Letter pubs.acs.org/NanoLett
Enhanced Neurite Outgrowth by Intracellular Stimulation Ilsoo Kim,† Hye Yeong Lee,⊥ Hyungsuk Kim,† Eungjang Lee,‡ Du-Won Jeong,¶ Ju-Jin Kim,¶ Seung-Han Park,‡ Yoon Ha,⊥ Jukwan Na,† Youngcheol Chae,§ Seong Yi,*,⊥ and Heon-Jin Choi*,† †
Department of Materials Science and Engineering, ‡Department of Physics, and §Department of Electrical and Electronic Engineering, Yonsei University, Seoul 120-749, Republic of Korea ⊥ Department of Neurosurgery, Spine, and Spinal Cord Institute, College of Medicine, Yonsei University, Seoul 120-752, Republic of Korea ¶ Department of Physics, Chonbuk National University, Jeonju 561-756, Republic of Korea S Supporting Information *
ABSTRACT: Electrical stimulation through direct electrical activation has been widely used to recover the function of neurons, primarily through the extracellular application of thin film electrodes. However, studies using extracellular methods show limited ability to reveal correlations between the cells and the electrical stimulation due to interference from external sources such as membrane capacitance and culture medium. Here, we demonstrate long-term intracellular electrical stimulation of undamaged pheochromocytoma (PC-12) cells by utilizing a vertical nanowire electrode array (VNEA). The VNEA was prepared by synthesizing silicon nanowires on a Si substrate through a vapor− liquid−solid (VLS) mechanism and then fabricating them into electrodes with semiconductor nanodevice processing. PC-12 cells were cultured on the VNEA for 4 days with intracellular electrical stimulation and then a 2-day stabilization period. Periodic scanning via two-photon microscopy confirmed that the electrodes pierced the cells without inducing damage. Electrical stimulation through the VNEA enhances cellular differentiation and neurite outgrowth by about 50% relative to extracellular stimulation under the same conditions. VNEA-mediated stimulation also revealed that cellular differentiation and growth in the cultures were dependent on the potential used to stimulate them. Intracellular stimulation using nanowires could pave the way for controlled cellular differentiation and outgrowth studies in living cells. KEYWORDS: Nanowires electrode, intracellular stimulation, cellular differentiation, enhanced neurite outgrowth
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provides direct electrical potential or current into cells through probe-shaped nanoelectrodes. In this approach, cell membrane resistivity and capacitance can be excluded as factors that influence a cell’s effective stimulation; thus, in this consideration, lower current and potential are required in intracellular ES. However, intracellular ES has not been reported because long-term stimulation using intracellular electrodes has not been successfully demonstrated. To deliver electricity intracellularly for cellular stimulation, durable, current-transparent electrodes that will not damage the cell are required. For this purpose, one-dimensional silicon nanowires (SiNWs) are promising. Previous studies indicate that SiNWs can be inserted into cells without damage by virtue of their small scale relative to that of cells.18−21 A semiconducting NW fabrication process for making such insertable electrode NWs has been established, and the process is scalable enough to allow larger arrays of SiNWs to be assembled, allowing stimulation of many cells simultaneously. In this study, we
he ephaptic interaction between living cells and electrical devices is relevant to understanding cellular function when viewing the cell as an electrochemical object. Recent studies showed that the functions of brain and spinal neurons can be controlled electrically.1−4 Neurons can be modulated by electrical stimulation (ES), which elicits cellular activity and can rehabilitate cellular functions (e.g., motor control, memory, audition, etc.).5−7 Indeed, a number of in vivo and in vitro studies have demonstrated the feasibility of ES for enhancing neurite extension and regeneration of transected nerve ends.8−13 Planar-type metallic films including those prepared from platinum, gold, and conducting polymers have been used thus far as electrodes for extracellular ES of neurons.11−15 To achieve effective stimulation, extracellular ES must provide electrical potential or current from outside the cell membrane through the external region of the cell, which includes passing these potential or current through whole cell as well as through culture medium and substrate. Furthermore, since extracellular ES passes through the resistivity and capacitance of the cell membrane to influence cell potential, it requires high current and potential.16,17 Because of these limitations, it is difficult to reveal correlations between cells and their electrical activation from this technique. Meanwhile, intracellular stimulation © XXXX American Chemical Society
Received: May 8, 2015 Revised: July 14, 2015
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DOI: 10.1021/acs.nanolett.5b01810 Nano Lett. XXXX, XXX, XXX−XXX
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Table 1. Defined Sample and Control Group for Electrical Stimulation Study sample VNEA (intracellular) VNEA (intracellular) Au film electrode (extracellular) Au film electrode (extracellular) ref. SiO2 substrate ref. cell culture plate
electrical stimulation (−50 mV, +50 mV, +100 mV) yes no yes no no no
fabrication as follows (Figure 1d−f). Double SiO2 passivation layers (each with a thickness of 300 nm) were used for electrode separation and for blocking the leakage of current from the substrate and culture media (Figure 1d,f). The SiO2 passivation layers were deposited by a high density plasma CVD process for anisotropic deposition of the NW surface and substrate. By using a sputtering process at a rate of 5 nm per minute, Au film with a thickness of 20 nm was then deposited onto the surface of the SiNWs to provide a conductive layer for electrical stimulation (Figure 1e). The second SiO2 passivation layer was deposited onto the substrate, and then the SiO2 layer of the NW electrodes was selectively etched out to expose the Au of the NW surface by a buffered oxide etchant (BOE) (Figure 1b,f). BOE used in this SiO2 etching process was a mixture of ammonium fluoride (NH4F), and hydrofluoric acid (HF) with a 6:1 volume ratio of 40% NH4F in water to 49% HF in water. Pheochromocytoma (PC-12) cells have been widely studied as a model for cellular function and cellular differentiation by external effects. PC-12 cells have some advantages as a cellular differentiation model including their ease of culture and the large amount of background knowledge on their mechanisms of proliferation and differentiation.22−24 Figure 2, panels a and b show scanning electron microscope (SEM) images of the PC12 cells grown on the VNEA, while Figure 2, panel c provides an illustration of these cells. Cell adhesion and growth on the electrodes are critical factors affecting electrical stimulation and thus were investigated together with the other planar-type substrates for comparison. The PC-12 cells on the VNEA grew well (about four times more densely) relative to the planar-type Au film or SiO2 substrates, and similarly to the density on standard cell culture plates (Supporting Information, Figure S1). The PC-12 cells on the VNEA exhibited a sticky
Figure 1. SEM images and scheme of the VNEA fabrication procedure. (a) 30° tilted SEM image of the vertical SiNWs on an Si (111) substrate. The length and diameter of an individual NW are about 3 μm and 60 nm, respectively. (b) 30° tilted SEM image of the SiNW-based VNEA. The VNEA was deposited with a double SiO2 passivation layer, and the Au-coated nanoelectrode tips were selectively exposed for electrical stimulation. (c−f) Schematic illustrations of the SiNW-based VNEA fabrication process: (c) synthesized vertical SiNW on Si substrate, (d) deposited first SiO2 passivation layer for preventing leakage current, (e) deposited Au electrode for electrical pathway, and (f) deposited second SiO2 passivation layer for separation between electrode and culture medium.
investigated intracellular stimulation of PC-12 cells using the SiNW-based vertical nanowire electrode array (VNEA). The fabrication process of the VNEA with conductive Au layer for ES is illustrated in Figure 1. The fabrication of this device was reported in detail in our previous study.21 Briefly, SiNWs are epitaxially grown on the silicon substrate by a vapor−liquid−solid (VLS) mechanism using chemical vapor deposition (CVD) (Figure 1a,c). The diameters, densities, and lengths of SiNWs can be controlled by the growth parameters, the size and concentration of Au colloid particles (colloid diameter 60 nm), and growth time, respectively. According to our previous studies, the optimum size of SiNWs for intracellular use with cells was 3−5 μm in height and 60 nm in diameter.21 A top-down complementary metal-oxide semiconductor (CMOS) device process was then used for electrode
Figure 2. SEM and z-step two-photon microscopy images of PC-12 cells on the VNEA. (a, b) 30° tilted SEM images of the PC-12 cells grown on the VNEA and the relative size of PC-12 cells and VNEA. (c) Illustrations of the PC-12 cells grown on the VNEA. (d, e) Z-step fluorescent scanning images collected through two-photon microscopy. (d) A 20° tilted three-dimensional (3-D) z-stack image with a 200 nm step size and (e) XZ and YZ cross-sectional images of a 3-D z-stack image of PC-12 cells invaginating the NW electrodes. B
DOI: 10.1021/acs.nanolett.5b01810 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 3. Equivalent circuit models for the electrical stimulation study and optical fluorescence images of PC-12 cells grown on substrates under various conditions (VVNEA, applied voltage in VNEA; VFilm, applied voltage in Au Film; Vm, membrane potential; Vrest, resting potential; RVNEA, NW electrode resistance; RFilm, Au filmresistance; Rm, membrane resistance; RL, electrode insulation resistance; Rs, seal resistance of NW electrode; Cm, membrane capacitance; Cp, parasitic capacitance). (a−c) PC-12 cells on the VNEA. (a) The equivalent circuit model of intracellular stimulation through the VNEA. (b) PC-12 cells on the VNEA with 50 mV of controlled electrical stimulation in the form of direct current voltage for 4 days, and an additional 2 days of stabilization. (c) PC-12 cells on the VNEA without electrical stimulation for 6 days in culture. (d−f) PC-12 cells on an Au film or SiO2 substrate. (d) The equivalent circuit model for extracellular stimulations through an Au film. (e) PC-12 cells on an Au film electrode with 50 mV of controlled electrical stimulation in the form of direct current voltage for 4 days, and an additional 2 days of stabilization. (f) PC-12 cells on a SiO2 substrate without electrical stimulation for 6 days in culture. Optical fluorescence images of PC-12 cells on the VNEA with electrical stimulation showed longer neurite outgrowth than the other experimental controls.
morphology with a mature appearance characterized by numerous long, adherent neurites growing out on the VNEA. Those growing on the flat substrates had a flat, spread-out morphology and little neurite outgrowth. Previous studies have shown that nanometer-scale topography of the substrates influences diverse cell behaviors including cell adhesion, cytoskeletal organization, apoptosis, macrophage activation, and gene expression.23,25−27 The VNEA device has a nanostructured surface topography via a vertical NW electrode of 3−5 μm in height, 60 nm in diameter, and 5 ea per 400 μm2 in density. In this study, the VNEA physically confined or penetrated the cells, while the neurites grabbed onto the VNEA. The interaction between the VNEA and cells is geared toward making it difficult for cells to move or detach from the VNEA surface and enhances cell adhesion.27−31 The PC-12 cells cultured on the VNEA were further characterized by z-step fluorescent scanning with two-photon microscopy (Figure 2d).19,32,33 The cross-sectional images (Figure 2e) clearly show that the cells invaginate the NW electrodes. We also observed several vertical NW electrode−cell interfaces by SEM and
confirmed that the electrodes penetrate and locate inside of cytoplasm of the cells by the cross-sectional SEM image (Supporting Information, Figure S2). Notably, images of the NW electrodes were obtainable without fluorescent dyes. NWs have strong and stable third-order nonlinear optical (NLO) signals, including four-wave mixing (FWM) and third-harmonic generation (THG) signals,34,35 which enable imaging of the NWs without any chemical dyeing treatment. An intracellular electrical stimulation system based on a VNEA is shown in Figure S3 of the Supporting Information. For the electrochemical characterization of the VNEA, cyclic voltammetry (CV) measurements were carried out using the Reference 600 controlled potentiostat in standard threeelectrode configuration. The VNEA, an Au film electrode, and Ag/AgCl electrode were used as working, counter, and reference electrodes, respectively, for the three-electrode configuration. CV was performed in the same media used for PC-12 cell culture (Supporting Information, Figure S4). The sample and control groups are listed in Table 1, including a VNEA, Au film electrode for extracellular stimulation, SiO2 C
DOI: 10.1021/acs.nanolett.5b01810 Nano Lett. XXXX, XXX, XXX−XXX
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fluorescence images of PC-12 cells in each group (Figure 3b,c,e,f) were taken after 6 days of culture. The images (Figure 3b) show longer neurite outgrowth in intracellular ES after 6 days of culture (4 days stimulation and an additional 2 days of stabilization) versus the other experimental controls with the same culture period (Figure 3c,e,f). The enhancement of neurite outgrowth by electrical stimulation on the VNEA was confirmed using two regions of the same substrate that were only partially stimulated through selective deposition of the Au film, and which showed active neurite outgrowth in only the stimulated region (Supporting Information, Figure S6). This selectively controlled experiment in same substrate suggests that intracellular ES intensively enhances the cellular outgrowth. Neurite outgrowth of the cells was statistically analyzed by measuring the length of cell neurites that were located on the substrate (Figure 4 and Supporting Information, Figure S7). The average increase in neurite length after intracellular ES was 40 μm (about 53%), which was greater than what had been obtained using the same device (i.e., same topology) without ES. This finding clearly indicates that intracellular ES enhanced neurite outgrowth. Moreover, intracellular ES through the VNEA produced about 75% longer neurite outgrowth than cells that had undergone extracellular ES on an Au thin film device. Regarding the topology, neurite length in both the nanostructured VNEA and flat SiO2 substrate without ES was so similar that they were within the same error range (Figure 4). These results indicate that surface topography may not have an effect on neurite outgrowth, and the main factor influencing neurite outgrowth in this study is intracellular ES. The distribution data of neurite length demonstrated reliability and the general tendencies of the cellular growth patterns in all samples (Supporting Information, Figure S7). Since there is a close relationship between cell growth and protein expression, quantitative analyses of β-actin, Tuj-1, and NF-66 proteins were carried out using Western blotting. βActin served as the housekeeping marker, while Tuj-1 is an early neuronal marker, and NF-66 is a mature neuronal marker (Figure 5a). Figure 5, panel b shows the relative amount of Tuj1 and NF-66 normalized to a standard control (culture plate), and β-actin, which reflected the relative quantity of cells in each sample. When PC-12 cells on the VNEA underwent intracellular ES, NF-66 was observed to be increased by about 3.7fold compared with the standard culture plate. On the other hand, these cells only exhibited a 2.4-fold increase in NF-66 on
Figure 4. Statistical analysis of the neurite length of PC-12 cells in different conditions. The statistical analysis was performed after 6 days of cell growth. Electrical stimulation was performed using 50 mV for 4 days. A 2-day stabilization period followed the 4 days of stimulation. Samples without electrical stimulation were cultured under same conditions and had the same 6-day growth period. All values are given as means ± standard deviation (as determined by an analysis of variance (ANOVA)/Tukey analysis). ∗ indicates statistical difference compared to VNEA with ES (p < 0.05 for all statistical comparisons and performed on 100 samples of neurite length in each condition).
substrate, and culture plate as reference groups. The potential used for stimulation was controlled at −50, 50, and 100 mV of direct current voltage for the 4-day stimulation period, followed by the 2-day stabilization period. The interface between the VNEA or Au film and the PC-12 cells for electrical stimulation was considered to be the equivalent circuit model.20,36 This modeling approach is useful for understanding the correlation between stimulated potential, membrane potential, and resting potential (Figure 3a,d). In the case of electrical stimulations through the VNEA as shown in Figure 3, panel a, we were able to only consider the intracellular stimulations by NW electrodes in a cell and were able to disregard the extracellular stimulations that were delivered by close, free NW electrodes that were uncovered with the cell. This is because the extracellular stimulation must be transferred through the crosstalk of the capacitance (Cc) and resistance (Rc) by the membrane capacitance (Cm), resistance (Rm), outside resting potential (Vrest), etc. In that case, Cc and Rc could be ignored in the parallel-connected VNEA device circuit with direct current (DC) voltage (Supporting Information, Figure S5). Optical
Figure 5. Western blot analysis of protein expression in PC-12 cells under different conditions. (a) Western blot data of β-actin, Tuj-1, and NF-66 proteins of PC-12 cells grown on a culture plate without electrical stimulation, on an Au film electrode with 50 mV of electrical stimulation, on the VNEA without electrical stimulation, and on the VNEA with 50 mV of electrical stimulation, respectively. (b) The relative amounts of Tuj-1 and NF66 normalized by a standard control (culture plate) and β-actin. All values are given as means ± standard deviation (as determined by an ANOVA/ Tukey analysis). ∗ indicates statistical difference compared with the culture plate (p < 0.05 for all statistical comparisons, statistical analysis was performed over eight sample groups in each condition). D
DOI: 10.1021/acs.nanolett.5b01810 Nano Lett. XXXX, XXX, XXX−XXX
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Au film with extracellular ES, and about a 1.6-fold increase when cells were on the VNEA without ES. These results indicate that the PC-12 cells that were grown on the VNEA and that underwent intracellular ES had differentiated to a more mature level than those in other control conditions. The increased NF-66 expression supports the hypothesis that intracellular ES enhances genetically organized cellular differentiation in PC-12 cells. Notably, neurite outgrowth and protein expression related to intracellular ES using the VNEA showed the same tendency toward enhancement, suggesting that intracellular stimulation and cellular activation were correlated. The membrane potential or resting potential is caused by specific changes in membrane permeability for nutrient ion including fomation ions of NGF, potassium, sodium, calcium, and chloride ions, which involves the functional activity of various ion channels and transporters. The resting potential of neurons is −60 to −70 mV, and ionic permeability is directly correlated to the resting potential.37−39 In this study, PC-12 cells were intracellularly stimulated with a constant 50 mV for 4 days. Intracellular stimulation can increase to the threshold of the resting potential in the cell and promote the release of the cell membrane because membrane permeabilization was typically accompanied by a depolarization of membrane potential with 10−20 mV.20,40,41 Therefore, these states enhance the ionic permeability that is critical to cell differentiation and neurite outgrowth. Our results also suggest that electrical stimulation produces an equivalent potential to that generated by receptor−ligand interactions mediating differentiation.38,42,43 The strength of electrical stimulation may also produce an effect on cellular differentiation through its effects on membrane potential or resting potential. While most previous studies support a positive role of electrical stimulation in neurite outgrowth, our data demonstrated that strong stimulation (100 mV), or negatively charged stimulation (−50 mV), did not enhance differentiation (Supporting Information, Figure S8). This suggests that membrane potential or resting potential of cells might be closely correlated with the intracellular stimulation potential and its effects on cellular differentiation and neurite outgrowth. In conclusion, a SiNW-based vertical nanoelectrode array was fabricated, and intracellular electrical stimulation was carried out in PC-12 cells. The array itself was revealed to be a good substrate for adhesion of the PC-12 cells, and we found that cell viability on a VNEA is better than that over standard substrates. It was also confirmed that the VNEA can penetrate PC-12 cells without causing obvious cell damage and can efficiently deliver intracellular electrical stimulation constantly over long periods (almost 100 h). Enhanced neurite outgrowth and cellular differentiation of PC-12 cells by intracellular electrical stimulation through the VNEA were verified statistically through neurite length measurements and Western blot analysis, respectively. The VNEA in this study implies that advanced nanoelectrodes are compatible with the study of neural cells and can be used to stimulate individual cells intracellularly for the regulation of cellular differentiation and outgrowth. We also demonstrated that NWs could be a suitable approach for long-term and real-time intracellular electrical stimulation of living cells. Given the potential of SiNWs for use in integrated circuit devices, the VNEA could create a pathway for the convergence of living cells and semiconductor technology.
Letter
ASSOCIATED CONTENT
S Supporting Information *
Cell culture condition and cell staining information. Additional optical and fluorescence microscope images of PC-12 cells grown on various substrates. Vertical NW electrode−cell interface image. Cyclic voltamogram data of VNEA. The device circuit with vertical NW electrode array. The distribution and statistical analysis of the neurite length of PC-12 cells. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b01810.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions
I.K. and H.Y.L. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2012R1A2A1A03010558 and No. 2014M3A7B4051594) and the Yonsei University Yonsei-SNU Collaborative Research Fund of 2014. H.Y.L., Y.H., and S.Y. were supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University.
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REFERENCES
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DOI: 10.1021/acs.nanolett.5b01810 Nano Lett. XXXX, XXX, XXX−XXX
Enhanced Neurite Outgrowth by Intracellular Stimulation Ilsoo Kim,† Hye Yeong Lee,⊥ Hyungsuk Kim,† Eungjang Lee,‡ Du-Won Jeong,¶ Ju-Jin Kim,¶ Seung-Han Park,‡ Yoon Ha,⊥ Jukwan Na,† Youngcheol Chae,§ Seong Yi,*,⊥ and Heon-Jin Choi*,† †
Department of Materials Science and Engineering, ‡Department of Physics, and §Department of Electrical and Electronic Engineering, Yonsei University, Seoul 120-749, Republic of Korea ⊥ Department of Neurosurgery, Spine, and Spinal Cord Institute, College of Medicine, Yonsei University, Seoul 120-752, Republic of Korea ¶ Department of Physics, Chonbuk National University, Jeonju 561-756, Republic of Korea S Supporting Information *
ABSTRACT: Electrical stimulation through direct electrical activation has been widely used to recover the function of neurons, primarily through the extracellular application of thin film electrodes. However, studies using extracellular methods show limited ability to reveal correlations between the cells and the electrical stimulation due to interference from external sources such as membrane capacitance and culture medium. Here, we demonstrate long-term intracellular electrical stimulation of undamaged pheochromocytoma (PC-12) cells by utilizing a vertical nanowire electrode array (VNEA). The VNEA was prepared by synthesizing silicon nanowires on a Si substrate through a vapor− liquid−solid (VLS) mechanism and then fabricating them into electrodes with semiconductor nanodevice processing. PC-12 cells were cultured on the VNEA for 4 days with intracellular electrical stimulation and then a 2-day stabilization period. Periodic scanning via two-photon microscopy confirmed that the electrodes pierced the cells without inducing damage. Electrical stimulation through the VNEA enhances cellular differentiation and neurite outgrowth by about 50% relative to extracellular stimulation under the same conditions. VNEA-mediated stimulation also revealed that cellular differentiation and growth in the cultures were dependent on the potential used to stimulate them. Intracellular stimulation using nanowires could pave the way for controlled cellular differentiation and outgrowth studies in living cells. KEYWORDS: Nanowires electrode, intracellular stimulation, cellular differentiation, enhanced neurite outgrowth
T
provides direct electrical potential or current into cells through probe-shaped nanoelectrodes. In this approach, cell membrane resistivity and capacitance can be excluded as factors that influence a cell’s effective stimulation; thus, in this consideration, lower current and potential are required in intracellular ES. However, intracellular ES has not been reported because long-term stimulation using intracellular electrodes has not been successfully demonstrated. To deliver electricity intracellularly for cellular stimulation, durable, current-transparent electrodes that will not damage the cell are required. For this purpose, one-dimensional silicon nanowires (SiNWs) are promising. Previous studies indicate that SiNWs can be inserted into cells without damage by virtue of their small scale relative to that of cells.18−21 A semiconducting NW fabrication process for making such insertable electrode NWs has been established, and the process is scalable enough to allow larger arrays of SiNWs to be assembled, allowing stimulation of many cells simultaneously. In this study, we
he ephaptic interaction between living cells and electrical devices is relevant to understanding cellular function when viewing the cell as an electrochemical object. Recent studies showed that the functions of brain and spinal neurons can be controlled electrically.1−4 Neurons can be modulated by electrical stimulation (ES), which elicits cellular activity and can rehabilitate cellular functions (e.g., motor control, memory, audition, etc.).5−7 Indeed, a number of in vivo and in vitro studies have demonstrated the feasibility of ES for enhancing neurite extension and regeneration of transected nerve ends.8−13 Planar-type metallic films including those prepared from platinum, gold, and conducting polymers have been used thus far as electrodes for extracellular ES of neurons.11−15 To achieve effective stimulation, extracellular ES must provide electrical potential or current from outside the cell membrane through the external region of the cell, which includes passing these potential or current through whole cell as well as through culture medium and substrate. Furthermore, since extracellular ES passes through the resistivity and capacitance of the cell membrane to influence cell potential, it requires high current and potential.16,17 Because of these limitations, it is difficult to reveal correlations between cells and their electrical activation from this technique. Meanwhile, intracellular stimulation © XXXX American Chemical Society
Received: May 8, 2015 Revised: July 14, 2015
A
DOI: 10.1021/acs.nanolett.5b01810 Nano Lett. XXXX, XXX, XXX−XXX
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Nano Letters
Table 1. Defined Sample and Control Group for Electrical Stimulation Study sample VNEA (intracellular) VNEA (intracellular) Au film electrode (extracellular) Au film electrode (extracellular) ref. SiO2 substrate ref. cell culture plate
electrical stimulation (−50 mV, +50 mV, +100 mV) yes no yes no no no
fabrication as follows (Figure 1d−f). Double SiO2 passivation layers (each with a thickness of 300 nm) were used for electrode separation and for blocking the leakage of current from the substrate and culture media (Figure 1d,f). The SiO2 passivation layers were deposited by a high density plasma CVD process for anisotropic deposition of the NW surface and substrate. By using a sputtering process at a rate of 5 nm per minute, Au film with a thickness of 20 nm was then deposited onto the surface of the SiNWs to provide a conductive layer for electrical stimulation (Figure 1e). The second SiO2 passivation layer was deposited onto the substrate, and then the SiO2 layer of the NW electrodes was selectively etched out to expose the Au of the NW surface by a buffered oxide etchant (BOE) (Figure 1b,f). BOE used in this SiO2 etching process was a mixture of ammonium fluoride (NH4F), and hydrofluoric acid (HF) with a 6:1 volume ratio of 40% NH4F in water to 49% HF in water. Pheochromocytoma (PC-12) cells have been widely studied as a model for cellular function and cellular differentiation by external effects. PC-12 cells have some advantages as a cellular differentiation model including their ease of culture and the large amount of background knowledge on their mechanisms of proliferation and differentiation.22−24 Figure 2, panels a and b show scanning electron microscope (SEM) images of the PC12 cells grown on the VNEA, while Figure 2, panel c provides an illustration of these cells. Cell adhesion and growth on the electrodes are critical factors affecting electrical stimulation and thus were investigated together with the other planar-type substrates for comparison. The PC-12 cells on the VNEA grew well (about four times more densely) relative to the planar-type Au film or SiO2 substrates, and similarly to the density on standard cell culture plates (Supporting Information, Figure S1). The PC-12 cells on the VNEA exhibited a sticky
Figure 1. SEM images and scheme of the VNEA fabrication procedure. (a) 30° tilted SEM image of the vertical SiNWs on an Si (111) substrate. The length and diameter of an individual NW are about 3 μm and 60 nm, respectively. (b) 30° tilted SEM image of the SiNW-based VNEA. The VNEA was deposited with a double SiO2 passivation layer, and the Au-coated nanoelectrode tips were selectively exposed for electrical stimulation. (c−f) Schematic illustrations of the SiNW-based VNEA fabrication process: (c) synthesized vertical SiNW on Si substrate, (d) deposited first SiO2 passivation layer for preventing leakage current, (e) deposited Au electrode for electrical pathway, and (f) deposited second SiO2 passivation layer for separation between electrode and culture medium.
investigated intracellular stimulation of PC-12 cells using the SiNW-based vertical nanowire electrode array (VNEA). The fabrication process of the VNEA with conductive Au layer for ES is illustrated in Figure 1. The fabrication of this device was reported in detail in our previous study.21 Briefly, SiNWs are epitaxially grown on the silicon substrate by a vapor−liquid−solid (VLS) mechanism using chemical vapor deposition (CVD) (Figure 1a,c). The diameters, densities, and lengths of SiNWs can be controlled by the growth parameters, the size and concentration of Au colloid particles (colloid diameter 60 nm), and growth time, respectively. According to our previous studies, the optimum size of SiNWs for intracellular use with cells was 3−5 μm in height and 60 nm in diameter.21 A top-down complementary metal-oxide semiconductor (CMOS) device process was then used for electrode
Figure 2. SEM and z-step two-photon microscopy images of PC-12 cells on the VNEA. (a, b) 30° tilted SEM images of the PC-12 cells grown on the VNEA and the relative size of PC-12 cells and VNEA. (c) Illustrations of the PC-12 cells grown on the VNEA. (d, e) Z-step fluorescent scanning images collected through two-photon microscopy. (d) A 20° tilted three-dimensional (3-D) z-stack image with a 200 nm step size and (e) XZ and YZ cross-sectional images of a 3-D z-stack image of PC-12 cells invaginating the NW electrodes. B
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Figure 3. Equivalent circuit models for the electrical stimulation study and optical fluorescence images of PC-12 cells grown on substrates under various conditions (VVNEA, applied voltage in VNEA; VFilm, applied voltage in Au Film; Vm, membrane potential; Vrest, resting potential; RVNEA, NW electrode resistance; RFilm, Au filmresistance; Rm, membrane resistance; RL, electrode insulation resistance; Rs, seal resistance of NW electrode; Cm, membrane capacitance; Cp, parasitic capacitance). (a−c) PC-12 cells on the VNEA. (a) The equivalent circuit model of intracellular stimulation through the VNEA. (b) PC-12 cells on the VNEA with 50 mV of controlled electrical stimulation in the form of direct current voltage for 4 days, and an additional 2 days of stabilization. (c) PC-12 cells on the VNEA without electrical stimulation for 6 days in culture. (d−f) PC-12 cells on an Au film or SiO2 substrate. (d) The equivalent circuit model for extracellular stimulations through an Au film. (e) PC-12 cells on an Au film electrode with 50 mV of controlled electrical stimulation in the form of direct current voltage for 4 days, and an additional 2 days of stabilization. (f) PC-12 cells on a SiO2 substrate without electrical stimulation for 6 days in culture. Optical fluorescence images of PC-12 cells on the VNEA with electrical stimulation showed longer neurite outgrowth than the other experimental controls.
morphology with a mature appearance characterized by numerous long, adherent neurites growing out on the VNEA. Those growing on the flat substrates had a flat, spread-out morphology and little neurite outgrowth. Previous studies have shown that nanometer-scale topography of the substrates influences diverse cell behaviors including cell adhesion, cytoskeletal organization, apoptosis, macrophage activation, and gene expression.23,25−27 The VNEA device has a nanostructured surface topography via a vertical NW electrode of 3−5 μm in height, 60 nm in diameter, and 5 ea per 400 μm2 in density. In this study, the VNEA physically confined or penetrated the cells, while the neurites grabbed onto the VNEA. The interaction between the VNEA and cells is geared toward making it difficult for cells to move or detach from the VNEA surface and enhances cell adhesion.27−31 The PC-12 cells cultured on the VNEA were further characterized by z-step fluorescent scanning with two-photon microscopy (Figure 2d).19,32,33 The cross-sectional images (Figure 2e) clearly show that the cells invaginate the NW electrodes. We also observed several vertical NW electrode−cell interfaces by SEM and
confirmed that the electrodes penetrate and locate inside of cytoplasm of the cells by the cross-sectional SEM image (Supporting Information, Figure S2). Notably, images of the NW electrodes were obtainable without fluorescent dyes. NWs have strong and stable third-order nonlinear optical (NLO) signals, including four-wave mixing (FWM) and third-harmonic generation (THG) signals,34,35 which enable imaging of the NWs without any chemical dyeing treatment. An intracellular electrical stimulation system based on a VNEA is shown in Figure S3 of the Supporting Information. For the electrochemical characterization of the VNEA, cyclic voltammetry (CV) measurements were carried out using the Reference 600 controlled potentiostat in standard threeelectrode configuration. The VNEA, an Au film electrode, and Ag/AgCl electrode were used as working, counter, and reference electrodes, respectively, for the three-electrode configuration. CV was performed in the same media used for PC-12 cell culture (Supporting Information, Figure S4). The sample and control groups are listed in Table 1, including a VNEA, Au film electrode for extracellular stimulation, SiO2 C
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fluorescence images of PC-12 cells in each group (Figure 3b,c,e,f) were taken after 6 days of culture. The images (Figure 3b) show longer neurite outgrowth in intracellular ES after 6 days of culture (4 days stimulation and an additional 2 days of stabilization) versus the other experimental controls with the same culture period (Figure 3c,e,f). The enhancement of neurite outgrowth by electrical stimulation on the VNEA was confirmed using two regions of the same substrate that were only partially stimulated through selective deposition of the Au film, and which showed active neurite outgrowth in only the stimulated region (Supporting Information, Figure S6). This selectively controlled experiment in same substrate suggests that intracellular ES intensively enhances the cellular outgrowth. Neurite outgrowth of the cells was statistically analyzed by measuring the length of cell neurites that were located on the substrate (Figure 4 and Supporting Information, Figure S7). The average increase in neurite length after intracellular ES was 40 μm (about 53%), which was greater than what had been obtained using the same device (i.e., same topology) without ES. This finding clearly indicates that intracellular ES enhanced neurite outgrowth. Moreover, intracellular ES through the VNEA produced about 75% longer neurite outgrowth than cells that had undergone extracellular ES on an Au thin film device. Regarding the topology, neurite length in both the nanostructured VNEA and flat SiO2 substrate without ES was so similar that they were within the same error range (Figure 4). These results indicate that surface topography may not have an effect on neurite outgrowth, and the main factor influencing neurite outgrowth in this study is intracellular ES. The distribution data of neurite length demonstrated reliability and the general tendencies of the cellular growth patterns in all samples (Supporting Information, Figure S7). Since there is a close relationship between cell growth and protein expression, quantitative analyses of β-actin, Tuj-1, and NF-66 proteins were carried out using Western blotting. βActin served as the housekeeping marker, while Tuj-1 is an early neuronal marker, and NF-66 is a mature neuronal marker (Figure 5a). Figure 5, panel b shows the relative amount of Tuj1 and NF-66 normalized to a standard control (culture plate), and β-actin, which reflected the relative quantity of cells in each sample. When PC-12 cells on the VNEA underwent intracellular ES, NF-66 was observed to be increased by about 3.7fold compared with the standard culture plate. On the other hand, these cells only exhibited a 2.4-fold increase in NF-66 on
Figure 4. Statistical analysis of the neurite length of PC-12 cells in different conditions. The statistical analysis was performed after 6 days of cell growth. Electrical stimulation was performed using 50 mV for 4 days. A 2-day stabilization period followed the 4 days of stimulation. Samples without electrical stimulation were cultured under same conditions and had the same 6-day growth period. All values are given as means ± standard deviation (as determined by an analysis of variance (ANOVA)/Tukey analysis). ∗ indicates statistical difference compared to VNEA with ES (p < 0.05 for all statistical comparisons and performed on 100 samples of neurite length in each condition).
substrate, and culture plate as reference groups. The potential used for stimulation was controlled at −50, 50, and 100 mV of direct current voltage for the 4-day stimulation period, followed by the 2-day stabilization period. The interface between the VNEA or Au film and the PC-12 cells for electrical stimulation was considered to be the equivalent circuit model.20,36 This modeling approach is useful for understanding the correlation between stimulated potential, membrane potential, and resting potential (Figure 3a,d). In the case of electrical stimulations through the VNEA as shown in Figure 3, panel a, we were able to only consider the intracellular stimulations by NW electrodes in a cell and were able to disregard the extracellular stimulations that were delivered by close, free NW electrodes that were uncovered with the cell. This is because the extracellular stimulation must be transferred through the crosstalk of the capacitance (Cc) and resistance (Rc) by the membrane capacitance (Cm), resistance (Rm), outside resting potential (Vrest), etc. In that case, Cc and Rc could be ignored in the parallel-connected VNEA device circuit with direct current (DC) voltage (Supporting Information, Figure S5). Optical
Figure 5. Western blot analysis of protein expression in PC-12 cells under different conditions. (a) Western blot data of β-actin, Tuj-1, and NF-66 proteins of PC-12 cells grown on a culture plate without electrical stimulation, on an Au film electrode with 50 mV of electrical stimulation, on the VNEA without electrical stimulation, and on the VNEA with 50 mV of electrical stimulation, respectively. (b) The relative amounts of Tuj-1 and NF66 normalized by a standard control (culture plate) and β-actin. All values are given as means ± standard deviation (as determined by an ANOVA/ Tukey analysis). ∗ indicates statistical difference compared with the culture plate (p < 0.05 for all statistical comparisons, statistical analysis was performed over eight sample groups in each condition). D
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Au film with extracellular ES, and about a 1.6-fold increase when cells were on the VNEA without ES. These results indicate that the PC-12 cells that were grown on the VNEA and that underwent intracellular ES had differentiated to a more mature level than those in other control conditions. The increased NF-66 expression supports the hypothesis that intracellular ES enhances genetically organized cellular differentiation in PC-12 cells. Notably, neurite outgrowth and protein expression related to intracellular ES using the VNEA showed the same tendency toward enhancement, suggesting that intracellular stimulation and cellular activation were correlated. The membrane potential or resting potential is caused by specific changes in membrane permeability for nutrient ion including fomation ions of NGF, potassium, sodium, calcium, and chloride ions, which involves the functional activity of various ion channels and transporters. The resting potential of neurons is −60 to −70 mV, and ionic permeability is directly correlated to the resting potential.37−39 In this study, PC-12 cells were intracellularly stimulated with a constant 50 mV for 4 days. Intracellular stimulation can increase to the threshold of the resting potential in the cell and promote the release of the cell membrane because membrane permeabilization was typically accompanied by a depolarization of membrane potential with 10−20 mV.20,40,41 Therefore, these states enhance the ionic permeability that is critical to cell differentiation and neurite outgrowth. Our results also suggest that electrical stimulation produces an equivalent potential to that generated by receptor−ligand interactions mediating differentiation.38,42,43 The strength of electrical stimulation may also produce an effect on cellular differentiation through its effects on membrane potential or resting potential. While most previous studies support a positive role of electrical stimulation in neurite outgrowth, our data demonstrated that strong stimulation (100 mV), or negatively charged stimulation (−50 mV), did not enhance differentiation (Supporting Information, Figure S8). This suggests that membrane potential or resting potential of cells might be closely correlated with the intracellular stimulation potential and its effects on cellular differentiation and neurite outgrowth. In conclusion, a SiNW-based vertical nanoelectrode array was fabricated, and intracellular electrical stimulation was carried out in PC-12 cells. The array itself was revealed to be a good substrate for adhesion of the PC-12 cells, and we found that cell viability on a VNEA is better than that over standard substrates. It was also confirmed that the VNEA can penetrate PC-12 cells without causing obvious cell damage and can efficiently deliver intracellular electrical stimulation constantly over long periods (almost 100 h). Enhanced neurite outgrowth and cellular differentiation of PC-12 cells by intracellular electrical stimulation through the VNEA were verified statistically through neurite length measurements and Western blot analysis, respectively. The VNEA in this study implies that advanced nanoelectrodes are compatible with the study of neural cells and can be used to stimulate individual cells intracellularly for the regulation of cellular differentiation and outgrowth. We also demonstrated that NWs could be a suitable approach for long-term and real-time intracellular electrical stimulation of living cells. Given the potential of SiNWs for use in integrated circuit devices, the VNEA could create a pathway for the convergence of living cells and semiconductor technology.
Letter
ASSOCIATED CONTENT
S Supporting Information *
Cell culture condition and cell staining information. Additional optical and fluorescence microscope images of PC-12 cells grown on various substrates. Vertical NW electrode−cell interface image. Cyclic voltamogram data of VNEA. The device circuit with vertical NW electrode array. The distribution and statistical analysis of the neurite length of PC-12 cells. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b01810.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions
I.K. and H.Y.L. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2012R1A2A1A03010558 and No. 2014M3A7B4051594) and the Yonsei University Yonsei-SNU Collaborative Research Fund of 2014. H.Y.L., Y.H., and S.Y. were supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University.
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REFERENCES
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