The cytological and electrophysiological effects of silver nanoparticles on neuron-like PC12 cells

doi:10.21203/rs.3.rs-1453101/v1

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The cytological and electrophysiological effects of silver nanoparticles on neuron-like PC12 cells

https://doi.org/10.21203/rs.3.rs-1453101/v1

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Background

Silver nanoparticles (SNPs) are widely used in a large number of products. SNPs can cross the blood-brain barrier and are subsequently deposited continuously in the brain, causing a variety of pathological responses and diseases. As a result, the toxic effects of SNPs on the nervous system have attracted considerable attention, but the mechanism of toxicity remains unclear. Moreover, neurons have different cytological and electrophysiological properties, and their functions are highly dependent on changes in electrophysiological properties.

Results

Different concentrations of SNPs (20 nm) were prepared, and the effects of different application durations on the cell viability and electrical excitability of rat adrenal pheochromocytoma (PC12) quasi-neuronal networks were investigated. The effects of 200 µM SNPs on the neurite length, cell membrane potential (CMP) difference, intracellular Ca²⁺ content, mitochondrial membrane potential (MMP) difference, adenosine triphosphate (ATP) content, and reactive oxygen species (ROS) content of networks were investigated. The results showed that 200 µM SNPs produced grade 1 cytotoxicity at 48 h of interaction, and the other concentrations of SNPs were noncytotoxic. Noncytotoxic 5 µM SNPs significantly increased the electrical excitability of PC12 quasi-neuronal networks, and noncytotoxic 100 µM SNPs led to an initial increase followed by a significant decrease in electrical excitability. While cytotoxic 200 µM SNPs significantly decreased the electrical excitability. 200 µM SNPs led to decreases in neurite length, MMP difference and ATP content and increases in CMP difference, and intracellular Ca²⁺ and ROS levels with increasing interaction time.

Conclusions

The results above revealed that using only cell viability to evaluate nanoparticles-induced neurotoxicity is partial. Therefore, not only cell viability but also electrophysiological properties should be considered when evaluating nanoparticles-induced neurotoxicity. The SNPs-induced cytotoxicity mainly originated from its effects on ATP content, cytoskeletal structure and ROS content. The SNPs-induced decrease in electrical excitability could be explained by a decrease in ATP content which could lead to cellular energy deficiency that opened K_ATP channels on the cell membrane, resulting in an increase in the CMP difference and hyperpolarization. ATP content may thus be an important indicator of both cell viability and electrical excitability of PC12 quasi-neuronal networks.

Silver nanoparticle

Cytotoxicity

Electrical excitability

Voltage threshold measurement method

High content analysis techniques

Due to their excellent antibacterial properties, silver nanoparticles (SNPs) are widely used in a large number of products such as food packaging, air filters, disinfectants, cosmetics, antibacterial gels, trauma dressings and cardiovascular implants [1, 2]. SNPs can enter the circulatory system through oral administration, inhalation, dermal contact and intravenous injection. SNPs smaller than 35 nm in diameter can cross the blood-brain barrier [3] and are subsequently deposited continuously in the brain, causing a variety of pathological responses and diseases [4]. As a result, the toxic effects of SNPs on the nervous system have attracted considerable attention, but the mechanism of toxicity remains unclear.

Unlike normal somatic cells, neurons have different cytological and electrophysiological properties, and their functions are highly dependent on changes in electrophysiological properties [5]. Therefore, we believe that to fully understand the toxicity of SNPs to neurons, both cytological and electrophysiological properties should be comprehensively investigated. However, most of the current studies have focused on the cytological effects of SNPs [6], while very few studies investigated their electrophysiological effects on neurons [7].

It is well known that reception, processing and transmission of information in neuronal networks is the basis of higher brain activities, so studying the electrical excitability of neuronal networks is an effective way for investigating the effects of exogenous factors on brain function in electrophysiological studies. A microelectrode array (MEA) can effectively record the electrical signals of neuronal networks without damaging the neurons [8]. Moreover, measuring electrophysiological characteristics, such as the discharge frequency, of neuronal networks cultured on MEAs has been proven to be an effective method for evaluating the toxic effects of exogenous factors (e.g. nanoparticles, etc.) on neurons [9]. Our group previously proposed an MEA-based voltage threshold measurement method (VTMM) to quantify the effects of exogenous factors on electrical excitability of neuronal networks. VTMM is similar to the rheobase measurement with patch clamp, and both evaluate the electrical excitability by measuring the minimum stimulus that causes excitation [10, 11]. Similar to the rheobase measurement, the lower the voltage threshold (V_Th) is in VTMM, the higher the electrical excitability is, and vice versa. However, in contrast to the rheobase method, which measures individual neurons, VTMM measures the electrical excitability of a neuronal network. The validity of the VTMM has been verified in our previous studies of hippocampal neuronal networks and hippocampal brain slices [11, 12].

However, the use of primary cultured neurons has several disadvantages such as complexity of extraction, individual variation and potential failure to comply with the 3R (Reduction, Refinement and Replacement) principles for animal protection. In contrast, differentiated rat adrenal pheochromocytoma (PC12) cells are more easily cultured and subcultured. Moreover, PC12 cells not only resemble neurons in terms of cell structure and electrical excitability [13, 14], but are also capable of forming quasi-neuronal networks [15]. Therefore, these cells have been widely used in nanoparticle-induced cytotoxicity and electrophysiology studies [16, 17]. Cui et al. used MEAs and fluorescence microscopy to simultaneously monitor action potentials and dopamine release from PC12 cells [18]. Our group demonstrated that the quasi-neuronal networks had similar electrical excitability changes as hippocampal neuronal networks and hippocampal brain slices to acetylcholine, ethanol, lidocaine hydrochloride, and changes in temperature. Therefore, PC12 cells are recommended as an alternative cell model for studying the cytotoxic and electrical excitability effects of exogenous factors on neurons under certain conditions [19].

The aim of this paper was to investigate the toxic effects of SNPs on PC12 cells in terms of changes in both cytological and electrophysiological properties as well as to determine the underlying mechanism of these effects. The effects of SNPs on the viability of PC12 cells and on the electrical excitability of quasi-neuronal networks were first evaluated by the methyl thiazolyl tetrazolium (MTT) method and VTMM respectively. Studies on six aspects of PC12 cells, namely, neurite length, cell membrane potential (CMP) difference, intracellular Ca²⁺ content, mitochondrial membrane potential (MMP) difference, reactive oxygen species (ROS) content and adenosine triphosphate (ATP) content, were performed subsequently using a high content analysis (HCA) system. The correlations among cell viability, electrical excitability and these six indicators after treatment with SNPs were jointly analysed to elucidate the different mechanisms underlying the SNPs-induced changes in cytological and electrophysiological properties.

Preparation and characterization of SNPs

The transmission electron microscopy (TEM) image (Fig. 1A) and absorption spectra (Fig. 1B) of SNPs showed that the SNPs were spherical with an average size of 21.45 ± 2.72 nm and a maximum absorption wavelength of 389 nm (The dataset is shown in Additional file 1) [20]. The original concentration of SNPs was 1.4 g/L.

Evaluation of SNP cytotoxicity in PC12 cells

The cell viability rates of PC12 cells treated with different concentrations (5, 25, 50, 100 and 200 µM) of 20 nm SNPs for 0.5, 1, 12, 24 or 48 h are shown in Table 1 and Fig. 2. The cell viability rates in all 5 µM SNPs-treated groups were higher than 93%. There was no significant difference between the cell viability rates in the 25, 50 and 100 µM SNPs-treated groups (P > 0.05), and the cell viability rates remained high after 48 h in these groups (88.45 ± 2.41%, 88.10 ± 3.61% and 88.27 ± 0.61%, respectively), indicating grade 0 cytotoxicity. In the 200 µM SNPs-treated groups, the cell viability rates were higher than 85% with grade 0 cytotoxicity when the duration of treatment was less than or equal to 24 h; after 48 h, the cell viability rate decreased to 71.29 ± 2.68%, indicating grade 1 cytotoxicity.

Table 1. Cell viability rates of PC12 cells treated with 20 nm SNPs at concentrations of 5, 25, 50, 100 and 200 μM for 0.5, 1, 12, 24 and 48 h. The results are presented as the mean ± standard deviation (SD) (n= 6).

Time (h) Concentration (μM)	0.5	1	12	24	48
5	95.54±2.78	95.86±4.02	93.35±4.20	94.31±2.23	93.74±1.86
25	92.89±5.37	93.91±1.41	89.96±3.07	88.59±2.40	88.45±2.41
50	95.07±1.66	93.14±3.95	89.61±6.55	88.46±2.05	88.10±3.61
100	92.01±1.00	91.36±4.36	89.10±4.55	88.31±3.27	88.27±0.61
200	91.86±2.09	91.29±3.62	87.12±3.90	85.21±0.60	71.29±2.68

Viability rate [81%, 100%] indicates grade 0 cytotoxicity, viability rate [61%, 80%] indicates grade 1 cytotoxicity, viability rate [41%, 60%] indicates grade 2 cytotoxicity, viability rate [21%, 40%] indicates grade 3 cytotoxicity, and viability rate [0, 20%] indicates grade 4 cytotoxicity.

Standard V_Th of PC12 quasi-neuronal networks

An image of PC12 quasi-neuronal networks on an MEA is shown in Fig. 3A, demonstrating that PC12 cells were tightly connected to each other on the MEA. One stimulating electrode and three detecting electrodes were randomly selected among the electrodes well covered by cells for experiments. Figure 3B shows the electrical signals of the stimulating and detecting electrodes displayed on the oscilloscope. As shown in Fig. 3B, 57.8 ms after stimulation (negative pulse amplitude of 37 mV) was applied to one normally cultured PC12 quasi-neuronal network, action potentials were recorded at each of the three detecting electrodes. Therefore, the standard V_Th of this specific PC12 quasi-neuronal network (shown in Fig. 3) was 37 mV.

According to our previous results, the minimum amplitude of the stimulation pulses that triggered responses from the networks in normal culture environment was defined as the standard V_Th. The V_Th of networks of the same kind of cells is a fixed value as the test environment remains unchanged [11, 12]. The standard V_Th of PC12 quasi-neuronal networks measured in this paper was 36.50 ± 0.58 mV, which was not significantly different (P > 0.05) from our previous result (36 ± 2.37 mV) [19].

V_Th of PC12 quasi-neuronal networks exposed to SNPs

The V_Th of PC12 quasi-neuronal networks treated with 20 nm SNPs at concentrations of 5, 100 or 200 µM for 0.5, 1, 12, 24 or 48 h are shown in Table 2 and Fig. 4.

Table 2

V_Th of PC12 quasi-neuronal networks treated with 20 nm SNPs at concentrations of 5, 100 and 200 µM for 0.5, 1, 12, 24 and 48 h. The results are presented as the mean ± SD (n = 4).
Time (h) Concentration(µM)	0 (Standard V_Th)	0.5	1	12	24	48
5	36.50 ± 0.58	32.00 ± 1.83	30.50 ± 1.29	29.00 ± 1.83	31.00 ± 2.16	27.25 ± 1.89
100		33.50 ± 0.58	36.25 ± 0.50	49.25 ± 0.50	75.75 ± 1.26	75.75 ± 0.96
200		37.80 ± 2.68	43.67 ± 1.51	52.00 ± 2.10	85.00 ± 0.71	94.80 ± 3.49

After treatment with 5 µM SNPs, the V_Th of PC12 quasi-neuronal networks was significantly lower than the standard V_Th at all five time points (P < 0.05), with a minimum value of 27.25 ± 1.89 mV at 48 h, which suggests that 5 µM SNPs led to a significant increase in the electrical excitability of PC12 quasi-neuronal networks.

After treatment with 100 µM SNPs, the V_Th of the networks was lower than the standard V_Th at 0.5 and 1 h and very significantly higher than the standard V_Th at 12 h and beyond (P < 0.01). This result suggests that 100 µM SNPs led to an increase followed by a significant decrease in the electrical excitability of PC12 quasi-neuronal networks.

After treatment with 200 µM SNPs, the V_Th of the networks was higher than the standard V_Th at each time point and was very significantly higher than the standard V_Th at 1 h and beyond (P < 0.01). This result suggests that 200 µM SNPs caused a significant decrease in the electrical excitability of PC12 quasi-neuronal networks after 1 h of treatment.

Neurite length of cells exposed to SNPs

The lengths of neurites of PC12 cells treated with 20 nm 200 µM SNPs for 0.5, 1, 12, 24 or 48 h are shown in Table 3 and Fig. 5. After treatment with 200 µM SNPs, the neurite length decreased gradually and was significantly (P < 0.05) lower than that of the control group after 12 h.

Table 3

Neurite length of PC12 cells treated with 20 nm 200 µM SNPs for 0.5, 1, 12, 24 and 48 h. The results are presented as the mean ± SD (n = 6).
Time (h)	0.5	1	12	24	48
Relative neurite length (%)	94.26 ± 14.05	84.93 ± 7.22	78.77 ± 5.74	78.90 ± 9.12	70.78 ± 5.90

CMP difference of cells exposed to SNPs

The CMP differences of PC12 cells treated with 20 nm 200 µM SNPs for 0.5, 1, 12, 24 or 48 h are shown in Table 4 and Fig. 6. The CMP differences in each SNPs-treated group were higher than those in the control group. Within 0–24 h, noncytotoxic concentrations of SNPs induced a sustained increase in CMP difference. At 48 h, a significant decrease in CMP difference was observed compared to that at 24 h (P < 0.05). However, at 48 h, the CMP difference was still very significantly higher than that of the control group (P < 0.01), and the V_Th was still very significantly higher than the standard V_Th (P < 0.01) (Fig. 4), which suggests that electrical excitability of these networks was still lower than normal.

Table 4

CMP difference of PC12 cells treated with 200 µM SNPs for 0.5, 1, 12, 24 and 48 h. The results are presented as the mean ± SD (n = 6).
Time (h)	0.5	1	12	24	48
Relative CMP difference (%)	102.33 ± 10.71	101.82 ± 2.80	107.40 ± 2.53	120.64 ± 2.45	114.11 ± 2.30

Intracellular Ca²⁺ content of cells exposed to SNPs

The intracellular Ca²⁺ content of PC12 cells treated with 20 nm 200 µM SNPs for 0.5, 1, 12, 24 or 48 h is shown in Table 5 and Fig. 7. The intracellular Ca²⁺ contents in all SNPs-treated groups were higher than those in the control group and were very significantly higher at 24 h (243.96%) and 48 h (221.50%) (P < 0.01). Moreover, the intracellular Ca²⁺ content at 48 h was significantly lower (P < 0.05) than that at 24 h.

Table 5

Intracellular Ca²⁺ content of PC12 cells treated with 20 nm 200 µM SNPs for 0.5, 1, 12, 24 and 48 h. The results are presented as the mean ± SD (n = 6).
Time (h)	0.5	1	12	24	48
Relative intracellular Ca²⁺ content (%)	105.70 ± 11.16	105.19 ± 13.57	116.35 ± 1.19	243.96 ± 10.01	221.50 ± 23.46

MMP difference, ATP content and ROS content of cells exposed to SNPs

The MMP difference, ATP content and ROS content of PC12 cells treated with 200 µM SNPs for 0.5, 1, 12, 24 or 48 h are shown in Table 6 and Fig. 8. The MMP difference and ATP content of cells exposed to SNPs were very significantly higher (P < 0.01) than those of the control groups at 0.5 h of treatment. The MMP difference and ATP content continuously decreased starting at 1 h and were very significantly lower than those of the control group at 24 h and 48 h and at 12 h, 24 h and 48 h, respectively (P < 0.01). The ROS content of cells exposed to SNPs continuously increased and was very significantly higher than that of the control group at 24 and 48 h (P < 0.01).

Table 6

MMP difference, ATP content and ROS content of PC12 cells treated with 20 nm 200 µM SNPs for 0.5, 1, 12, 24 and 48 h. The results are presented as the mean ± SD (n = 6).
Time (h)	0.5	1	12	24	48
Relative MMP difference (%)	110.68 ± 5.00	96.91 ± 3.92	96.53 ± 0.59	91.98 ± 2.93	91.28 ± 3.18
Relative ATP content (%)	113.92 ± 2.80	98.53 ± 1.58	72.33 ± 5.03	62.82 ± 3.41	55.78 ± 3.82
Relative ROS content (%)	108.55 ± 30.08	110.22 ± 37.44	120.92 ± 17.85	226.78 ± 4.81	510.69 ± 76.18

Conjoint analysis between cytological indicators, cell viability and V_Th

A correlation analysis of cell viability and V_Th of cells exposed to SNPs revealed a high correlation (r= -0.95). Thus, to discuss the molecular mechanisms of cell viability and V_Th variation, the results of cell viability and V_Th were analysed conjointly with the results of the above six cytological indicators, and Pearson correlation coefficients were calculated for the relationships between cell viability, V_Th and each indicator (Table 7).

Table 7

Pearson correlation coefficients (r) between each cytological indicators, cell viability and V_Th
	V_Th	ATP content	Neurite length	ROS content	Intracellular Ca²⁺ content	MMP difference	CMP difference
Cell viability	-0.95	0.95	0.93	-0.90	-0.83	0.77	-0.79
V_Th		-0.92	-0.85	0.87	0.96	-0.79	0.91

|r| = 0.8 to 1.0 indicates high correlation, |r| = 0.5 to 0.8 indicates moderate correlation, |r| = 0.3 to 0.5 indicates low correlation and |r| = 0 to 0.3 indicates very weak or no correlation. Both PC12 cell viability and V_Th were highly correlated with ATP content, neurite length, ROS content and intracellular Ca²⁺ content (|r|>0.8), and both were moderately correlated with MMP difference (r = 0.77 and − 0.79). Additionally, V_Th was highly correlated with CMP difference (r = 0.91), while cell viability was only moderately correlated with CMP difference (r=-0.79).

SNPs smaller than 35 nm are able to cross the blood-brain barrier [3]. The size of SNPs commonly used in clinical practice is generally ~ 20 nm, and SNPs of this size have been used in neurotoxicity studies by several research groups [21, 22]. Therefore, 20 nm SNPs were selected in this paper to investigate their toxic effects.

In the current paper, the MTT results showed that 200 µM SNPs produced grade 1 cytotoxicity at 48 h of interaction, and the other concentrations of SNPs were noncytotoxic. To investigate the effects of noncytotoxic and cytotoxic concentrations of SNPs on PC12 quasi-neuronal networks and to compare our current findings with our previous work [23], three concentrations of SNPs (5, 100 and 200 µM) were selected for VTMM experiments.

Figure 4 suggests that 5 µM SNPs led to a significant increase in the electrical excitability of PC12 quasi-neuronal networks. And 100 µM SNPs led to an increase followed by a significant decrease in the electrical excitability of the networks. It also suggests that 200 µM SNPs caused a significant decrease in the electrical excitability of the networks after 1 h of treatment.

When comparing Fig. 2 and Fig. 4, it was evident that the noncytotoxic 5 µM SNPs led to a significant decrease (P < 0.05) in the V_Th of PC12 quasi-neuronal networks at 0.5 h, while noncytotoxic 100 µM SNPs led to a significant increase (P < 0.01) in the V_Th of the networks at 12 h. This result indicates that SNPs were still able to alter the electrical excitability of PC12 quasi-neuronal networks at noncytotoxic concentrations. Grade 1 cytotoxicity appeared after 48 h of treatment with 200 µM SNPs, yet the V_Th was very significantly (P < 0.01) higher than the standard V_Th after only 1 h, which indicated a reduction in the electrical excitability of PC12 quasi-neuronal networks. Taken together, these results show that the SNPs-induced changes in electrophysiological properties of PC12 cells appeared before the changes in cell viability, suggesting that using cell viability alone to evaluate nanoparticles-induced neurotoxicity is partial. Therefore, not only cell viability, but also electrophysiological properties should be considered when evaluating nanoparticles-induced neurotoxicity.

To further investigate the mechanisms of SNPs-induced cytotoxicity and changes in electrical excitability, changes in six aspects of PC12 cells, namely, neurite length, CMP difference, intracellular Ca²⁺ content, MMP difference, ROS content and ATP content were studied under the effect of 200 µM cytotoxic SNPs.

On the one hand, the presence of neurites is a prerequisite for the formation of synapses, which are the basis of signaling between neurons. In addition, a relatively high density of voltage-gated sodium channels, which play an important role in the production and conduction of neural signals, are distributed along the axon initiation segment of neurons [24]. Decreased neurite length might shorten the axon initiation segment, therefore leading to a decrease in the number of sodium channels, which in turn would affect neurotransmitter release and reduce the electrical excitability of neuronal networks [12]. On the other hand, decreased neurite length correspond to cell damage, particularly cytoskeletal damage [25]. Thus, the SNPs-induced decrease in neurite length (Fig. 5) suggests that SNPs caused cytoskeletal damage and this decrease might affect signaling between neuron-like PC12 cells.

The CMP is one of the most important indicators of cell survival, as a decrease in CMP difference is usually accompanied by toxic effects, apoptosis and necrosis [26]. Additionally, the CMP difference directly influences the resting potential and polarization of neurons, rendering it a pivotal factor affecting the electrical excitability of neurons [27]. Within 0–24 h, noncytotoxic concentrations of SNPs induced a sustained increase in CMP difference (Fig. 6), which indicated cell hyperpolarization [28], thereby contributing to reduced electrical excitability (Fig. 4). At 48 h, a significant decrease in CMP difference was observed compared to that at 24 h (P < 0.05). This might be due to the cytotoxicity of SNPs at 48 h, as cell death is usually accompanied by a decrease in CMP difference [29].

The intracellular Ca²⁺ contents in all SNPs-treated groups were higher than those in the control group (Fig. 7). These results are consistent with the finding of Elżbieta Ziemińska et al. that 75 µg/mL SNPs that were 5–35 nm in size could lead to elevated Ca²⁺ levels in cerebellar granule cells due to overactivation of Ca²⁺ channels [30]. Moreover, the intracellular Ca²⁺ content at 48 h was significantly lower (P < 0.05) than that at 24 h. This might be attributed to the very significant increase in intracellular Ca²⁺ content at 24 h, which initiated negative feedback regulation, followed by inactivation of Ca²⁺ channels [31], resulting in a decrease in intracellular Ca²⁺ content at 48 h. Overload of intracellular Ca²⁺ leads to mitochondrial and cytoskeletal damage and even apoptosis [32, 33]. Moreover, Ca²⁺ is an important intracellular signaling molecule that can control neurotransmitter release, regulate the expression of various proteins and influence the excitability of neurons [34]. In the current paper, 200 µM SNPs applied to PC12 cells for 48 h were found to simultaneously induce grade 1 cytotoxicity (Fig. 2), very significantly increase V_Th (Fig. 4) and very significantly increase intracellular Ca²⁺ content (Fig. 7).

The mitochondrion is an important organelle in the cell, as it is the site of energy production and one of the important targets of nanoparticles to induce toxicity [2]. The MMP difference is an important indicator of mitochondrial function. A decreased MMP difference implies mitochondrial damage and is one of the early signs of apoptosis [35]. Additionally, impaired mitochondria can lead to a decrease in ATP (an important cellular energy source) content, which subsequently can lead to cellular energy deficiency [36]. A previous study by our group found that the ATP content of human dermal fibroblasts decreased under the effect of SNPs [37]. Moreover, electrons escaping from a damaged mitochondrial electron transport chain might directly react with substances such as oxygen and generate ROS [38]. Damage to mitochondria therefore leads to increases in ROS content, and excessive ROS levels might contribute to further cellular damage (e.g., DNA, protein, and synaptic damage) [39]. Thus, ROS content is one of the most important indicators of cellular damage and the effect of nanoparticles on cells. The results displayed in Table 6 and Fig. 8 indicate that treatment with SNPs for 24 and 48 h caused mitochondrial damage which reduced the MMP difference, contributing to decreases in ATP content and increases in ROS content. Additionally, the MMP difference and ATP content of cells exposed to SNPs were very significantly higher (P < 0.01) than those of the control groups at 0.5 h of treatment, which could have been caused by the metabolic adaptation to the presence of SNPs. The metabolic adaptation might increase the MMP difference and enhance tricarboxylic acid cycle of mitochondria, resulting in increased ATP production [40].

The three indicators most highly correlated with cell viability in Table 7 were ATP content (r = 0.95), neurite length (r = 0.93), and ROS content (r=-0.90). SNPs have been found to simultaneously decrease ATP content and suppress neurite growth in human embryonic stem cell-derived neurons [41], and to decrease MMP difference, increase ROS content and decrease viability in A549 cells [42]. The current paper demonstrated that ATP content, neurite length and ROS content were important indicators of cellular damage caused by nanoparticles, and that the main cause of SNPs-induced cytotoxicity was the detrimental effects on cellular energy supply, cytoskeletal integrity and the ROS content.

Elevated intracellular Ca²⁺ content have been found to contribute to reduced ATP synthesis [32] and cellular energy deficiencies, which in turn open ATP-sensitive potassium (K_ATP) channels on the cell membrane, resulting in increases in CMP difference and hyperpolarization [28]. This paper showed that SNPs increased intracellular Ca²⁺ content (Fig. 7), decreased ATP content (Fig. 5), increased CMP difference (Fig. 6) and increased V_Th (Fig. 4) in PC12 cells. Furthermore, the three indicators most highly correlated with V_Th (displayed in Table 7) were intracellular Ca²⁺ content (r = 0.96), ATP content (r=-0.92) and CMP difference (r = 0.91). The above results suggest that the SNPs-induced decrease in electrical excitability may be explained by a decrease in ATP content due to an increase in intracellular Ca²⁺ content, which led to cellular energy deficiency that opened K_ATP channels on the cell membrane, resulting in an increase in CMP difference and hyperpolarization.

Additionally, ATP content was the only cytological indicator that correlated with both cell viability and V_Th, with correlation coefficients above 0.9. This result indicates that the ATP content was the main cytological indicator that affected both cytotoxicity and electrical excitability in the presence of SNPs, and illustrates the importance of energy supply for the maintenance of neuronal cell structure and function.

Possible mechanisms for the SNPs-induced changes in cytotoxicity and electrical excitability of PC12 cells are summarized in Fig. 9. SNPs decreased neurite length in PC12 cells, suggesting that SNPs caused cytoskeletal damage [25] and cytotoxicity. In addition, decreased neurite length might lead to a reduction in voltage-gated sodium channels and diminish the electrical excitability of PC12 quasi-neuronal networks [12]. The SNPs-induced decrease in MMP difference led to an increase in ROS content, which might damage biomolecules such as DNA and proteins, leading to cell death [39]. Moreover, increased ROS content might impair synaptic structures, which could affect intercellular signaling [43] and reduce the electrical excitability of PC12 quasi-neuronal networks. Both the decrease in MMP difference and the increase in intracellular Ca²⁺ content could lead to a decrease in ATP content. Decreased ATP content could lead to cellular energy deficiency, which both could activate apoptotic pathways [44], causing a decrease in cell viability, and could open K_ATP channels [28], causing an increase in CMP difference and hyperpolarization, eventually resulting in reduced electrical excitability.

In this paper, the effects of SNPs on the viability and electrical excitability of PC12 cells were investigated using the MTT method and VTMM. SNPs were found to alter the electrical excitability of PC12 quasi-neuronal networks at noncytotoxic concentrations. At the cytotoxic concentration, SNPs altered electrical excitability before they altered cell viability, suggesting that using only cell viability to evaluate nanoparticles-induced neurotoxicity is partial. Therefore, not only cell viability but also electrophysiological properties should be considered when evaluating nanoparticles-induced neurotoxicity. Further experiments revealed that the main reason for SNPs-induced cytotoxicity was the detrimental effects on cellular energy supply, cytoskeletal integrity and ROS content. The SNPs-induced decrease in electrical excitability could be explained by a decrease in ATP content caused by mitochondrial damage and caused by increased intracellular Ca²⁺ content. A decrease in ATP content could lead to cellular energy deficiency that opened K_ATP channels on the cell membrane, resulting in an increase in the CMP difference and hyperpolarization. ATP content was the main cytological indicator of both cytotoxicity and electrical excitability in the presence of SNPs.

Preparation and characterization of SNPs

SNPs were prepared by the sodium borohydride reduction of silver nitrate [20]. The morphology of SNPs was obtained using TEM (JEOL JEM-2100, Japan), and size analysis was performed using Image-Pro Plus software v 6.0 (Media Cybernetics, Inc., USA). The concentration of SNPs was measured using an inductive coupled plasma-optical emission spectrometer (Optima 5300DV, Perkin Elmer, USA).

Cell culture

Differentiated PC12 cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences. The cells were cultured in high-glucose DMEM (HyClone, United States) plus 10% fetal bovine serum (Biological Industries, Israel) and 1% (v/v) penicillin-streptomycin (Biological Industries, Israel), and were incubated in a 37°C, 5% CO₂ incubator with saturated humidity (Thermo Forma 3111, Thermo Fisher Scientific, United States). The experiments were performed with cells in logarithmic growth phase.

Evaluation of SNPs-induced cytotoxicity in PC12 cells

PC12 cells (100 µL/well) at a concentration of 6 × 10⁴ cells/ml were added to a 96-well plate. After 24 h, the culture medium was aspirated, 5, 25, 50, 100 or 200 µM SNPs were added, and PC12 cells were then treated for another 0.5, 1, 12, 24 or 48 h. The cytotoxicity of SNPs was evaluated by the MTT method [45]. Cells cultured in medium without SNPs were used as the negative control, and cells cultured in medium containing 0.7% acrylamide were used as the positive control. Six parallel experiments were conducted for each concentration at each time point.

Preparation of PC12 quasi-neuronal networks on MEAs

At the beginning of each experiment, the MEA (60MEA100/10iR-Ti, Multi Channel Systems MCS GmbH, Germany) was immersed in 75% ethanol for 30 min, dried, and sterilized using ultraviolet light for 8 h. Next, the MEA chamber was filled with sufficient 0.1 mg/mL poly-L-lysine solution (Sigma-Aldrich, U.S.) to cover all the electrodes. Then, the MEA was placed in an incubator at 37°C with 5% CO₂ and saturated humidity for 24 h. Finally, the poly-L-lysine was removed, and the MEA was rinsed with sterilized ultra-pure water and dried in a laminar flow cabinet.

To build PC12 quasi-neuronal networks, 20 µL of differentiated PC12 cells with quasi-neuronal features were seeded onto the surface of the prepared MEA at a cell density of 1×10⁶ cells/mL. Then, the MEA with PC12 cells was placed in an incubator and cultured with the culture medium for approximately 24 h at 37°C with 5% CO₂ and saturated humidity. The following experiments could be performed when the PC12 quasi-neuronal networks developed well and covered most of the electrode area on the MEA.

Measurement of standard V_Th

The experimental procedure and the selection of the voltage stimulation waveform for measuring the normal V_Th through VTMM are detailed in our previous paper [11, 12]. In brief, voltage stimulation from one selected stimulating electrode was generated by a voltage pulse generator (Agilent 33220A, USA), and used to trigger responses from the networks (Fig. 10A). The stimulation was an asymmetric charge-balanced biphasic pulse at 50 Hz with a positive phase of 2.00 ms and a negative phase of 0.20 ms (Fig. 10B). The amplitude of the negative pulse started at 0 mV and increased in steps of 1 mV. An oscilloscope (Agilent 2024A, USA) was used to supervise, recognize, and record the stimulation from the stimulating electrode and the responses from three selected detecting electrodes in real time (Fig. 10A). The minimum amplitude of the negative phase of the stimulation pulses that triggered responses from the networks in normal culture environment (with no exogenous factors) was defined as the standard V_Th. Each MEA was used only once. Four parallel experiments were conducted.

Measurement of V_Th of networks exposed to SNPs

The original culture medium in the MEA chamber was replaced with fresh test culture medium containing 5, 100 or 200 µM SNPs. After 0.5, 1, 12, 24 or 48 h of incubation, the V_Th of the networks was measured as described in Section 2.5. Four parallel experiments were conducted to measure the V_Th for each time point under each SNP concentration.

Measurement of neurite length of cells exposed to SNPs

PC12 cells (100 µL/well) at a concentration of 6 × 10⁴ cells/ml were added to a 96-well plate. After 24 h, the culture medium was aspirated, and 200 µM SNPs were added. After PC12 cells were treated for another 0.5, 1, 12, 24 or 48 h, the culture medium was removed, and the cells were stained with TRITC-conjugated phalloidin and DAPI (Millipore, USA) [46]. The average neurite length was analysed with a HCA system (Array XTI, Thermo Fisher, USA). Cells cultured in medium without SNPs were used as the negative control. Six parallel experiments were conducted for each time point.

Measurement of CMP difference of cells exposed to SNPs

The CMP difference was monitored by bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3)) (B438, Molecular Probes, United States). DiBAC₄(3) stock solution (20 mM, dissolved in dimethylsulfoxide) was diluted in Hank's balanced salt solution (C0218, Beyotime, China) to working solution (40 µM). PC12 cells were stained with DiBAC₄(3) working solution in 96-well plates (100 µL/well, 1 h) after 0.5, 1, 12, 24 or 48 h treatment with 200 µM SNPs [47]. The average fluorescence intensity of DiBAC₄(3) was measured with the HCA system. Cells cultured in medium without SNPs were used as the negative control. Six parallel experiments were conducted for each time point.

Measurement of intracellular Ca²⁺ content of cells exposed to SNPs

PC12 cells were stained using Fluo-3 AM (S1056, Beyotime, China) in 96-well plates after treatment with 200 µM SNPs for 0.5, 1, 12, 24 or 48 h [48]. The average fluorescence intensity of the probes was measured with the HCA system. Cells cultured in medium without SNPs were used as the negative control. Six parallel experiments were conducted for each time point.

Measurement of MMP difference, ATP content, and ROS content of cells exposed to SNPs

JC-1 (C2006, Beyotime, China) was used to stain PC12 cells after treatment with 200 µM SNPs for 0.5, 1, 12, 24 or 48 h [49]. The average fluorescence intensity of JC-1 aggregates (which produce red fluorescence) and monomers (which produce green fluorescence) was measured with the HCA system. Cells cultured in medium without SNPs were used as the negative control. Six parallel experiments were conducted for each time point.

The ATP content was measured using an ATP assay kit (S0026, Beyotime, China). PC12 cells (1 ml/well) at a concentration of 6×10⁴/ml were cultured in a 12-well plate. After 24 h, the culture solution was aspirated, and 1 ml of 200 µM SNPs was added. After 0.5, 1, 12, 24 or 48 h of treatment, the cells were lysed, and the supernatants were collected. The ATP concentrations (C_ATP) and protein concentrations (C_Protein) were then detected, and the ATP content was determined as C_ATP/C_Protein and expressed in nmol/mg [50]. Cells cultured in medium without SNPs were used as the negative control. Six parallel experiments were conducted for each time point.

An ROS Assay Kit (S0033S, Beyotime, China) was used to stain PC12 cells in 96-well plates after treatment with 200 µM SNPs for 0.5, 1, 12, 24 or 48 h [50]. The average fluorescence intensity of dichlorofluorescein generated by the oxidation of ROS was measured with the HCA system. Cells cultured in medium without SNPs were used as the negative control. Six parallel experiments were conducted for each time point.

Data processing and analysis

The Shapiro-Wilk test was employed to test the distribution of the results from parallel groups for normality via SPSS 20.0 software (IBM, Armonk, NY, USA). All experimental data are expressed as the mean ± SD. Student’s t test was performed unless otherwise noted. P < 0.05 was considered to indicate a significant difference, and P < 0.01 was considered to indicate a very significant difference. All experiments were repeated at least three times. Pearson correlation analysis was used for correlation analysis of datasets. The correlation coefficient is abbreviated as “r”; |r| = 0.8 to 1.0 indicates high correlations, |r| = 0.5 to 0.8 indicates moderate correlations, |r| = 0.3 to 0.5 indicates low correlations and |r| = 0 to 0.3 indicates very weak or no correlations [51].

SNPs

Silver nanoparticles

MEA

Microelectrode array

VTMM

Voltage threshold measurement method

V_Th

Voltage threshold

PC12 cells

Rat adrenal pheochromocytoma cells

CMP

Cell membrane potential

MMP

Mitochondrial membrane potential

ATP

Adenosine triphosphate

ROS

Reactive oxygen species

HCA

High content analysis

TEM

Transmission electron microscopy

Standard deviation

K_ATP channels

ATP-sensitive potassium channels

DiBAC4(3)

Bis-(1,3-dibutylbarbituric acid) trimethine oxonol

C_ATP

ATP concentrations

C_Protein

Protein concentrations.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This study was supported by the National Natural Sciences Foundation of China (grant no. 61874024, 61534003, 61076118).

Authors’ contributions

Ze-Qun Zhang: Formal analysis, Data curation, Validation, Investigation, Visualization, Writing – original draft and Writing – review & editing.

Chen Meng: Investigation and Validation.

Kun Hou: Investigation and Validation.

Zhi-Gong Wang: Supervision, Conceptualization, Methodology, Project administration and Funding acquisition.

Yan Huang: Supervision, Methodology, Validation and Writing – review & editing.

Xiao-Ying Lü: Supervision, Conceptualization, Methodology, Validation, Project administration, Funding acquisition and Writing – review & editing.

Zhi-Gong Wang, Yan Huang and Xiao-Ying Lü are Corresponding authors.

All authors read and approved the final manuscript.

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No competing interests reported.

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The cytological and electrophysiological effects of silver nanoparticles on neuron-like PC12 cells

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Preparation and characterization of SNPs

Evaluation of SNP cytotoxicity in PC12 cells

Standard V_Th of PC12 quasi-neuronal networks

V_Th of PC12 quasi-neuronal networks exposed to SNPs

Neurite length of cells exposed to SNPs

CMP difference of cells exposed to SNPs

Intracellular Ca²⁺ content of cells exposed to SNPs

MMP difference, ATP content and ROS content of cells exposed to SNPs

Conjoint analysis between cytological indicators, cell viability and V_Th

Discussion

Conclusion

Methods

Preparation and characterization of SNPs

Cell culture

Evaluation of SNPs-induced cytotoxicity in PC12 cells

Preparation of PC12 quasi-neuronal networks on MEAs

Measurement of standard V_Th

Measurement of V_Th of networks exposed to SNPs

Measurement of neurite length of cells exposed to SNPs

Measurement of CMP difference of cells exposed to SNPs

Measurement of intracellular Ca²⁺ content of cells exposed to SNPs

Measurement of MMP difference, ATP content, and ROS content of cells exposed to SNPs

Data processing and analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

The cytological and electrophysiological effects of silver nanoparticles on neuron-like PC12 cells

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Preparation and characterization of SNPs

Evaluation of SNP cytotoxicity in PC12 cells

Standard VTh of PC12 quasi-neuronal networks

VTh of PC12 quasi-neuronal networks exposed to SNPs

Neurite length of cells exposed to SNPs

CMP difference of cells exposed to SNPs

Intracellular Ca2+ content of cells exposed to SNPs

MMP difference, ATP content and ROS content of cells exposed to SNPs

Conjoint analysis between cytological indicators, cell viability and VTh

Discussion

Conclusion

Methods

Preparation and characterization of SNPs

Cell culture

Evaluation of SNPs-induced cytotoxicity in PC12 cells

Preparation of PC12 quasi-neuronal networks on MEAs

Measurement of standard VTh

Measurement of VTh of networks exposed to SNPs

Measurement of neurite length of cells exposed to SNPs

Measurement of CMP difference of cells exposed to SNPs

Measurement of intracellular Ca2+ content of cells exposed to SNPs

Measurement of MMP difference, ATP content, and ROS content of cells exposed to SNPs

Data processing and analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Standard V_Th of PC12 quasi-neuronal networks

V_Th of PC12 quasi-neuronal networks exposed to SNPs

Intracellular Ca²⁺ content of cells exposed to SNPs

Conjoint analysis between cytological indicators, cell viability and V_Th

Measurement of standard V_Th

Measurement of V_Th of networks exposed to SNPs

Measurement of intracellular Ca²⁺ content of cells exposed to SNPs