In vitro toxicity of PEG/Cor Nps to PC-12 cells
PC-12 cells were co-cultured with PEG/Cor Nps for 4h and observed under confocal microscopic images after removing redundant PEG/Cor Nps. The results showed PEG/Cor Nps distributed in the cytoplasm but did not penetrate the nucleus (Fig. 2a, c).Comparing with previous studies, PEG/Cor Nps have stronger auto-fluorescence intensity than FITC-labeled mesoporous silica nanoparticles[19] and Texas Red-labeled fullerenes nanoparticles[21]. It means that the Cor have potential values in cell imaging and photodynamic therapy(Fig. 2c)[4, 12, 22]. S. Liu et.al. supported that the Cor preferred mitochondria accumulation due to the large negative membrane potential of mitochondria. In this study, however, we did not co-locate Cor with mitochondria[22].
The viability of PC-12 cells was determined by the standard cytotoxicity test. The viability and inhibiting rate were presented time-dependent and dose-dependent manners after PEG/Cor Nps treatment. The half maximal inhibitory concentration (IC50) was calculated as 10 µg/mL (72h co-culture), 20 µg/mL (50h co-culture), 40 µg/mL (4h co-culture) and 50 µg/mL (3h co-culture) (Fig. 2b). It means that the cytotoxicity of PEG/Cor Nps dependent on adding concentration and culturing time. Regarding 5 µg/mL, cell viability did not show half maximal inhibitory rate after 72h co-culture (Fig. 2b). L. Zhang and S. Liu reported that Cor has functioned in controlling ROS production[22]. According to this study, Cor could induce a higher extent of cytotoxicity and thus more satisfactory therapeutic outcomes than perylene[22]. Nevertheless, our results showed that PEG/Cor Nps have characteristics of low cytotoxicity, high rate of cell uptake and fluorescence intensity at 5µg/ml concentration. Therefore, we speculated that Cor-based nanoparticles have potential values in cell imaging and drug carries at low concentration.
The dose-dependent effects of PEG/Cor Nps on whole-cell ion currents
Carbon nanomaterials have been evidenced to have impacts on regulating ion channels[15, 16]. For example, SWCNTs can physically occlude the potassium voltage-gated channel[15] and inhibit calcium ion channel activation through releasing yttrium[16]. Carbon materials were therefore used to regulate physical functions and cell behaviors, such as calcium-dependent cellular functions of growing neurons [23] and cell death controlling[16]. A highlight study demonstrated the pore occlusion mechanism and suggested the diversity of the topological structure of carbon materials can induce different ion channel behaviors[15]. Cor as novel carbon nanomaterials, the effects on ion channel behaviors was examined by whole cell patch clamp.
According to our present study, PEG/Cor Nps concentration at 5µg/mL was selected to investigate the behaviors of ion channels. All test cells were held at -60 mV and the current traces were evoked by using 300 ms constant depolarizing pulse from − 50 mV to + 90 mV in increments of 10 mV during the recording of whole-cell ion currents. The inward currents and outward currents of the registered cells showed typical voltage-gated Na+ currents and K+ currents. Electrophysiological behavior is similar to what was reported in other studies(Fig. 3a)[24]. The activated inward current could be completely blocked by bath application of 0.5 mM TTX (Fig. 3a), indicating that voltage-gated sodium current carries the largest inward currents. The application of PEG/Cor Nps in different dose produced obvious inhibiting effects on inward currents (voltage-gated Na+ currents) (Fig. 3a, c) but no significant changes on outward currents (voltage-gated K+ currents) (Fig. 3b, e). The activation curves of inward currents produced depolarization shifts with the increasing of PEG/Cor Nps(Fig. 3d). It means that PEG/Cor Nps reduce the opening number of voltage-gated Na+ currents at each command potential in PC-12 cells as the dose increased[25]. These results suggested the PEG/Cor Nps have dose-dependent effects on inhibiting voltage-gated Na+ currents activation.
Molecular dynamics (MD) simulations for pore occlusion mechanism
All-atom molecular dynamics (MD) simulations were carried out to capture the undying mechanism of Cor molecule blocking the sodium channel[26–28]. As shown in Fig. 5a, we construct a cell membrane embedding a sodium protein in the center, while two sides are covers by enough water molecules representing the real environment in the life system. The number of Cor (N) molecules will be placed in the inner side of the cell membrane (Fig. 5b) considering that the Cor will be delivered into the cell directly as the experimental results shown that. To measure the structure change of protein induced by the Cor, we calculate the Root-Mean-Square Deviation (RMSD) via the flowing equation[29],
$${\text{RMSD=}}\left\langle {\sqrt {\frac{1}{M}\sum\limits_{{i=1,M}} {\delta _{i}^{2}} } } \right\rangle$$
3
Where M is the total number of atoms, δi is the distance between the ith atom and the reference structure, the angular bracket representing the average of the total simulation time. As shown in Fig. 5c, the RMSD will increase firstly and then up to the convergence stage at about 1 ns. The average of RMSD in the last 1 ns was summarized to quantificationally describe the influence of Cor on the protein (Fig. 5d). The fact can be concluded that the influence will be larger when more Cor molecules accumulated in the system, which will introduce a significant effect on blocking the pore of sodium protein.
We further summarized the configurations of Corto describe the mechanism of Cor molecule blocking the protein channel. From the results, the Cor will accumulate near the pore of the channel protein at lower concentration (N = 8) to block the pore of the channel protein (Fig. 6a, b). As the time increases, the concentration of Cor will be increased and prefer aggregate to a cluster near the inner surface of the protein (Fig. 6c, d) (N = 80). Both cases would block the protein channel and inhibit sodium ion channel functions, which agrees well with the above experimental results. These two different mechanisms can be explained well via the analysis of energy. First, the small molecules prefer to aggregate in the water environment, upping to the lowest total energy. So, Cor will form a cluster at higher concentration (Fig. 6c, d). However, at lower concentration, the interfacial energy between Cor and protein will be larger than the interfacial energy within the Cor, and then Cor will be absorbed onto the surface of the protein and even enter the channel of protein. Besides, the result also explains the time-dependent manner of blocking effects. As the time increasing, Cor will be absorbed by the protein and increases concentration of Cor while causing inhibition from low to high concentration stage.
Moreover, we found that the effects of Cor on blocking sodium ion channel function dependents on the orientation angle (θ) configuration of Cor in the system, which is defined as the included angle between the z-direction and the normal direction of Cor (Fig. 7a). For the first, the orientation angle has an almost uniform distribution ranging from 0° to 180° for different concentration of Cor. The time evolution of the orientation angle is very important for the configuration of Cor in the system and the blocking mechanism of protein. The orientation angle as a function of time is summarized, implying that the change of angle depends greatly on the concentration (Fig. 7b). At low concentration (N = 8), the orientation angle can change greatly between “rim-to-protein” and “bottom-to-protein” states. As the time increase, Cor will be enriched leading to increase of concentration, the orientation angle will have small variation and maintain the initial state.
In present study, we demonstrated PEG/Cor complexes could accumulate in cytoplasm of PC-12 cells. It is important that the accumulated PEG/Cor complexes could dose-dependent and time-dependent inhibit voltage-gated Na+ currents but pure mPEG-DSPE did not have this function. Thus we speculated voltage-gated Na+ currents were inhibited as a result of releasing and accumulating of Cor. Many of the most common diseases involve abnormalities of voltage-gated Na+ currents, such as cancer[18], neurological disorder[17, 30] and cardiovascular diseases[27, 31]. These functions of voltage-gated Na+ currents make it easy targets for external agents such as natural toxins and synthetic drugs that react with them by establishment electrochemical interactions[15, 28]. Therefore, these findings postulate new uses for Cor in biological applications.
The mechanisms of Cor inhibiting voltage-gated Na+ currents were discussed according to previous studies. First, the voltage-gated Na+ currents were reduced during oxidative stress[32]. Eliminating ROS could decrease the response of inhibiting voltage-gated Na+ currents[33]. Curved Cor induced dipole moment aids electron transfer and enhance enables ROS generation[4, 22]. These studies suggested the Cor-induced production of ROS play a key role in inhibiting voltage-gated Na+ currents(Fig. 8b). Moreover, topological structure of carbon materials may influenced the response of ion channels[15]. K. H. Park et.al. made a study which compared the ion channels functions of PC-12 cells after treatment with SWCNTs, MWCNTs and fullerenes[15]. They suggested that the carbon materials have effects on blocking K+ channels and the blockage was dependent on the shape and dimension of materials used[15]. Cor as a bowl-shape three dimension carbon materials, may inhibited voltage-gated Na+ currents by blocking the pore of sodium ion channel protein as similar as SWCNTs[15]. However, this effects were decided by the sodium ion channel protein subunit induced pore regulating mechanisms[34]. For example, Arcobacterbutzleri voltage-gated Na+ channel protein (NaVAb)with an orifice of ~ 4.6×4.6 Å moved only ~ 1 Å(Fig. 8a red)[30, 34, 35]. Therefore, the Cor (~ 7.2 Å ) cannot passed the pore and blocked on it, such as pore-blocking toxin tetrodotoxin[30]. Regarding Magnetococcussp voltage-gated Na+ channel protein (NaVMs), the pore S6 segments displaced at ~ 2.5 Å scale, would result in an aperture in the activation gate with a diameter > 8 Å(Fig. 8a blue)[34].The aperture was easily sufficient for Cor to pass through. These general pore architecture reveals the structural basis for gated access of blocking ions and drugs to the lumen of the pore observed in classical studies of ion selective and pore block[15, 34, 35]. This mechanism appears to be governed by geometrical factors as a “physical barrier” [15]and lacks any other biological/chemical component that usually required by conventional agents[36]. However, the specific experiment and molecular simulation needs to be studied in the future to reveal the really mechanisms[14, 16].