The future of neural stimulation

The non-invasive modulation of neuronal functions represents a highly critical issue for the treatment of symptoms of different neurological pathologies, such as Parkinson’s disease and epilepsy [1,2], as well as for an accurate investigation of neural networks. Non-invasive stimulation of the nervous system is mostly limited by its restricted accessibility [3]. To date, the main techniques adopted for a selective and deep stimulation of the nervous system are optogenetics [4], deep brain stimulation (DBS) [5], trans-cranial direct current stimulation (tDCS) [6], and trans-cranial magnetic stimulation (TMS) [7]. Major drawbacks of these techniques include the scarce penetration of light in the visible spectrum through the nervous tissue [8], the onset of inflammation and gliosis due to the invasiveness of implanted electrodes [9], and the low spatial resolution of both tDCS and TMS (about 1 cm) [10]. For these reasons, innovative approaches are under investigation, aiming at remotely activating neurons in depth and without the aforesaid disadvantages.

A promising approach for wireless and non-invasive neural stimulation is represented by the exploitation of piezoelectric nanomaterials [11]. When subjected to mechanical deformations, piezoelectric nanotransducers are able to generate electric potentials on their surface by virtue of the direct piezoelectric effect [12]; moreover, their activation can successfully be obtained by choosing various sources of mechanical stimulation, including vibration plates [13], sounds [14], and ultrasounds (USs) [15]. In this context, USs are able to penetrate biological tissues at a considerable depth (even tens of centimeters, depending on tissue acoustic impedance and US frequency [16]), and can be efficiently focalized through hyperlenses in the specific region where piezoelectric nanoparticles are located, thus allowing for a remote stimulation with high spatial resolution [17]. Recently, exciting findings reported successful neural stimulation in vitro by synergic exploitation of piezoelectric nanotransducers and USs [18–20]. The reduced size and the biocompatibility of piezoelectric nanoparticles, that will extensively be described in this paper, thus promise improved performances with respect to the invasiveness of the traditional mesoscale electrodes.

Piezoelectric nanomaterials in biomedicine

Piezoelectric nanomaterials show great potential in nanomedicine, not only concerning the activation of the nervous tissue, but also for the stimulation of other electrically excitable cells (e.g., cardiomyocytes, skeletal myotubes, osteoblasts). In general terms, the electric stimulation of excitable cells allows for the fine modulation of tissue functionality, and plays an important role in many medical treatments for addressing a wide range of pathological conditions, such as cardiac arrhythmia, chronic pain, Parkinson’s disease, etc. Traditional stimulation methodologies usually exploit wires to carry current to the intended sites of action, with highly invasive surgical procedures and persistent risks of infection. The possibility of achieving an indirect electric stimulation, by exploiting piezoelectric nanoparticles that act as 'nanotransducers', is therefore of outstanding interest for all the biomedical research, and could represent a realistic approach in the near future clinical practice. Further investigations on the efficiency of this approach in vivo could definitively pave the way for the exploitation of these nanomaterials in clinics.

The nanomaterials intended to work as piezoelectric nanotransducers can be distinguished according to their chemical composition (e.g., boron nitride, zinc oxide, barium titanate, etc.) and shape (e.g., nanotubes, nanowires, nanospheres, etc.). The choice of the most appropriate nanomaterial to be used for piezoelectric stimulation must take into account its efficiency in mechano-electrical transduction (expressed as piezoelectric coefficient, d), the level of biocompatibility, the material purity, and the commercial availability.

Among piezoelectric nanomaterials, boron nitride nanotubes (BNNTs) are characterized by high biocompatibility, which was confirmed with different in vitro [21] and in vivo [22,23] investigations, and good piezoelectric properties [24,25]. Moreover, in the latest few years different companies started to provide BNNTs commercially with different purity grades and sizes [26]. Other nanotransducers with good piezoelectric properties are zinc oxide nanowires (ZnO NWs; d₃₃ about 10 pm/V) [27]. The synthesis procedures of ZnO NWs are well established, and large quantities of this nanomaterial with great purity levels can be easily obtained. Despite this, ZnO NW toxicity limits their use for biomedical applications [28]. Barium titanate nanoparticles (BTNPs) are characterized by excellent piezoelectricity owing to their perovskite structure (d₃₃ is about 30 pm/V in the case of tetrahedral configuration of the crystal lattice) [29]. Furthermore, BTNPs can be used at relatively high concentration due to their great biocompatibility [30], and highly pure BTNPs (characterized by various specific sizes) are available on the market from different providers. Interestingly, the availability of non-piezoelectric BTNPs (characterized by cubic configuration of the crystal lattice), makes it possible to carry out proper negative controls [19].

Finally, piezoelectric polymers, such as poly(vinylidene fluoride) (PVDF) and poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE), are of great interest in the field of neural tissue engineering due to their optimal biocompatibility. Indeed, different PVDF and P(VDF-TrFE) substrates, such as thin films and micro/nanofibers, were successfully used for cell culturing/stimulation [13,31,32]. Furthermore, the possibility of enhancing their piezoelectricity by annealing [33] or by loading with piezoelectric nanoparticles [20] makes these polymers good candidates for further in vivo investigations.

Neural stimulation assisted by piezoelectric nanomaterials

We have pioneered research on US-mediated stimulation of piezoelectric nanoparticles, by first demonstrating that BNNT administration and US exposure synergistically promote a significantly improved axonal sprouting in PC12-derived neurons (30% longer with respect to the controls). Furthermore, we provided the first indirect indication of the involvement of Ca²⁺ influx and TrkA receptor in PC12 cells exposed to BNNTs and USs [18].

A more detailed study on the mechanisms of the piezoelectric stimulation with SH-SY5Y neuroblastoma cells was subsequently conducted by using tetragonal BTNPs. In this study we demonstrated for the first time that when actuated by USs, BTNPs can elicit a significant cellular response in terms of Ca²⁺ and Na⁺ influxes. The lack of cellular response when USs were applied in combination with non-piezoelectric nanoparticles further supported the hypothesis of piezoelectric stimulation, which was finally corroborated by an electroelastic model of BTNPs exposed to USs. Other indirect evidences of successful piezoelectric neural stimulation have been reported by many other groups by taking advantage of different piezoelectric systems, such as piezoelectric nano/microfibers and films [13,19,20,31,33].

Among these studies, extremely interesting results were obtained by Arinzeh and co-workers, who exploited piezoelectric scaffolds to promote in vitro neural differentiation of human neural stem cells, thus demonstrating their potential in regenerative medicine and neural tissue engineering [31]. In another pioneering work, Royo-Gascon and co-workers were able to generate electric fields on piezoelectric PVDF substrates by applying mechanical vibration [13]. Chronic application of piezoelectric stimulation to rat spinal cord neurons induced the enhancement of number, length, and branching of neural processes with respect to non-stimulated controls. No effects on neurite regeneration were observed when vibrations were applied to non-piezoelectric films, thus excluding the involvement of other unspecific phenomena (e.g. mechanical stimulation of neurons).

Characteristics of piezoelectric materials, possible methods of stimulation, and resulting effects on neuronal models are summarized in Table 1.

Future directions and conclusions

A translation of piezoelectric stimulation into the medical practice is the final goal of many research efforts. Main current limitations are represented by the lack of extensive in vivo biocompatibility testing of piezeoelectric nanomaterials, that should be absolutely addressed in the next close future. The evaluation of the safety and of the biocompatibility of these nanomaterials is in fact of extreme importance: long-term adverse effects, benefit/risk ratio related to their use, biodistribution and tissue accumulation are essential aspects to be clarified before any realistic application, especially at the level of nervous system. In particular, the investigation all of these safety issues at an early stage of research is mandatory to collect important data about the suitability of the proposed nanomaterials, thus allowing, if necessary, to tailor their properties in order to maximize the chance of translational success.

Moreover, direct evidences of neural activities following piezoelectric stimulation are still missing, and object of intense research by our group, despite the intrinsic difficulties due to the interferences caused by the mechanical stimulation with traditional recording systems. This notwithstanding, we are fully confident that devices based on the proposed stimulation strategy could find soon space in the clinical field. As an example, our group very recently contributed to moving the first steps towards the development of a self-powered cochlear implant based on piezoelectric nanocomposite films [20], that can be used to transduce pressure stimulations from sound waves into electrical signals, and thus to stimulate the acoustic nerve.

The potentialities of the piezo-nanotransduction are of outstanding interest to biomedical research, and the described preliminary successes strongly encourage to push our research efforts towards a realistic approach in the near future for clinical practice.

Acknowledgments 

Author affiliations: Center for Micro-BioRobotics @SSSA, Istituto Italiano di Tecnologia, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy; Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy.

This research was partially supported by the Italian Ministry of Health Grant Number RF-2011-02350464.

This article was originally published in Nano Today 14 (2017) 9-12.

References

[1] Y. Chen, M. Xiong, Y. Dong, A. Haberman, J. Cao, H. Liu, W. Zhou, S.-C. Zhang. Cell Stem Cell, 18 (2016), 817-826.

[2] E. Auffenberg, A. Jurik, C. Mattusch, R. Stoffel, A. Genewsky, C. Namendorf, R.M. Schmid, G. Rammes, M. Biel, M. Uhr, S. Moosmang, S. Michalakis, C.T. Wotjak, C.K. Thoeringer. Sci. Rep., 6 (2016), 19293.

[3] J.-Y. Chang. J. Pharmacol. Exp. Ther., 309 (2004), 1-7.

[4] O. Yizhar, L.E. Fenno, T.J. Davidson, M. Mogri, K. Deisseroth. Neuron, 71 (2011), 9-34.

[5] A.L. Benabid, S. Chabardes, J. Mitrofanis, P. Pollak. Lancet Neurol., 8 (2009), 67-81.

[6] C.J. Stagg, M.A. Nitsche. Neuroscientist, 17 (2011), 37-53.

[7] P.M. Rossini, S. Rossi. Neurology, 68 (2007), 484-488.

[8] S.A. Stanley, J.E. Gagner, S. Damanpour, M. Yoshida, J.S. Dordick, J.M. Friedman. Science, 336 (2012), 604-608.

[9] W.M. Grill. Expert Rev. Med. Devices, 2 (2005), 409-420.

[10] U. Najib, S. Bashir, D. Edwards, A. Rotenberg, A. Pascual-Leone. Neurosurg. Clin. N. Am., 22 (2011), 233–ix.

[11] A.H. Rajabi, M. Jaffe, T.L. Arinzeh. Acta Biomater., 24 (2015), 12-23.

[12] X. Wang, J. Liu, J. Song, Z.L. Wang. Nano Lett., 7 (2007), 2475-2479.

[13] N. Royo-Gascon, M. Wininger, J.I. Scheinbeim, B.L. Firestein, W. Craelius. Ann. Biomed. Eng., 41 (2013), 112-122.

[14] T. Inaoka, H. Shintaku, T. Nakagawa, S. Kawano, H. Ogita, T. Sakamoto, S. Hamanishi, H. Wada, J. Ito. Proc. Natl. Acad. Sci. U.S.A., 108 (2011), 18390-18395.

[15] X. Wang, J. Song, J. Liu, Z.L. Wang. Science, 316 (2007), 102-105.

[16] V. Chang, A. Perlas. S.N. Narouze (Ed.), Atlas of Ultrasound-Guided Procedures in Interventional Pain Management, Springer, US (2011), 13-19.

[17] S. Zhang, L. Yin, N. Fang. Phys. Rev. Lett., 102 (2009), 194301.

[18] G. Ciofani, S. Danti, D. D’Alessandro, L. Ricotti, S. Moscato, G. Bertoni, A. Falqui, S. Berrettini, M. Petrini, V. Mattoli, A. Menciassi. ACS Nano, 4 (2010), 6267-6277.

[19] A. Marino, S. Arai, Y. Hou, E. Sinibaldi, M. Pellegrino, Y.-T. Chang, B. Mazzolai, V. Mattoli, M. Suzuki, G.Ciofani. ACS Nano, 9 (2015), 7678-7689.

[20] G.G. Genchi, L. Ceseracciu, A. Marino, M. Labardi, S. Marras, F. Pignatelli, L. Bruschini, V. Mattoli, G.Ciofani. Adv. Healthc. Mater., 5 (2016), 1808-1820.

[21] G. Ciofani, S. Del Turco, A. Rocca, G. de Vito, V. Cappello, M. Yamaguchi, X. Li, B. Mazzolai, G. Basta, M. Gemmi, V. Piazza, D. Golberg, V. Mattoli. Nanomedicine, 9 (2014), 773-788.

[22] G. Ciofani, S. Danti, S. Nitti, B. Mazzolai, V. Mattoli, M. Giorgi. Int. J. Pharm., 444 (2013), 85-88.

[23] A. Salvetti, L. Rossi, P. Iacopetti, X. Li, S. Nitti, T. Pellegrino, V. Mattoli, D. Golberg, G. Ciofani. Nanomedicine, 10 (2015), 1911-1922.

[24] E.J. Mele, P. Král. Phys. Rev. Lett., 88 (2002), 56803.

[25] S.M. Nakhmanson, A. Calzolari, V. Meunier, J. Bernholc, M. Buongiorno Nardelli. Phys. Rev. B, 67 (2003), 235406.

[26] G.G. Genchi, A. Rocca, A. Grillone, A. Marino, G. Ciofani. Boron Nitride Nanotubes in Nanomedicine, G. Ciofani, V. Mattoli (Eds.), Elsevier, UK (2016), 201-218.

[27] E. Broitman, M.Y. Soomro, J. Lu, M. Willander, L. Hultman. Phys. Chem. Chem. Phys. PCCP, 15 (2013), 11113-11118.

[28] R.J. Vandebriel, W.H. De Jong. Nanotechnol. Sci. Appl., 5 (2012), 61-71.

[29] Z. Deng, Y. Dai, W. Chen, X. Pei, J. Liao. Nanoscale Res. Lett., 5 (2010), 1217.

[30] G.G. Genchi, A. Marino, A. Rocca, V. Mattoli, G. Ciofani. Nanotechnology, 27 (2016), 232001.

[31] Y.-S. Lee, T.L. Arinzeh. Tissue Eng. Part A, 18 (2012), 2063-2072.

[32] H.-F. Guo, Z.-S. Li, S.-W. Dong, W.-J. Chen, L. Deng, Y.-F. Wang, D.-J. Ying. Colloids Surf. B Biointerfaces, 96 (2012), 29-36.

[33] Y.-S. Lee, G. Collins, T.L. Arinzeh. Acta Biomater., 7 (2011), 3877-3886.