This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.
Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.
Recent studies have demonstrated that the transient heating associated with the absorption of infrared light by water (wavelength >1,400 nm) can be used to induce action potentials in nerve tissue1 and intracellular calcium transients in cardiomyocytes2. The use of infrared light has raised great interest for applications in neural prostheses, due to the potential finer spatial resolution, lack of direct contact with the tissue, minimization of stimulation artifacts, and removal of the need to genetically modify the cells prior to stimulation (as required in optogenetics)1. Despite all of these benefits, recently developed thermal models suggested that the target tissue/cells may be affected by cumulative heating effects, when multiple stimulus sites and/or high repetition rates are used3,4.
In response to these challenges, researchers have recognized the potential to use extrinsic absorbers for nerve stimulation to produce more localized heating effects in the tissue. Huang et al. demonstrated this principle by using superparamagnetic ferrite nanoparticles to remotely activate the temperature-sensitive TRPV1 channels in HEK 293 cells with a radio-frequency magnetic field5. Although this technique may allow for deeper penetration (magnetic fields interact relatively weakly with tissue), the responses were only recorded over periods of seconds, rather than the millisecond durations required in bionic devices5. Similarly, Farah et al. demonstrated electrical stimulation of rat cortical neurons with black micro-particles in vitro. They showed cell-level precision in stimulation using pulse durations on the order of hundreds of µs and energies in the range of µJ, potentially allowing for faster repetition rates6.
The use of extrinsic absorbers has also been applied to induce morphological changes in vitro. Ciofani et al. showed a ~40% increase in neuronal cell outgrowth using piezoelectric boron nitride nanotubes excited by ultrasound7. Similarly, endocytosed iron oxide nanoparticles in PC12 cells have been reported to enhance neurite differentiation in a dose-dependent manner, due to the activation of cell adhesion molecules with the iron oxide8.
Recently, the interest in extrinsic absorbers to assist neural stimulation has also focused on the use of gold nanoparticles (Au NPs). Au NPs have the ability to efficiently absorb laser light at the plasmonic peak and to dissipate it into the surrounding environment in the form of heat9. Amongst all of the available particle shapes, the optical absorption of gold nanorods (Au NRs) conveniently matches the therapeutic window of biological tissues (near infrared – NIR, wavelength between 750-1,400 nm)10. Moreover, in the context of neural stimulation, the use of Au NRs provides relatively favorable biocompatibility and a wide range of surface functionalization options11. Recent studies have shown that a stimulatory effect on differentiation can be induced after continuous laser exposures of Au NRs in NG108-15 neuronal cells12. Similarly, intracellular calcium transients were recorded in neuronal cells cultured with Au NRs after laser irradiation modulated with variable frequencies and pulse lengths13. Cell membrane depolarization was also recorded after NIR laser illumination of Au NRs in primary cultures of spiral ganglion neurons14. The first in vivo application with irradiated Au NRs has been demonstrated just recently. Eom and coworkers exposed Au NRs at their plasmonic peak and recorded a six-fold increase in the amplitude of compound nerve action potentials (CNAPs) and a three-fold decrease in the stimulation threshold in rat sciatic nerves. The enhanced response was attributed to local heating effects resulting from the excitation of the NR plasmonic peak15.
In the present paper, protocols for investigating the effects of laser stimulation in NG108-15 neuronal cells cultured with Au NRs are specified. These methods provide a simple, yet powerful, way to irradiate cell populations in vitro using standard biological techniques and materials. The protocol is based on a fiber-coupled laser diode (LD) that allows safe operation and repeatable alignment. The Au NR sample preparation and laser irradiation methods can be further extended to different particle shapes and neuronal cell cultures, providing that the specific synthesis and culture protocols are known, respectively.
1. Au NRs Preparation
Note: Au NRs can be synthesized by a number of recipes16, or purchased from commercial vendors.
2. NG108-15 Neuronal Cell Line Culture and Differentiation
3. Neurite Outgrowth Enhancement
4. Laser-induced Intracellular Ca2+ Imaging
By using Protocols 1, 2, and 3 described here, a stimulatory effect on differentiation was observed in NG108-15 neuronal cells cultured with Au NPs (Au NRs, poly(styrenesulfonate)-coated Au NRs and silica-coated Au NRs) after laser exposures between 1.25 and 7.5 W·cm−2 . Confocal images of rhodamineB-labelled Au NRs demonstrated that the particles were internalized from day 1 of incubation12. The localization was predominantly observed in the cell cytoplasm, indicating that the preferred mechanism of uptake was via the cell body membrane12. The main morphological changes detected after inducing differentiation in NG108-15 neuronal cells were the arrest of proliferation, the expression of βIII-tubulin protein and the outgrowth of neurites, which were analyzed in terms of maximum length and number22.
Samples cultured with NRs showed a neurite length increase at the laser irradiance levels measured here (between 1.25 and 7.5 W·cm−2). Control samples (cells cultured without NPs and irradiated with the same laser power) showed no significant change in length. Using an irradiation dose of 7.5 W·cm−2, the final neurite length of NG108-15 cultured with Au NRs was roughly 36% higher (p <0.01) than the non-laser-irradiated samples. This behavior was not specifically linked to the surface chemistry of the NRs. These values were almost 20% greater than the neurites developed by NG108 -15 alone and exposed to the same value of laser exposure (p <0.05)12. These results are in line with previously published studies on PC12 neuronal cells cultured with piezoelectric nanotubes and irradiated with ultrasound radiation7.
Control experiments without Au NRs also showed some stimulatory effects of the 780 nm light in terms of percentage of neurons with neurites and number of neurites per neuron. This stimulation was more effective at lower laser powers (1.25 W∙cm-2) with a subsequent decrease at the highest laser energy (7.5 W∙cm-2)12. A moderate stimulation caused by the NPs without any laser irradiation (poly(styrenesulfonate)-coated and silica-coated only) was detected in the percentage of neurons with neurites12. These results are in line with recently published observations that gold nanoparticles can increase neuronal activity in vitro23,24. Figure 2 shows an example of epifluorescence images of differentiated NG108-15 neuronal cells cultured alone (Figure 2A) or with Au NRs (Figure 2B) and irradiated with different laser powers (indicated in the figure).
The potential for photo-generated intracellular Ca2+ release was assessed using pulsed NIR light in accordance with Protocols 1, 2, and 4. Calcium ions play an important role in different cellular activities, such as mitosis, muscle contraction and neurite extension25,26. In response to a stimulus, Ca2+ increases, oscillates and decreases, leading to the activation, modulation or termination of a specific cell function. Recently, Ca2+ transients have also been observed as a consequence of IR laser exposure in cardiomyocytes. In that work, the Ca2+ responses evoked after IR exposure exhibited lower amplitudes and faster recovery times than the spontaneous Ca2+ transients2. Figure 3 shows an example of NG108-15 neuronal cells loaded with Fluo-4 AM and imaged with a confocal microscope. Fluo-4 AM was observed to enter the cell membrane in a non-disruptive manner, resulting in a generally uniform distribution of the indicator across the cell cytoplasm. Only minor nuclear or cytoplasmic organelle staining was detected. As previously indicated, NRs were expected to be located intracellularly12. NG108-15 neuronal cells alone or cultured with Au NRs and poly(styrenesulfonate)-coated-Au NRs were exposed thereafter to laser irradiances between 0.07 J∙cm-2 and 370 J∙cm-2, with the laser frequency modulated in the range of 0.5-2 Hz.
Figure 4 shows representative examples of how the amplitude of the responses was mapped as a function of time. The amplitude of the Ca2+ response varied with the radiant exposure (Figure 4A–C) and was observed not to be consistently triggered by the laser pulses (Figure 4C)13. The most likely explanations were the transient depletion of intracellular Ca2+ stores attributed to incomplete Ca2+ loading2 or the different efficiency of NR internalization in the neuronal cells. When NRs were not used in culture (control experiments, Figure 4D), a stimulatory effect of the 780 nm light was also observed. However, this produced lower fluorescence amplitude peaks and lower probability of activation (detected in only 16% of the analyzed samples). Overall, a 48% probability of NR laser-induced cell activation was achieved and despite the background events due to the 780 nm light, exposure of NR-treated cells demonstrated higher stimulation efficiency with lower laser energy and higher peaks of response13. In fact, the calcium response was found to peak at 0.33 J∙cm-2 in the NR-treated cells. This was attributed to thermal inhibition13. During the experiments, no evidence of blebbing or any other form of cell membrane disruption was detected, which is consistent with the results of Huang et al. that reported cellular photodestruction with a relatively high power density of 19 W∙cm-2 applied for 4 min27. No spontaneous activity in the NR-treated cells was recorded without laser exposure.
Figure 1: Optical fiber experimental setup (A) and average laser irradiances as a function of the laser power for a laser beam of area equals to 0.4 mm2 (B). Beam parameters are (A): the half-angle of the maximum cone of light exiting the fiber (θ), the beam radius (r) and the distance between the optical fiber and the sample (d).
Figure 2: Examples of epifluorescence images of differentiated NG108-15 neuronal cells cultured alone (A) or with Au NRs (B) and irradiated with different laser powers (indicated in the figure). Samples were incubated for one day before laser irradiation. Cells were fixed and labeled for anti-β-III tubulin (red) and DAPI (blue) three days after laser irradiation. Scale bars are 100 µm.
Figure 3: Example of differentiated NG108-15 neuronal cells loaded with Fluo4-AM Ca2+ indicator. The image was taken using an inverted confocal microscope with a 40X oil-immersion objective.
Figure 4: Representative examples of laser-induced Ca2+ variations as a function of time in NG108-15 neuronal cells cultured in serum-free conditions for three days with (A) poly(styrenesulfonate)-Au NRs, (B, C) Au NRs, and (D) without NRs (control sample). These results were obtained with laser pulses of 100 msec (A, C, D) and 50 msec (B). The frequencies used for the experiments (dashed vertical lines) were 1 Hz (A–C) and 0.5 Hz (D). The calculated radiant exposures were 69.4 J∙cm-2 (A), 34.7 J∙cm-2 (B), 0.37 J∙cm-2 (C) and 138.87 J∙cm-2 (D). Fmax /F0 indicates the maximum fluorescence increase detected in NG108 -15 neuronal cells, from calibration with ionomycin (reproduced with permission13).
The protocols outlined in this presentation describe how to culture, differentiate and optically stimulate neuronal cells using extrinsic absorbers. The NR characteristics (e.g. dimensions, shape, plasmon resonance wavelength and surface chemistry) and the laser stimulation parameters (such as wavelength, pulse length, repetition rate, etc.) can be varied to match different experimental needs. The effects on cell behavior can be monitored using standard biological assays and materials. Overall the approach provides a simple, yet powerful, way to irradiate cell populations in vitro and could be extended to primary cells, tissue samples and in vivo studies.
The main requirements that Au NRs need to satisfy to be used for biological applications are stability (both chemical and physical) and biocompatibility. The latter is particularly critical, due to the presence of a cationic surfactant (commonly CTAB) on the surface of the Au. CTAB is known to induce cytotoxicity both in vitro28 and in vivo29 and it is commonly used during the synthesis process to drive the NR-shape formation30. Stability and biocompatibility are often improved by depositing additional coatings on the Au NR surface (e.g. polyethylene glycol, silica)31. To assess the biocompatibility, different assays can be used in vitro (e.g. live/dead, MTS, MTT, etc.) while histological analysis is often performed during tissue experiments7,8,12,15.
Another challenge to face when working with nanomaterials is the difficulty of correctly determining their molar concentration. The huge variety of NPs in terms of shape, size, and chemical properties makes the techniques currently available suitable for only certain classes of particles. For example, the standard dynamic light scattering method assumes that NPs have a spherical shape and scatter light isotropically17. Therefore, application of this method to Au NRs results in discrepancies between the measured concentration and the real one. The issue of NP concentration is particularly problematic if related to the nanomedicine field, where the concentration administered needs to be precisely controlled in order to maximize the efficacy of the process (e.g. for drug delivery applications) and minimize the toxicity of the nanomaterials17. In the studies reported here, the optical density used gave good results in terms of cell viability and particle number.
Due to their intrinsic absorption properties, Au NPs are often used in combination with a laser source. During the exposure, absorption and scattering are the two main processes likely to occur at the surface of the NPs. If the laser wavelength matches the plasmon resonance wavelength, absorption often prevails over scattering, exciting the conduction electrons at the NP surface. These form an electron gas that moves away from its equilibrium position and creates a resonant coherent oscillation called the localized surface plasmon resonance (LSPR). The energy is then transferred to the NP crystal lattice as heat, which is thereafter dissipated rapidly into the surrounding environment32. Since heat is the main effect observed after the excitation of the NR LSPR, it is advisable to monitor cell viability after laser exposure.
It has been hypothesized that the observed effects on the neurite outgrowth and the intracellular Ca2+ pathway were due to the transient heating arising from excitation of the LSPR. This hypothesis is in line with the activation of the temperature-sensitive TRPV1 channel after magnetic exposure of ferrite NPs5. This process is also consistent with observations that thermally sensitive TRPV4 channels play an important role in infrared nerve stimulation33. At a molecular level, it has been recently shown that TRPV4 is thermosensitive only after the interaction of phosphoinositide -4,5 -biphosphate (PIP2 ) with the channel34. Therefore, it is possible that the transient heating arising after NR excitation could serve to accelerate and/or induce the opening of the TRPV4 channels. This hypothesis can be confirmed by future Ca2+ experiments by using Ca2+– free medium and/or molecular controls over the PIP2 depletion.
Different groups have also demonstrated that small temperature gradients over the physiological temperature range can be used to induce other responses, such as neuronal growth cone guiding25 or depolarizing currents in human embryonic kidney cells35. Yong and coworkers recorded a significant increase in electrical signal activity in primary auditory neurons cultured with silica-coated Au NRs after laser irradiation of the NRs at the LSPR. The heating produced by the laser-irradiated particles was measured using patch-clamp techniques14. More recently, Eom et al. recorded an increase in the amplitude of CNAPs after laser exposure of 3.4 × 109 Au NRs when continuously perfused in the vicinity of the sheath of the nerve bundle of a rat sciatic nerve15.
The stimulatory effect of the 780 nm laser diode observed during the experiments was not linked to heat generated by water absorption, as this is known to be negligible at 780 nm36. Previously published results have also reported stimulatory effects of 780 nm light on neuronal tissue in vivo37,38. In vitro studies have demonstrated the involvement of reactive oxygen species in the process39. However, the detailed mechanisms by which NIR stimulation affects gene transcription and other cellular activities are currently unresolved. Current data suggests the interplay of different effects in the stimulation, including photon absorption within chromophores in the mitochondria (almost 50% of the energy at 780 nm might be absorbed by cytochrome c oxidase)37,39-41, changes in membrane permeability to calcium37 and inhibition of inflammatory activity in the cells37.
The results presented in this manuscript demonstrate that nanoparticle absorbers hold great promise for future applications in optical stimulation of neuronal cells. The main advantage lies in the ability of NIR light to penetrate deep into tissues (therapeutic window). These results also show that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption. For future applications in neural prostheses, it would be of interest to investigate different surface functionalizations with chemical affinity for neuronal axons, which are the main targets for many optical stimulation applications.
The authors have nothing to disclose.
The authors would like to acknowledge NanoVentures Australia for travel funding support and Prof. John Haycock for having partially hosted this research at the University of Sheffield and Ms. Jaimee Mayne for her help during the filming.
Au NR | Sigma Aldrich | 716812 | |
NG108-15 | Sigma Aldrich | 8811230 | |
DMEM | Sigma Aldrich | D6546 | |
FCS | Life Technologies | 10100147 | |
L-glutamine | Sigma Aldrich | G7513 | |
Penicillin/streptomycin | Life Technologies | 15140122 | |
Amphotericin B | Life Technologies | 15290018 | |
Formaldehyde | Sigma Aldrich | F8775 | |
Triton X-100 | BDH | T8532 | |
BSA | Sigma Aldrich | A2058 | |
Anti-βIII-tubulin | Promega | G7121 | |
TRITC-conjugated anti-mouse IgG antibody | Sigma Aldrich | T5393 | |
DAPI | Invitrogen | D1306 | |
Fluo-4 AM | Invitrogen | F14201 | |
DMSO | Sigma Aldrich | 472301 | |
Pluronic F-127 | Invitrogen | P6867 | |
Equipment name | Company | Catalogue Number | |
UV-Vis spectrometer | Varian Medical Systems Inc. | Cary 50 Bio | |
Mini centrifuge | Eppendorf | Mini Spin | |
Sonic bath | Unisonics Australia | FPX 10D | |
Cell culture incubator | Kendro | Hera Cell 150 | |
Cell culture centrifuge | Hettich | Rotofix 32A | |
Laser diode | Optotech | 780 nm single mode fibre – coupled LD | |
Optical fiber | Thorlabs | 780 HP | |
Power meter | Coherent | Laser Check | |
ImageJ | http://rsb.info.nih.gov/ij/index.html | ||
Epifluorescent microscope | Axon Instruments | ImageX-press 5000A | |
μ-slide well | Ibidi | 80826 | |
Inverted confocal microscope | Carl Zeiss Microscopy Ltd. | LSM 510 meta-confocal microscope | |
Oscilloscope | Tektronix | TDS210 |