significant difference (p < 0.05) from control.

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3.4 Cobalt-based NPs induce high concentrations of O₂^•– and H₂O₂ within cells

The production of reactive oxygen species (ROS) is considered the primary mechanism of nanoparticle toxicity, and this can occur as early as 1–4 h into the incubation period. The degree of ROS induction and the cell type can affect the length of time it takes for ROS production to occur. To accurately measure ROS, a short 4-hour exposure was included in addition to the final 24-hour exposure time. Dyes specific to O₂^•– and H₂O₂ were co-labeled and imaging was conducted (Fig. 4A and B) The fluorescent data extracted from images (Fig. 4C and D) suggest that Co NPs have the potential to induce O₂^•– at 4 h of exposure but not H₂O₂. However, at 24 h of exposure, Co NPs caused the induction of both O₂^•– and H₂O₂. In the case of iron-containing Co-Fe NPs, H₂O₂ was significantly higher than the control at both 4- and 24-hour exposures. Co-Fe NPs also generated O₂^•– at both exposure periods, but with lower intensity than Co NPs. The iron in Co-Fe may play a catalyzing role (like the Fenton reaction) in the higher generation of H₂O₂ compared to only Co NPs.

3.5 NPs induce apoptosis mode of cell death

The A549 cells that were treated underwent extensive staining with PI and annexin V as evidenced in live-cell images provided in (Fig. 5). It is important to note that cells undergoing apoptosis display intact cell membranes with blebs, as well as a reduction in volume and tightly packed chromatin that emits bright and clumped fluorescence which is present in images of cells treated with both types of NPs. In contrast, cells undergoing necrosis exhibit early cell membrane damage, an increase in cell volume, and the dilated nuclear periphery with circumscribed chromatin that gives off diffused fluorescence and a preferential uptake of PI over annexinV-FITC (Fig. 5A). In live cell images of triple staining, it is hard to observe the aforementioned evidence of necrosis. Additionally, activated levels of caspases 9 (5B) and 3 (5C) serve as further evidence of apoptosis in A549 cells. Overall, data suggest cobalt-based-NPs be apoptotic in A549 cells.

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Fig. 5

Shows a potential disbalance between oxidants generation and antioxidant depletion due to exposure to cobalt-based nps. this canlead to lipid peroxidation (LPO) and lactate dehydrogenase (LDH) release, as seen in Fig A and B, respectively. The antioxidant capacity of N-acetylcysteine (NAC) as a precursor of glutathione (GSH) and chelator of oxidant-inducing metal ions is shown in Fig C. Figure D demonstrates the chelation of metal ions by NAC, which prevents quenching of CalceinAM fluorescence by Co²⁺, as quantified in Figure E. The data represented are mean ± SD of three identical experiments (n = 3) made in triplicates, with * denoting a statistically significant difference from the control (p < 0.05). The α and β symbols indicate significant prevention in quenching of CalceinAM fluorescence in the presence of 2 mM exogenously applied NAC along with NPs as compared to that by NPs of Co and Co-Fe alone, respectively.

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3.6 Co NPs induced oxidative stress, and exogenous NAC prevented GSH exhaustion and inhibited quenching of CalceinAM fluorescence

In addition to inducing ROS, NPs (NPs) also cause lipid peroxidation (as shown in Fig. 6A) and degradation of membrane integrity (as shown in Fig. 6B). However, the addition of N-acetylcysteine (NAC) significantly prevented the depletion of the intracellular antioxidant glutathione (GSH) caused by NPs exposure (as shown in Fig. 6C). It is worth noting that NAC has been reported to chelate metal ions, which could explain the significant restoration of CalceinAM fluorescence (as shown in Fig. 6D and E) that was previously quenched by Co²⁺. This finding suggests that NAC can protect cells by reducing oxidative stress, restoring GSH levels, and chelating metal ions such as Co²⁺. Therefore, the application of NAC is crucial for managing and designing experiments involving metal ions like Co²⁺.

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Fig. 6

shows the mode of cell death induced by each variant of cobalt-based NPs in A549 cells. Triple staining (A) and detection of caspase 9 (B) and caspase 3 (C) activities were used to determine cell death. The data represented are mean ± SD of three identical experiments (n = 3) made in triplicates, with * denoting a statistically significant difference from the control (p < 0.05 for each).

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