The use of engineered gold nanoparticles (Au NPs) in neuroscience has increased considerably over the past decade. Au NPs can easily be bio-conjugated for cell-specific targeting, can be delivered by injection, and match the dimensions of subcellular components, such as those of the cell receptors and ion channels [ 5 ]. In the context of stimulation and modulation of neural activity, Au NPs have already been successfully employed for several applications including: enhancement of neurite outgrowth [ 6 7 ], modulation of intracellular calcium signaling [ 8 9 ], neuron depolarization [ 10 11 ], and suppression of neuronal activity [ 12 ]. The focus of this review is to provide a critical perspective on the use of Au NPs as an interface to modulate the activity of neuronal tissue. The topics of gene-therapy and cellular uptake and neural toxicity of NPs have been extensively discussed in other recent publications [ 1 13 ] and therefore have not been included here.

The nervous system is essential to the functional transmission and processing of information within the human body. It consists of two main parts: the central nervous system (CNS), which includes the brain and spinal cord, and the peripheral nervous system (PNS) that comprises all other neural tissues in the body [ 1 ]. The basic unit of the nervous system is the neuron, a sophisticated biological machine capable of receiving and sending electrical signals on millisecond time-scales [ 2 ]. The complex electrical network that neurons form throughout the body constitutes the key mechanism for organ communication and for maintaining all physiological functions. Under pathological conditions, this pathway can be partially or totally disrupted, resulting in the loss of electrical transmission. Clinical therapies to restore the damaged neuronal network range from axonal gap connections (<25 mm) [ 3 ] to neural prostheses and neural interfaces for non-treatable conditions (e.g., neuro-degenerative diseases or spinal cord injuries). In this context, nanomaterials are expected to introduce new opportunities and provide improvements in current cell-based or immunological therapies [ 1 ]. Due to their small size, nanotechnology-based devices can interact with biological systems at the molecular level, with a high degree of spatial and temporal specificity. They can penetrate the blood-brain barrier and deliver specific therapeutic agents, probes, or biological materials to targeted cells and tissues [ 4 ]. The availability of new experimental techniques and tools also allows complex biological processes to be monitored in real time at the single cell level.

Interestingly, the rapid heating of the lattice generated by fs laser pulses also leads to the impulsive excitation of low frequency acoustic breathing modes of the Au particles (electron–phonon coupling) [ 35 ]. The volume of the NPs increases and decreases with a period of about 4–5 ps, which in turn leads to a periodic change in the free-electron density and thus an observable oscillation in the transient absorption [ 33 ]. The frequency of these acoustic modes is inversely proportional to the particle radius. A further consequence of the rapid heating is laser-induced reshaping, if the temperature of the NP lattice reaches the melting temperature of Au (pulse energies ~1–10 mJ·cm). In spherical NPs, the melting may remain unnoticed, but it has been shown that NRs melt into NSs as the most thermodynamically favorable shape within a transformation time of at least 30 ps [ 36 ]. This leads to significant bleaching of the longitudinal absorption mode [ 37 ]. At higher laser fluences fragmentation of the NPs may also occur, either through vaporization [ 38 ] or through ejection of photoelectrons and subsequent electrostatic fragmentation [ 39 ]. Although the risk of particle reshaping is reduced when longer laser pulses are applied, it has been shown that ns pulses can produce many partially melted particles, where the shape remains cylindrical but with a rounded mid-section [ 40 ]. Depositing additional surface coatings could be a strategy to improve the photothermal stability under ns laser pulse irradiation [ 41 ].

Once the excitation is removed, heat conduction into the surrounding medium will lead to the electron gas cooling via the curves shown in Figure 1 c, until the system returns to initial state in Figure 1 a. If the laser pulse energy is sufficiently high, particle melting (≈ 1337 K in gold) and explosive boiling can occur and may be observed in an aqueous medium. Computational modeling of the thermal relaxation process has shown that water at the surface of a 48 × 14 nmAu NR reaches the critical point (= 647 K) for laser pulses of 250 fs and an average fluence above about 0.47 mJ·cm 31 ]. Particle reshaping appears to start just above this energy, possibly due to reduced heat dissipation within the gas bubble. For fluences below this level, the temperature across the metal–water boundary typically equilibrates within about 1 ns, with a heated zone of some tens of nanometers [ 31 34 ]. Interestingly, Ekici et al. [ 31 ] found that exposure to a 1 µs stream of 250 fs pulses at 80 MHz (i.e., 80 laser pulses), generated an overall rise of only 3 K in the water at the particle surface, and this rise was attained during the first few pulses. Larger temperature rises of tens of K can be achieved with continuous wave laser exposure at fluxes of 10–10W·cmand in NP clusters [ 34 ].

A range of energy conversion processes occur when an Au NP is irradiated by laser light, and it is useful to understand how these give rise to the various phenomena discussed in this review. Au NPs are typically exposed to a laser source in four distinct time regimes: (i) low-energy femtosecond (fs, ultrafast) laser pulses; (ii) high-energy fs laser pulses; (iii) nanosecond (ns) laser pulses; and (iv) continuous irradiation. The irradiation of metal NPs with an fs pulse leads to a rapid increase in electron energy. For low pulse energies, the temperature of the NP lattice rises by only a few tens of degrees (depending on particle size, optical density, and laser irradiance), while for high pulse energies the temperature of the metal can be raised above its melting point [ 31 ]. When ns pulses are applied, the energy threshold for the complete melting of the NRs is effectively reduced due to surface diffusion [ 32 ]. In the case of continuous laser irradiation, the particles are constantly saturated, thus reducing their absorption efficiency and the overall photothermal energy conversion.

Au NPs also have several attractive features as “high precision” photothermal agents for in vivo neural modulation. As a result of their very small size relative to mammalian cells, Au NPs only heat their immediate environment. This allows the overall heat delivery to be reduced, as long as the particles are strategically positioned close to the target cell. It also leads to a reduction in the diffusion path length for cooling. Consequently, Au NP photothermal modulation acts on sub-millisecond timescales (see the following section), which is critical for temporally precise stimulation of neuronal activity. Moreover, accurate targeting of NPs to the neurons, together with removal of excess particles by the circulation of interstitial fluids, allows off-target environmental heating to be minimized. These properties are likely to be critically important for avoiding damage to thermally sensitive tissues and limiting toxicity due to high concentrations of exogenous particles. Table 1 summarizes the main findings that will be described in the following sections.

The vast number of applications of Au NPs in biology and medicine is closely related with their unique optical properties. When Au NPs are perturbed by an external light field in the visible or near infrared (NIR) domain, the conduction electrons move away from their equilibrium position, creating a resonant coherent oscillation called the localized surface plasmon resonance (LSPR) [ 22 ]. LSPR wavelengths typically fall in the visible to NIR range, with the precise position depending on the particle morphology, interparticle distance, and refractive index of the surrounding medium [ 23 ]. For many biological applications, the plasmon absorption peak is selected to match the transparency window of biological tissues (600–1200 nm), meaning that NRs, nanoshells, nanostars, and nanocages appear to be the most suitable morphologies [ 24 ]. Despite this, to date only NRs and nanospheres (NSs) have been used for modulation of neuronal activity (see Table 1 ). Au NRs have proven to be particularly useful, as their resonance wavelength can be tuned by modification of the NR aspect ratio. In addition, they possess two distinct plasmon excitation bands corresponding to the excitation of the short and long axes of the NRs [ 25 ].

The typical size of Au NPs ranges from approximately 1 to 100 nm, which is comparable to large biological molecules. This favors the interaction with cells, both at the surface and at a fundamental molecular level. In this context, Au NPs have already been used in several biomedical applications, such as biosensing, bioimaging, drug delivery, therapy, and tissue engineering [ 13 ]. However, all biological applications require a careful control over biocompatibility. It is well known that some of the most commonly used capping ligands for the fabrication of Au NPs are toxic to cells. A prominent example is the cationic surfactant cetyltrimethylammonium bromide (CTAB), which is commonly used in the preparation of gold nanorods (Au NRs) [ 16 ]. CTAB is known to induce cytotoxicity both in vitro [ 17 ] and in vivo [ 18 ] and to interfere with the surface hydration of the particles [ 19 ]. Depositing additional surface coatings has been one of the main strategies to reduce the negative effects caused by residual chemicals used during particle synthesis [ 20 21 ].

The integration of Au NPs in neurological research has the potential to find new strategies for diseases that are not currently treatable. This perspective arises from their unique properties, including optical response, chemical and physical stability, relatively low toxicity, and wide range of possible surface functionalizations [ 13 14 ]. For example, functionalization with specific ligands allows cellular and molecular specificity, which enables the interaction with target cells and tissues in controlled ways. Thus, Au NPs have been engineered to bind to voltage-gated sodium channels, transient receptor potential vanilloid member 1 (TRPV1) channels, and P2X3 receptor ion channel in dorsal root ganglion neurons [ 15 ].

Although the interest in Au NPs for applications in nerve regeneration is expanding, in vivo studies are still limited by a lack of knowledge about the consequences of nanomaterials on intracellular pathways and inflammatory responses. It is known that a high concentration of metal nanoparticles in living organisms can cause cell oxidative stress and reactive oxygen species production, leading to other serious cellular dysfunctions, such as inflammation, cell membrane disruption, DNA damage, cancer, or apoptosis [ 45 ]. Söderstjerna et al. recorded a significantly higher number of apoptotic and oxidatively-stressed cells after exposing Au NPs in a primary tissue model of the mouse retina [ 46 ]. In our laboratory, we detected a significant oxidative stress increase after exposing NG108-15 neuronal cells to Au NRs for one hour [ 47 ]. This result confirmed a previously published report showing oxidative stress generated in the rat brain [ 48 ]. Au NPs have also been observed to cause a significant decrease in the levels of dopamine and serotonin in vivo [ 48 ]. Moreover, Au NPs have been imaged not only intracellularly, but also intranuclearly, raising questions of whether these nanomaterials can cause DNA damage and/or alter gene expression [ 46 ]. However, these effects can generally be minimized by reducing the concentration of NPs and using particles larger than about 15 nm [ 25 ].

Au NPs were also used for integration into nanocomposite nerve conduits. Recently Baranes et al. reported a nerve guide fabricated with electrospun nanofibers doped with 10 nm Au NPs (shown schematically in Figure 2 c). The scaffolds encouraged a longer outgrowth of the neurites in primary neurons of the medicinal leech, preferring axonal elongation over the formation of complex networks [ 27 ]. Similarly, Das and coworkers reported on a nerve guide fabricated by adsorbing Au NPs onto silk fibers. This nano-hybrid material was successfully tested in a neurotmesis grade injury (complete axonal loss and conduction failure) of a sciatic nerve of Sprague-Dawley rats over a period of 18 months. The nano-composites were found to promote adhesion and proliferation of Schwann cells in vitro and did not elicit any toxic or immunogenic responses in vivo [ 28 ]. Lin and colleagues tested chitosan-AuNP microgrooved nerve conduits both in vitro and in vivo. The results showed that the conduits preseeded with primary neuronal stem cells were able to support regeneration of the sciatic nerve better than the controls [ 29 ]. Taken together, these studies clearly show that neural regeneration is also influenced by the mechanical support of the guides. Nanoparticle-doped scaffolds open up new strategies to combine bio-materials and nanoparticles for providing physical and/or bioactive environments for neural regeneration. There is also potential to combine the electrical properties of Au NP and bio-materials to promote peripheral nerve elongation [ 44 ].

In our laboratory, we discovered that the heat released by plasmon excitation of Au NRs can be used to stimulate neurite outgrowth in NG108-15 neuronal cells ( Figure 2 a). The greatest outgrowth was observed after irradiating the endocytosed particles with the highest laser dose (7.5 W/cm), obtaining an average increase in neurite length of almost 36% compared to the non-irradiated sample [ 6 ]. We hypothesized that the mechanism underlying the outgrowth involves the activation of one or more transcription factors, supporting previous studies on iron oxide nanoparticles. Indeed, Kim and colleagues performed gene expression analysis in PC12 cells, observing changes in genes related to the cytoskeleton, signaling molecules, receptors for growth hormones, and ion channels [ 43 ]. These genes are known to be involved in neuronal differentiation [ 42 ]. Papastefanaki et al. used PEG-coated Au NPs after mouse spinal cord injury, showing hind limb motor recovery, attenuation of microglial response, enhanced motor neuron protection, and increased remyelination eight weeks after treatment ( Figure 2 b) [ 7 ]. In a different approach, Bhang and coworkers doped spherical Au NPs with manganese, which allows pH-triggered released of manganese ions after the endocytosis of the particles. They observed neurite outgrowth 24 hours after treatment, showing an increase of roughly 70% compared to control samples. They speculated that changes in intracellular signaling pathways were responsible for the outgrowth increase [ 26 ].

The primary function of a peripheral nerve is to transmit signals from the CNS to the rest of the body, or to convey sensory information from the rest of the body to the CNS. In the case of injury or a health disorder, this pathway can be partially or totally disrupted, resulting in pain, loss of sensation, reduced muscular strength, poor coordination, atrophy, or complete paralysis. Even if peripheral nerves have the capacity of spontaneously regenerating following traumatic injuries, a clinical operation must be performed in case of a complete nerve transaction. Current clinical strategies include autografts, allografts, and nerve guides, yet the maximum regeneration distance is limited to 25 mm [ 3 ]. Researchers are currently focused on finding new methods and materials to improve this nerve regeneration distance. Even though the process of neural regeneration is well-known, nerve regeneration following injury remains a great challenge for neuroscientists and neurologists. The process involves outgrowth of neuronal branches (neurites) from the cell body. The neurites elongate, bifurcate, and connect to neighboring neurons to form an electrically functional network. Typically, one of the neurites differentiates into an axon, while the others either turn into dendrites or fail to become functional and retract [ 42 ].

4. Modulation of Nerve Electrical Activity

The use of light to modulate the electrical activity of neuronal cells, as shown schematically in Figure 3 a, has attracted growing interest, due to the potential for less invasive neuronal interfaces, improved spatial resolution of stimulation and avoiding electrical artifacts in associated neural recordings [ 50 51 ]. The potential to use Au NPs as an exogenous light absorber in neural stimulation appears to have been first identified by [ 52 ], but we are not aware of any published demonstration by these workers, who have subsequently focused on the use of black photo-absorbers of ~6 μm diameter [ 53 ]. The initial suggestion was based on an analogy with infrared neural stimulation [ 54 ], where pulsed laser wavelengths in the range of approximately 1–6 μm have been used to stimulate action potentials in neurons. The primary mechanism in infrared neural stimulation appears to be the transient heating associated with absorption of light by water in the tissue [ 55 56 ]. However, water absorption also limits the penetration depth of the infrared light to a few hundred microns [ 57 ], while cumulative heating effects tend to limit the stimulation site density and maximum repetition rates [ 58 ]. As discussed above, Au NRs allow highly localized photothermal heating through the absorption of wavelengths in the water transmission window from 600 to 1200 nm.

Yong et al. [ 10 ] first confirmed that Au NRs can be used to stimulate cultured rat primary auditory neurons with near-infrared (780 nm) illumination. The laser-induced cell electrical activity was observed using whole cell patch clamp electrophysiology, as shown in Figure 3 b. The open patch technique was used to show that action potentials were associated with transient temperature increases of about 6 °C. The NRs were endocytosed by the neurons after 15–17 h incubation, as shown by dark field microspectroscopy. This work was soon followed by a demonstration that Au NRs could be used to elicit compound action potentials in the rat sciatic nerve in vivo (shown schematically in Figure 3 c) [ 11 ]. The NRs with peak absorption at 977 nm were introduced to the nerve bundle by micro-injection. Subsequent TEM analysis of fixed cross-sectional slices showed Au NRs located near the surface of the axon plasma membrane. In contrast, Yoo et al. [ 12 ] found that Au NRs inhibited neural activity in networks of primary cultured hippocampal neurons. This inhibitory effect was associated with longer laser exposures (1–30 min) and the NPs were coated with positively-charged amine-terminated polyethylene glycol, which may have had an increased affinity to attach to the cell membrane. The increased exposure time led to a sustained temperature rise of as much as 10 °C at the plasma membrane. It is well known that a sustained increase in environmental temperature can have an inhibitory effect on neural activity [ 59 ] and similar effects have been observed in infrared neural stimulation [ 60 ].

2+ dynamics in the soma may also be involved [2+ influx by TRPV1 activation. The surface chemistry of the NPs was modified with a cationic lipoprotein for non-cytotoxic targeting of the plasma membrane and the effect was also demonstrated in primary cultured dorsal root ganglion cells from wild type mice. Although the detailed mechanism is not yet understood, the broad principles for these effects do indeed appear to be analogous to infrared neural stimulation. In particular, the local increase in temperature due to plasmonic heating produces a change in the electrical capacitance of the plasma membrane [ 10 ], in agreement with the observations of Shapiro et al. [ 55 ]. In isolation, these changes in cell capacitance are unlikely to act as an excitatory stimulus, except in the most voltage sensitive cells [ 61 62 ]. However, infrared-induced temperature changes have also been shown to modulate the responses of voltage- and temperature-sensitive (TRPV) ion channels [ 63 64 ], as shown in Figure 3 d. Modulation of Cadynamics in the soma may also be involved [ 8 ], again in analogy with effects observed for infrared neural stimulation [ 65 ]. Nakatsuji et al. [ 9 ] have subsequently confirmed that laser heating of Au NRs causes Cainflux by TRPV1 activation. The surface chemistry of the NPs was modified with a cationic lipoprotein for non-cytotoxic targeting of the plasma membrane and the effect was also demonstrated in primary cultured dorsal root ganglion cells from wild type mice.

2+ uptake and cellular swelling [11,2 for ~1 ms [ Finally, it is has been shown that relatively high levels of infrared laser exposure can lead to nanoporation of the cell membrane, with concomitant Cauptake and cellular swelling [ 66 ]. Once again, similar disruptions of the cell membrane have been observed in the presence of Au NPs, across a wide range from continuous wave to fs laser pulse lengths [ 67 68 ]. However, it appears unlikely that this level of disruption is generated by the relatively modest laser irradiances involved in NR-mediated neural modulation [ 10 12 ]. On the other hand, it is known that nanoscale heating with Au NSs can induce a gel-fluid phase transition in phospholipid giant unilamellar vesicles [ 69 ]. Subsequent studies have shown that membrane conductance can be controlled by plasmonic heating of single Au NPs over periods of several seconds, without a phase transition or nanopore formation. It was proposed that this effect is due to an increase in phospholipid mobility with increasing temperature and was observed in both artificial lipid bilayers formed on a planar patch clamp system and in HEK293 cells that lack temperature-sensitive ion channels [ 70 ]. Similar experiments with artificial membranes composed of asolectin have interpreted the transient current during initial heating in terms of capacitance changes, with a single NP producing a capacitive current of 0.75 pA under irradiance of 18 kW/cmfor ~1 ms [ 15 ]. Further work is needed to clarify the relative importance of these various contributions, their relationship to the ensuing biochemical pathways, and any long term deleterious effects that may arise.

2+ transients in astrocytes [ Recently, it has been shown that less than 1 ms of NIR stimulation combined with Au NRs reliably produces strong Catransients in astrocytes [ 30 ]. While this interaction may help to facilitate minimally invasive studies of astrocyte function, it also points to the importance of targeting NPs to specific locations in the tissue. Eom et al. [ 30 ] targeted the astrocyte surface with biotinylated anti-thymocyte antigen-1 antibody and streptavidin-coated Au NRs. Carvalho-de-Souza et al. have demonstrated that Au NSs can be conjugated with functional groups that target voltage-gated sodium, TRPV1, and P2X3 ion channels, all of which are known to be expressed in the membrane of dorsal root ganglion neurons [ 15 ]. In each case, it was found that the Au NPs bound to the cultured neurons without impeding their excitatory capability and generated optically-evoked action potentials at relatively low NP concentrations. In comparison, unconjugated Au NPs required higher concentrations to support optical stimulation and were readily washed out on solution exchange. Targeting membrane receptors is an important approach, as cells of different sensory specialization can express very different profiles of membrane receptors. However, to our knowledge, neuronal selectivity has yet to be demonstrated in mixed cultures or in vivo. Interestingly, it has been found that organically-modified silica (ORMASIL) nanoparticles are preferentially taken up by neurons in vivo [ 71 ], but it is not yet clear what mechanism is involved and whether it can be extended to Au NPs.