Wireless Brain Oscillation Control via Magnetoelectric Stimulation with Millisecond Precision

Wireless Brain Oscillation Control via Magnetoelectric Stimulation with Millisecond Precision | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Wireless Brain Oscillation Control via Magnetoelectric Stimulation with Millisecond Precision Chao-Chun Cheng, Li-Ling Chen, Mu-Yun Huang, Chih-Ning Tseng, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3959025/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Electrical deep brain stimulation (DBS) is a pivotal technology in treating neurological disorders and advancing neuroscience research. Minimizing the invasiveness of conventional DBS can reduce the risk of large hardware implants. Our study introduces a novel wireless magnetoelectric neuromodulation method termed Magnetic-driven Torque-Induced Electrical Stimulation (MagTIES). Diverging from traditional magnetostriction-based magnetoelectric stimulation approaches, we utilized the torque force from magnetic nanodiscs during alternating magnetic fields (AMF) to induce a piezoelectric effect on piezoelectric nanoparticles. This technique triggered neuronal activity in vitro and in vivo at millisecond-scale temporal precision using weak AMF at slow frequency. Importantly, it allows fine-tuning brain oscillations in deep brain areas through AMF frequency adjustments. MagTIES represents a significant advancement in neuromodulation, providing a minimally invasive, transgene-free approach for precise and wireless brain activity control, with vast potential for neurological therapies and neuroscience research. Biological sciences/Neuroscience Biological sciences/Biotechnology/Nanobiotechnology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Regulating brain activity through electrical stimulation is a pivotal technique in modern neuroscience research and clinical neurological therapy. It holds the key to unlocking treatments for a spectrum of neurological disorders that affect millions worldwide, ranging from Parkinson's disease and epilepsy to chronic pain. Deep Brain Stimulation (DBS), a prominent treatment using electrode implants, has relieved over 160,000 patients globally with various neurological disorders in the last 30 years 1 . Despite its clinical success, electrical implants for DBS face risks such as complications from surgery, infection, and potential damage due to micromotions during daily activities 2 . Consequently, the development of less invasive methods for electrical brain stimulation is essential. In pursuit of minimizing invasiveness, recent advances have shifted towards magnetic approaches 3 – 6 , utilizing the ability of magnetic fields to penetrate the skull, bones, and tissues without interference, thus offering an ideal solution for remotely manipulating neuronal activity 5 . Transcranial Magnetic Stimulation (TMS), the most prominent among these methods, employs strong magnetic fields more significant than 1.5 T to induce electrical currents non-invasively within the brain. However, TMS's spatial and depth precision is limited in its capacity to stimulate the deeper brain regions without affecting the surface areas 7 , thereby restricting its scope of application. The limitations of those neuromodulation methods catalyzed the exploration of alternative magnetic-based neuromodulation strategies capable of reaching deeper neural targets with better spatial precision. In this context, magnetic nanoparticle-based neuromodulation emerges as a promising approach. These magnetic nanoparticles can be manipulated remotely with much fewer magnetic fields than TMS, offering a potentially transformative solution for deep brain stimulation 5 , 8 . Over the last decade, various magnetic nanoparticle-based neuromodulation approaches have been invented for wirelessly stimulating deep brain neurons with minimal invasiveness, such as magnetothermal stimulation 8 , 9 , magnetomechanical stimulation 3 , 4 , 10 , magnetochemogenetics 11 and magnetoelectric stimulation 12 – 17 . Among them, magnetoelectric stimulation is the only approach that doesn’t require expressing specific thermosensitive ion channels, mechanosensitive ion channels, or receptors at target neurons. This approach not only bypasses the complexities associated with gene delivery and overexpression of exogenous genes but also sidesteps the challenge of variable and poorly understood intrinsic mechanosensor expression, complicating the predictability of neuromodulation effects in diverse neuronal types. While magnetoelectric stimulation offers these advantages, it, too, faces its own set of challenges. Current magnetoelectric nanomaterials for neuromodulation predominantly focus on magnetostriction-based approaches, utilizing superparamagnetic and piezoelectric materials 12 – 18 . The effectiveness of these methods largely depends on the coupling efficiency between the magnetic and piezoelectric elements. Core-shell architectures are frequently employed in these systems to optimize this interaction. Previous studies have explored various nanomaterial composites in spherical and disc shapes, such as CoFe 2 O 4 -BaTiO 3 nanoparticles 13 , 17 , CoFe 2 O 4 -BiFeO 3 nanoparticles 14 , 15 and Fe 3 O 4 -CoFe 2 O 4 -BaTiO 3 nanodiscs 12 , demonstrating their potential in applications ranging from DBS 12 , 17 , dissociation of Aβ 14 to tumor cell apoptosis induction 15 . For effective DBS, these technologies often require a significant static magnetic field, approximately 220 mT, coupled with an alternating magnetic field of more than 6 mT at frequencies beyond 140 Hz 12 , 17 . However, these systems primarily offer second-scale temporal precision. This limitation underscores a significant need for advancements toward millisecond-scale precision to achieve more precise modulation of neuronal activity. Recognizing these challenges in the current state of the art, we turn our attention to the untapped potential of Magnetite Nanodiscs (MNDs), which possess the capability to transduce magnetic fields into mechanical forces 3 , 4 , 10 , 19 (Fig. 1 a). However, the prospect of harnessing the mechanical force generated by MNDs for magnetoelectric stimulation has yet to be explored. We hypothesize that adapting MND-mediated magnetic-driven torque force for magnetoelectric stimulation could lower the magnetic field requirements and enable the upscaling of magnetic apparatus for broader applications. Meanwhile, BaTiO 3 nanoparticles (BTOs) are the commonly used piezoelectric nanoparticles with a high piezoelectric coefficient 20 . These have been demonstrated for ultrasound-based wireless stimulation on neuron-like SH-SY5Y cells18 (Fig. 1 b). However, the potential of utilizing magnetic-driven torque forces to induce a dielectric effect in BTOs remains uncharted. Building upon this concept, our study introduces an innovative magnetoelectrical neuromodulation technique named Magnetic-driven Torque Induced Electric Stimulation (MagTIES). This approach utilizes uniquely configured piezoelectric BTOs and MNDs. By applying these nanoparticles sequentially to cells or tissues and conjugating BTOs and MNDs by biotin-avidin linkage, we propose that torque forces induced in MNDs by an alternating magnetic field can stimulate the release of electric fields from BTOs, which was strategically positioned between cell membranes and MNDs (Fig. 1 c). We hypothesize that this electric field can trigger voltage-gated ion channels on cell membranes, inducing action potentials in neurons. This innovative approach reduces the required magnetic intensity and frequency compared to previous magnetoelectric stimulation methods, potentially offering a more precise, less invasive, and deeper-reaching neuromodulation technique. Results Preparation of nanomaterials Magnetite nanodiscs (MNDs) were synthesized following a two-step solvothermal synthesis procedure 3 , 4 . In the first step, hematite nanodiscs (HNDs) were produced by solvothermal synthesis. In the second step, HNDs were reduced to MNDs while maintaining the morphology. The crystal structures of these nanodiscs were characterized using Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) (Fig. 1 d, e, g). The XRD and VSM results indicate that HNDs are fully reduced to MNDs after a two-step synthesis process (Fig. 1 g, S1a). HNDs, which served as our control group, were not magnetized by external magnetic fields. Additionally, BaTiO 3 nanoparticles (BTO), sourced from US Research Nanomaterial, Inc., were characterized by TEM and XRD (Fig. 1 f, h). All nanomaterials were functionalized for cellular applications; BTOs were coated with PEG to facilitate attachment to cell membranes 18 and further functionalized with neutravidin for linkage with nanodiscs. Consistent with previous studies 3 , 4 , both MNDs and HNDs were coated with PMAO. The PMAO-coated nanodiscs were conjugated with biotin for linkage with BTOs. MagTIES induced neuronal responses in vitro To validate our hypothesis regarding the efficacy of MagTIES, we initially applied the functionalized BTOs to primary cultured hippocampal neurons. Subsequently, we introduced the functionalized MNDs or HNDs to these neurons. The biotinylated MNDs or HNDs were linked to the neutravidin-conjugated BTOs through biotin-neutravidin binding (Fig. 1 i). To visually confirm the binding of BTOs and MNDs in the primary cultured cells, we employed Förster Resonance Energy Transfer (FRET). For this purpose, the functionalized 100 nm BTOs (BTO 100 ) and 250 nm MNDs (MND 250 ) were conjugated with Alexa-488 and Alexa-594, respectively. Following applying BTO 100 and MND 250 to the cultured neurons, illumination with blue light at 470 nm was used to excite the Alexa-488 on the BTO 100 . This resulted in the emission of green fluorescence from Alexa-488, which subsequently transferred energy to the nearby Alexa-594 on the MND 250 (Fig. 1 i). The FRET ratio was significantly increased after the application of MNDs, indicating successful binding (Fig. 1 j, S1b). Moreover, the Scanning Electron Microscopy (SEM) image of a neuron in the BTO 100 /MND 250 group shows the BTOs were attached to the cell membrane, and the MNDs were sitting on top of BTOs (Fig. 1 k). Similar results can be observed in SEM image of neuron with BTO 100 and 250 nm HNDs (HND 250 ) (Fig. 1 l) We then measured the magnetoelectric stimulated Ca 2+ responses in the cultured hippocampal neurons with a custom-made air-core coil in the upright fluorescence microscope (Fig. 2 a, S2). Upon applying the functionalized BTO 100 and MND 250 , magnetic field-induced Ca 2+ responses were observed during the application of slow and weak AMF of 50 mT at 10 Hz (Fig. 2 b). Notably, repeated AMF at this intensity and frequency elicited multiple Ca 2+ responses (Fig. 2 e). Such responses were not observed in the control group with BTO 100 and HND 250 (Fig. 2 c, f). The fluorescence responses were significantly higher in the BTO 100 /MND 250 group (Fig. 2 h), indicating the specificity of the MagTIES-induced responses. Interestingly, if we rearranged the application sequence to place the MND 250 between BTO 100 and the cell membrane, the AMF-induced responses were significantly smaller (Fig. 2 c, g-h). This result indicates that the arrangement of BTOs between MNDs and cell membranes is critical for inducing neuronal activity. To further elucidate the temporal precision of MagTIES, we employed Di-8-ANEPPS, a radiometric voltage-sensitive dye (VSD), to measure action potentials in cultured neurons at a temporal resolution of 1 kHz. Di-8-ANEPPS shifts its emission spectrum upon neuronal depolarization when excited at 470 nm, enabling the quantification of membrane potential changes by comparing green to red emission light (Fig. 2 i). 21 , 22 Fluorescence imaging revealed that Di-8-ANEPPS predominantly localizes at the plasma membrane (Fig. 2 j), ensuring the accuracy of potential change measurements. By applying short alternating magnetic field (AMF) pulses of 100 ms at 50 mT and 10 Hz, we observed MagTIES-induced neuronal spikes within milliseconds (Fig. 2 k). Each AMF cycle alternates the external magnetic field between two directions. This bidirectional alternation in a cycle can generate two distinct torque forces by the magnetite nanodiscs (MNDs). Consequently, we recorded one or two spikes per 10 Hz stimulation cycle over 100 ms periods, demonstrating the millisecond-scale temporal precision enabled by MagTIES. MagTIES with nanoparticles in different sizes Unlike bulk BaTiO3, the piezoelectric coefficients of single BTOs have been shown to correlate with their size inversely 20 . Smaller BTOs, under 120 nm, possess higher piezoelectric coefficients, which have a piezoelectric coefficient (d 33 ) more significant than 1500 pC/N 20 . BaTiO 3 nanoparticles larger than 300 nm had d 33 smaller than 300 pC/N 20 . In this context, we further investigated neuronal responses by combining MND 250 with BTOs of different sizes, including 100 nm (BTO 100 ), 300 nm (BTO 300 ), and 500 nm (BTO 500 ) (Fig. 3 a-c). We found that BTO 100 /MND 250 groups elicited significantly stronger neuronal responses than BTO300/MND250 and BTO500/MND250 groups (Fig. 3 d-f). These results indicate that stimulated response is inversely correlated to the size of BTOs. Conversely, the size of MNDs might also affect the efficacy of MagTIES. The torque force generated by MNDs is positively correlated to the size of nanodiscs. We hypothesized that smaller MNDs might induce less piezoelectric responses from BaTiO 3 nanoparticles (BTOs) and thereby affect the neuronal activity induced by MagTIES. To test this hypothesis, we compared the stimulated neuronal responses using MagTIES with BTO 100 combined with MND 250 , 220 nm MNDs (MND 220 ), or 135 nm MNDs (MND 135 ) (Fig. 3 g-i). The cultured neurons activated in response to all combinations (Fig. 3 g-i). We found that the BTO100/MND250 group had a significantly more robust response than the BTO100/MND135 group (Fig. 3 i). The BTO100/MND250 group had a slightly more robust response than the BTO 100 /MND 220 group. However, these two groups have no significant difference (Fig. 3 i). These results show that MagTIES with larger MNDs can induce more effective responses. In addition, the cell viability test shows that the cell death rate was meager in both BTO 100 /MND 250 and BTO 100 /HND 250 groups after MagTIES (Fig. S3). These results collectively suggest the effectiveness and biosafety of MagTIES when employing BTO 100 combined with MND 250 ; this combination was used in the following stages of this study. A previous study demonstrated that applying MND 250 alone during AMF can activate neuronal activity by triggering the intrinsic mechanosensitive ion channel, TRPC, in cultured neurons 4 . In MagTIES, a layer of BTOs is positioned between the MNDs and cell membranes, which reduces the direct mechanical force transduced from MNDs to membranes. We found that applying the TRPC-specific antagonist, SKF96365, does not reduce MagTIES-induced neuronal activity (Fig. 4 a, d). We further confirmed that MagTIES-induced Ca 2+ responses depended on voltage-gated channels (Fig. 4 b-e) by using antagonist of voltage-gated Na + channels, tetrodotoxin (TTX), and the antagonist of voltage-gated Ca 2+ channels, mibefradil. These results indicate that TRPC is not critical for the MagTIES-induced activity of voltage-gated ion channels. MagTIES Induced Neuronal Activity In Vivo To evaluate the in vivo efficacy of MagTIES, particularly its ability to target deep brain regions, we selected the amygdala, a crucial area for emotion processing located deep within the brain 23 , 24 . We utilized stereotactic injection to introduce a combination of BTO 100 and MND 250 into the amygdala of mice (Fig. 4 a). Following a day of recovery, mice were exposed to MagTIES within an 11 cm coil, designed to generate an AMF tailored explicitly for our experiment. The mice were acclimated to the chamber, measuring 10 cm in diameter and 9 cm in height, for 30 minutes before stimulation (Fig. 5 a-d). The stimulation protocol consisted of 10 times 30 s stimulation periods interspersed with 30 s rest intervals, with the AMF set at 50 mT and 10 Hz. To systematically evaluate the effect of MagTIES on neuronal activity, we varied the ratios and amounts of BTOs and MNDs injected. The experimental groups included varying ratios such as 40 µg BTOs with 4 µg MNDs (40B/4M), 20 µg BTOs with 4 µg MNDs (20B/4M), and 4 µg BTOs with 4 µg MNDs (4B/4M). We also examined different total amounts at the same ratio, including combinations like 20 µg BTOs with 20 µg MNDs (20B/20M) and 40 µg BTOs with 40 µg MNDs (40B/40M). Before craniotomy with stereotactic injection, functionalized BTOs and functionalized MNDs were characterized by FRET in vitro to confirm the function of biotin-avidin linkage in each batch of materials (Fig. 1 i-j). Intriguingly, we observed that the expression of c-Fos, an immediate early gene marker for neuronal activation, was significantly higher in the injected hemisphere compared to the contralateral side in the 40B/4M and 20B/4M groups (Fig. 4 b, d). However, this trend was not as pronounced in the 4B/4M group. No significant differences were observed between the contralateral and ipsilateral sides (Fig. 4 d). Interestingly, increasing the total amount of BTO and MND injections in the 20B/20M and 40B/40M groups did not significantly enhance c-Fos expression in the ipsilateral amygdala (Fig. 4 d). These findings indicate that MagTIES with a specific amount of 20 µg BTOs and 4 µg MNDs injection can effectively induce neuronal activity in vivo . In contrast, the control group with 20 µg BTOs and 4 µg HNDs injections (20B/4H) showed no significant changes in c-Fos expression (Fig. 4 c, e), underscoring the specificity of the MagTIES-induced response. Modulating the neural oscillation by MagTIES Brain oscillations are crucial for various functions across different brain regions 25 – 28 . Neuromodulation technologies with high temporal precision, such as electrical DBS and optogenetics, can target and modulate these oscillations to specific frequencies 27 – 29 . This capability is essential for manipulating neuronal circuitry by controlling neuronal activity with precise timing. However, tuning the frequency of brain oscillation via magnetic nanoparticle-based neuromodulation technologies has not been previously reported. Here, we utilized fiber photometry for recording real-time neuronal activity in vivo 30 , circumventing interference from magnetic stimulation to the measurements (Fig. 4 f). AAV-hSyn-GCaMP7s-WPRE were unilaterally injected into the basolateral amygdala (BLA). After 3 to 7 weeks, the 20 µg BTOs and 4 µg MNDs (20B/4M) or 20 µg BTOs and 4 µg HNDs (20B/4H) were stereotaxic injected into the same location. The optical fiber with 400 µm diameter was implanted into the BLA. Post-surgical recovery, the Ca 2+ responses in vivo were measured by fiber photometry in a magnetic apparatus with a diameter of 10 cm and a height of 19 cm (Fig. S5e-g). First, we perform the MagTIES using 50 mT AMF at 10 Hz for 30 s. In the 20B/4M group, we observed a significant increase in fluorescence change (dF/F) compared to pre-stimulation levels (Fig. 4 g-h). In contrast, there was no notable fluorescence change in the control group with 20B/4H (Fig. 4 g-h). In 20B/4M group, reducing the magnetic field intensity to 40 mT, 30 mT, and 20 mT still resulted in an observable trend of increased fluorescence, but without statistical significance (Fig. 4 i). Interestingly, further analysis using Fast Fourier Transform (FFT) revealed increased brain oscillations at 20 Hz during 10 Hz AMF application in the 20B/4M group (Fig. 4 j), a phenomenon not seen in the 20B/4H group. The power spectrum intensity at 20 Hz was significantly increased in 20B/4M injected mice when applying 50 mT AMF (Fig. 4 k), which cannot be observed in 20B/4H injected mice. (Fig. 4 k). The increased power spectrum intensity trend was also notable when reducing the magnetic field intensity to 40 mT, 30 mT, and 20 mT, but without statistical significance (Fig. 4 l). Different AMF frequencies were used for MagTIES to determine whether the oscillation frequency corresponded to the MagTIES frequency. While we increased the frequency of AMF from 10 Hz to 11 Hz and 12 Hz, we can observe the apparent increment of power intensity at 20 Hz, 22 Hz, and 24 Hz, respectively (Fig. 5 a, S6a). Align with our hypothesis, the oscillation of Ca 2+ responses was precisely two times the applied frequency of AMF. When using 10 Hz AMF to the 20B/4M group, the change of power intensity at 20 Hz was ~ 7-fold more prominent than the baseline intensity (Fig. 5 b, e), which is significantly higher than the change of power intensity in 20B/4H group (Fig. 5 b, e). But the shift in power intensity at nearby frequencies doesn’t show the difference between the 20B/4M group and 20B/4H group (Fig. 5 e). Similarly, by using MagTIES with 11 Hz, 50 mT AMF, The increase of power spectrum intensity at the 22 Hz was significantly larger in the 20B/4M group than 20B/4H group (Fig. 5 c, f). The power intensity was not changed at 20 H and 24 Hz (Fig. 5 f). We can observe similar results using MagTIES with 12 Hz and 50 mT AMF. Only a change of power intensity at 24 Hz was increased in the 20B/4M group compared to the 20B/4H group (Fig. 5 d), which cannot be observed in other frequencies (Fig. 5 g). Finally, we observed that MagTIES induced responses even when the AMF stimulation period was reduced to 5 seconds or 1 second. Specifically, applying AMF for a 5-second stimulation period, interspersed with 5-second intervals for three cycles, led to a marked increase in power spectrum intensity, notably at 20 Hz during the stimulation periods (Fig. 5 h-i). The change in power intensity at 20 Hz, observed 5 seconds before and after stimulation, was significantly greater in the 20B/4M group than in the 20B/4H group (Fig. 5 j, S6b). A similar pattern emerged when AMF was applied for 1-second periods, followed by 1-second resting periods for 15 cycles. A significant increase in power spectrum intensity at 20 Hz was noted during these 1-second stimulation periods (Fig. 5 k-l). The rise in power intensity at 20 Hz was also significantly more significant in the 20B/4M group (Fig. 5 m, S6c). These fiber photometry results indicate the unique capability of MagTIES to manipulate neural oscillations in the amygdala with a high degree of temporal and frequency specificity, marking a significant advancement in the field of neuromodulation. Discussion This study is the first to demonstrate a non-magnetostrictive method for magnetoelectric stimulation at the nanoscale, utilizing two separate nanomaterials: BTOs and MNDs. This approach offers a more straightforward yet effective method for stimulating biological tissues and cells. Unlike core-shell magnetoelectric nanoparticles that rely on a tight interface between magnetostrictive and piezoelectric materials, our use of separate BTO and MND materials, linked via biotin-avidin, simplifies the synthesis process and broadens potential applications. Our findings demonstrate that magnetic-driven torque forces from MNDs effectively induce a dielectric field in BTOs, triggering neuronal activity. This approach has been successfully implemented in vitro and in vivo , showcasing the potential of MagTIES in neuromodulation. Using voltage imaging and fiber photometry, we revealed that this approach can trigger neuronal activity within milliseconds in vitro and precisely modulate brain oscillations to specific frequencies in vivo . Significantly, MagTIES marks the first instance of a magnetic nanoparticle-based technology achieving modulation of brain oscillations with millisecond-scale temporal precision, positioning it at the forefront of advancements in neuromodulation technology. Furthermore, we reveal a nanomaterial size-dependent response in the MagTIES approach, where smaller BTOs and larger MNDs produce more robust neuronal responses. This finding adds an essential dimension to the design considerations for optimizing MagTIES systems. Considering that the electric field decreases with the square of the distance from its source, the effectiveness of the electric field from BTOs in influencing membrane potential would diminish with increased distance from the cell membrane. Intriguingly, our findings highlight the critical importance of the spatial arrangement of BTOs and MNDs in the effectiveness of MagTIES. When BTOs were directly attached to the cell membrane in the BTO 100 /MND 250 group, the magnetically induced responses were significantly more pronounced (Fig. 2 ). Conversely, when MNDs were positioned between the BTOs and the cell membrane, the neuronal responses were substantially weaker. This underscores the importance of optimizing the spatial configuration of these nanomaterials for maximized neuromodulation efficacy. The varied expression levels of intrinsic mechanosensitive ion channels across different cell types and species have been a limiting factor in applying transgene-free magnetomechanical stimulation 4 . Our findings indicate that the intrinsic mechanosensitive ion channel, TRPC, is not essential for MagTIES-induced neuronal activity (Fig. S3a, d). In addition, our approach has demonstrated the ability to induce brain oscillations in vivo with alternating magnetic fields (AMF) below 50 mT, a threshold lower than the one needed for activating intrinsic TRPC 4 . In contrast to approaches that depend on mechanosensitive ion channels, electric stimulation is already widely accepted for triggering neuronal activity. While we cannot entirely discount the involvement of intrinsic mechanosensitive ion channels, the effectiveness of MagTIES, regardless of innate mechano-sensitivity in neurons, significantly broadens its applicability. This magnetoelectric stimulation approach enables neuronal stimulation without necessitating knowledge of their specific mechanosensitive ion channel expression profiles, thus offering a more universally applicable neuromodulation strategy. Regarding biosafety and clinical relevance, both BTO and MNDs have previously been reported as biocompatible materials 31 , 32 . BTO is one of the most biocompatible piezoelectric materials with the highest piezoelectric coefficient 20 , 32 . On the other hand, MNDs are made of Fe 3 O 4 , a biocompatible material similar to Ferumoxytol, an FDA-approved magnetic nanoparticle for clinical use 31 . As a transgenic-free approach, MagTIES sidesteps the complexities and potential side effects associated with gene delivery methods 33 . Future studies are necessary to understand the long-term biosafety of these materials and their feasibility for clinical use. Exploring combinations of various piezoelectric and superparamagnetic materials could further advance the capabilities of MagTIES. Additionally, considering the scalability and adaptability of the magnetic apparatus used in MagTIES, this technology holds promise for a wide range of applications in both fundamental research and clinical therapies. In conclusion, MagTIES introduces an innovative, less invasive, and precise approach to neuromodulation, particularly in deep brain regions. Its ability to target specific frequencies with high temporal accuracy opens new avenues for neurological research and treatment, setting a foundation for future explorations in this exciting field. Declarations Acknowledgments: We thank the National Science and Technology Council (NSTC) for funding (NSTC 110-2636-B-A49-003, NSTC 111-2636-B-A49-008, NSTC 112-2636-B-A49-006). Schematics in Fig. 2 were created with BioRender.com. Author contributions: PC. Formal Analysis: CC, PC. Funding acquisition: PC. Investigation: CC, LC, MH, CT, YT, GT, PC. Methodology: CC, LC, JX, PC. Project administration: PC. Supervision: PC. Validation: CC, LC, MH, CT, YT, GT. Visualization: CC, PC. Writing – original draft: PC. Writing – review & editing: CC, PC Competing Interests: PC and CC have filed a patent in Taiwan (application No. 112130503) and in the U.S. (application No. 18/540750) describing magnetic field-induced electrical stimulation of cells, which is related to this research. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3959025","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":275461524,"identity":"20d03763-95c2-4099-b31d-48493a7931f2","order_by":0,"name":"Chao-Chun Cheng","email":"","orcid":"","institution":"Institute of Biomedical Engineering, National Yang Ming Chiao Tung University, Taiwan (R.O.C.)","correspondingAuthor":false,"prefix":"","firstName":"Chao-Chun","middleName":"","lastName":"Cheng","suffix":""},{"id":275461525,"identity":"c11dedcb-e1dc-4f18-8436-f13065f7975b","order_by":1,"name":"Li-Ling Chen","email":"","orcid":"","institution":"Institute of Biomedical Engineering, National Yang Ming Chiao Tung University, Taiwan (R.O.C.)","correspondingAuthor":false,"prefix":"","firstName":"Li-Ling","middleName":"","lastName":"Chen","suffix":""},{"id":275461527,"identity":"b21ccde7-9d57-43f6-bc12-6740b0db4f8e","order_by":2,"name":"Mu-Yun Huang","email":"","orcid":"","institution":"Institute of Biomedical Engineering, National Yang Ming Chiao Tung University, Taiwan (R.O.C.)","correspondingAuthor":false,"prefix":"","firstName":"Mu-Yun","middleName":"","lastName":"Huang","suffix":""},{"id":275461526,"identity":"f91d8871-0b8f-42b2-876d-4af9639d8276","order_by":3,"name":"Chih-Ning Tseng","email":"","orcid":"","institution":"Institute of Biomedical Engineering, National Yang Ming Chiao Tung University, Taiwan 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National Yang Ming Chiao Tung University, Taiwan (R.O.C.)","correspondingAuthor":false,"prefix":"","firstName":"Jun-Xuan","middleName":"","lastName":"Huang","suffix":""},{"id":275461531,"identity":"4ebcb3ba-99ad-4929-bdb2-b9cafde5dd73","order_by":7,"name":"Chih-Hsuan Wu","email":"","orcid":"","institution":"Institute of Biomedical Engineering, National Yang Ming Chiao Tung University, Taiwan (R.O.C.)","correspondingAuthor":false,"prefix":"","firstName":"Chih-Hsuan","middleName":"","lastName":"Wu","suffix":""},{"id":275461523,"identity":"9a28bd1e-003c-4f9e-b8d0-8c7717438298","order_by":8,"name":"Po-Han Chiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYDACCQbGB1CmAdFamGFKgXQCcVrYJEjTYi7d/Ky6sM0usYG9eZsE44/DhLVYzjlmdntmW3JiA8+xMgmGBCK0GNzIYbvN23YgsUEixwyo5TZxWorBWuTfkKCFGWILD5FaLGekGUvznEs2buNJK7ZISPtPWIu5RPLDzzxldrL97Ic33vhgk0aEw0AEIxsDAxuIkUBYAyyR/CFG6SgYBaNgFIxYAAAw2TRjNAN7VQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4100-4051","institution":"Institute of Biomedical Engineering, National Yang Ming Chiao Tung University, Taiwan (R.O.C.)","correspondingAuthor":true,"prefix":"","firstName":"Po-Han","middleName":"","lastName":"Chiang","suffix":""}],"badges":[],"createdAt":"2024-02-15 15:21:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3959025/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3959025/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52187459,"identity":"e0b4d825-40ff-4287-91a4-1ca2b455ff47","added_by":"auto","created_at":"2024-03-07 18:52:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1049624,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArrangement of nanomaterials in MagTIES\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e \u003cstrong\u003ea,\u003c/strong\u003e Schematic of torque force generated by MNDs when applying a magnetic field. \u003cstrong\u003eb, \u003c/strong\u003eSchematic of piezoelectric effect generated by BTOs when applying an external force. \u003cstrong\u003ec,\u003c/strong\u003e Experimental Scheme. When an external AMF was applied, the torque force generated by the MNDs was transduced to the BTOs, triggering piezoelectric responses. The electric field released from the BTOs could further activate the neurons by triggering voltage-gated channels. \u003cstrong\u003ed \u003c/strong\u003eto\u003cstrong\u003e f\u003c/strong\u003e, TEM image of MND\u003csub\u003e250\u003c/sub\u003e (\u003cstrong\u003ed\u003c/strong\u003e), HND\u003csub\u003e250\u003c/sub\u003e (\u003cstrong\u003ee\u003c/strong\u003e). BTO\u003csub\u003e100\u003c/sub\u003e (\u003cstrong\u003ef\u003c/strong\u003e). \u003cstrong\u003eg \u003c/strong\u003eand\u003cstrong\u003e h\u003c/strong\u003e, XRD traces of MNDs, HNDs (\u003cstrong\u003eg\u003c/strong\u003e), and BTOs (\u003cstrong\u003eh\u003c/strong\u003e). \u003cstrong\u003ei,\u003c/strong\u003e Schematic of FRET. Alexa-488-conjugated BTOs and Alexa-594-conjugated MNDs were binding by biotin-avidin linkage. When 470 nm blue light was applied, the green fluorescence from Alexa-488 was transferred to Alexa-594, emitting red fluorescence. \u003cstrong\u003ej,\u003c/strong\u003e FRET ratio (red intensity/ green intensity) of Alexa-488-conjugated BTOs treated neurons with and without Alexa-594-conjugated MNDs. n = 10 in both groups. ***p \u0026lt; 0.001. \u003cstrong\u003ek\u003c/strong\u003e and \u003cstrong\u003el\u003c/strong\u003e, SEM images show neurons in the BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e group (\u003cstrong\u003ek\u003c/strong\u003e) and in the BTO\u003csub\u003e100\u003c/sub\u003e/HND\u003csub\u003e250\u003c/sub\u003e group (\u003cstrong\u003el\u003c/strong\u003e). White arrow, BTOs. White arrowhead, nanodiscs.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3959025/v1/e00bc62d4681949ace79008c.png"},{"id":52187462,"identity":"8f5c4b52-4f39-4c17-84b9-776f985cb886","added_by":"auto","created_at":"2024-03-07 18:52:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":576200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWireless neuromodulation by MagTIES \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a,\u003c/strong\u003e Schematic of magnetic apparatus for applying slow AMF in fluorescence microscopy. \u003cstrong\u003eb\u003c/strong\u003e to \u003cstrong\u003ed\u003c/strong\u003e, Fluorescence change in different groups. Top, Schematics of the nanomaterials arrangements in BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250 \u003c/sub\u003egroup (\u003cstrong\u003eb\u003c/strong\u003e), BTO\u003csub\u003e100\u003c/sub\u003e/HND\u003csub\u003e250 \u003c/sub\u003egroup (\u003cstrong\u003ec\u003c/strong\u003e), and MND\u003csub\u003e250\u003c/sub\u003e/BTO\u003csub\u003e100 \u003c/sub\u003egroup (\u003cstrong\u003ed\u003c/strong\u003e). Bottom, Color maps of fluorescence intensity in neurons before, during, and after AMF stimulation. \u003cstrong\u003ee\u003c/strong\u003e to \u003cstrong\u003eg\u003c/strong\u003e, Fluorescence changes (top) and cell response rates (bottom) were observed in the BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e group (\u003cstrong\u003ee\u003c/strong\u003e), BTO\u003csub\u003e100\u003c/sub\u003e/HND\u003csub\u003e250\u003c/sub\u003e group (\u003cstrong\u003ef\u003c/strong\u003e), and MND\u003csub\u003e250\u003c/sub\u003e/BTO\u003csub\u003e100\u003c/sub\u003e group (\u003cstrong\u003eg\u003c/strong\u003e) following the application of AMF (light orange) at 50 mT, 10 Hz. \u003cstrong\u003eh\u003c/strong\u003e, Maximum fluorescence change in BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e group (n = 139), BTO\u003csub\u003e100\u003c/sub\u003e/HND\u003csub\u003e250\u003c/sub\u003e group (n = 34) and MND\u003csub\u003e250\u003c/sub\u003e/BTO\u003csub\u003e100\u003c/sub\u003e group (n = 53), ***p ≤ 0.001. Tukey post-hoc test after One-way ANOVA. \u003cstrong\u003ei\u003c/strong\u003e, Schematic representation of emission spectrum shift of Di-8-ANEPPS by increasing membrane potential (+V\u003csub\u003em\u003c/sub\u003e). \u003cstrong\u003ej\u003c/strong\u003e, Green (top), red (middle), and merged (bottom) fluorescence image for a neuron with Di-ANEPPS. \u003cstrong\u003ek\u003c/strong\u003e, Traces of green/red ratio of Di-8-ANEPPS from two individual neurons in BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e group.\u003c/p\u003e","description":"","filename":"Fig2di8.png","url":"https://assets-eu.researchsquare.com/files/rs-3959025/v1/dad68731a75e13982a1a640a.png"},{"id":52187457,"identity":"e3539733-8056-4faf-84b0-4b6cc095d864","added_by":"auto","created_at":"2024-03-07 18:52:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":525946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMagTIES with different sizes of BTOs and MND\u003c/strong\u003es. \u003cstrong\u003ea\u003c/strong\u003e, TEM images of BTO\u003csub\u003e300\u003c/sub\u003e. \u003cstrong\u003eb,\u003c/strong\u003e TEM images of BTO\u003csub\u003e500.\u003c/sub\u003e \u003cstrong\u003ec\u003c/strong\u003e, Diameters of BTOs. \u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003ee\u003c/strong\u003e, The calcium responses in the BTO300/MND250 group (d, n = 34) and the BTO\u003csub\u003e500\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e group (\u003cstrong\u003ee\u003c/strong\u003e, n = 34) following the application of AMF at 50 mT, 10 Hz. Top, Schematics of BTOs/MNDs combination with different sizes of BTOs. Middle, Averaged fluorescence changes. Bottom, Cell response rates. Light orange areas, periods of AMF application. \u003cstrong\u003ef,\u003c/strong\u003e Maximum fluorescence changes in neurons with MND\u003csub\u003e250\u003c/sub\u003e and different sizes of BTOs from (\u003cstrong\u003ed\u003c/strong\u003e), (\u003cstrong\u003ee\u003c/strong\u003e), and Figure 2E. \u003cstrong\u003eg\u003c/strong\u003e, TEM images of MND\u003csub\u003e220\u003c/sub\u003e. \u003cstrong\u003eh\u003c/strong\u003e, TEM images of MND\u003csub\u003e135.\u003c/sub\u003e \u003cstrong\u003ei\u003c/strong\u003e, Diameters of MNDs. \u003cstrong\u003ej\u003c/strong\u003e and \u003cstrong\u003ek\u003c/strong\u003e, The calcium responses in the BTO100/MND220 group (j, n = 50) and the BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e135\u003c/sub\u003e group (\u003cstrong\u003ek\u003c/strong\u003e, n = 76) following the application of AMF at 50 mT, 10 Hz. Top, Schematics of BTOs/MNDs combination with different sizes of MNDs. Middle, Averaged fluorescence changes. Bottom, Cell response rates. Light orange areas, periods of AMF application. \u003cstrong\u003el\u003c/strong\u003e, Maximum fluorescence changes in neurons with BTO\u003csub\u003e100\u003c/sub\u003e and different sizes of MNDs from (\u003cstrong\u003ej\u003c/strong\u003e), (\u003cstrong\u003ek\u003c/strong\u003e), and Fig. 2e. ***p ≤ 0.001, *p ≤ 0.05. Tukey post-hoc test after One-way ANOVA.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3959025/v1/5c0d79641118883fa59d22e6.png"},{"id":52187463,"identity":"ff661442-2eec-4272-ae30-209cc288b4fe","added_by":"auto","created_at":"2024-03-07 18:52:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":764148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal responses \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eby MagTIES with different amounts of BTOs and MNDs\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e a\u003c/strong\u003e, Experimental scheme for investigating MagTIES by immunostaining the c-fos. During the MagTIES procedure, an AMF of 50 mT at 10 Hz was applied ten times at 30 s intervals. \u003cstrong\u003eb \u003c/strong\u003eand\u003cstrong\u003ec\u003c/strong\u003e, Amygdala slices from the 20B/4M (\u003cstrong\u003eb\u003c/strong\u003e) and 20B/4H (\u003cstrong\u003ec\u003c/strong\u003e) groups. Bright-field images (top) and c-fos staining (bottom) of the ipsilateral (left) and contralateral (right) BLA. \u003cstrong\u003ed\u003c/strong\u003e, The c-fos expression level of BLA in 40B/4M (n = 8), 20B/4M (n = 6), 4B/4M (n = 6), 20B/20M (n = 6), and 40B/40M (n = 6) group. **p ≤ 0.01. \u003cstrong\u003ee\u003c/strong\u003e, The c-fos expression level in the 40B/4H group. n = 5. \u003cstrong\u003ef\u003c/strong\u003e, Experimental scheme for investigating MagTIES by fiber photometry. \u003cstrong\u003eg\u003c/strong\u003e, Average trace of ΔF/F in 20B/4M (top, n = 5) and 20B/4H (bottom, n = 6) group. The bar indicates the period when the 50 mT, 10 Hz AMF was applied. \u003cstrong\u003eh\u003c/strong\u003e, Averaged ΔF/F before and during stimulation with 50 mT 10 Hz AMF. 20B/4M, n = 5; 20B/4H, n = 6. *p ≤0.05. \u003cstrong\u003ei\u003c/strong\u003e, Averaged ΔF/F before and during stimulation with 10 Hz AMF at different intensities from the mice in the 20B/4M group. n = 5. *p ≤ 0.05. \u003cstrong\u003ej\u003c/strong\u003e, the Power spectrum of 10 Hz AMF at different intensities from a mouse in the 20B/4M group. The stimulation periods are indicated by the bar. \u003cstrong\u003ek\u003c/strong\u003e, Averaged power intensity before and during 50 mT, 10 Hz AMF stimulation in 20B/4M (top, n = 5) and 20B/4H (bottom, n = 6) group. *p ≤0.05. \u003cstrong\u003el\u003c/strong\u003e, Averaged power intensity before and during stimulation with 10 Hz AMF at different intensities in the 20B/4M group. n = 5. *p ≤ 0.05.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3959025/v1/c1ff4017959face8ec6857b1.png"},{"id":52188167,"identity":"d27ec43f-2ff3-46a4-9525-a585f34b3f3b","added_by":"auto","created_at":"2024-03-07 19:00:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":497454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControlling the brain oscillation by MagTIES with high temporal precision. a\u003c/strong\u003e, Power spectrum of MagTIES with 50mT AMF at 10, 11, and 12 Hz for 30 s. \u003cstrong\u003eb \u003c/strong\u003eto\u003cstrong\u003e d\u003c/strong\u003e, Averaged traces of power intensity at 20 Hz (\u003cstrong\u003eb\u003c/strong\u003e), 22 Hz (\u003cstrong\u003ec\u003c/strong\u003e), and 24 Hz (\u003cstrong\u003ed\u003c/strong\u003e) were obtained using MagTIES with 10 Hz (\u003cstrong\u003eb\u003c/strong\u003e), 11 Hz (\u003cstrong\u003ec\u003c/strong\u003e), and 12 Hz (\u003cstrong\u003ed\u003c/strong\u003e) AMF stimulations, respectively. Orange (\u003cstrong\u003eb\u003c/strong\u003e), green (\u003cstrong\u003ec\u003c/strong\u003e), and blue (\u003cstrong\u003ed\u003c/strong\u003e) traces are in the 20B/4M group (n = 5). Black traces are from 20B/4H group (n = 6). \u003cstrong\u003ee \u003c/strong\u003eto \u003cstrong\u003eg\u003c/strong\u003e, Change in power intensity at 20 Hz, 22 Hz, and 24 Hz from the 20B/4M group (n = 5) and 20B/4H group (n = 6) was observed following MagTIES with 10 Hz (\u003cstrong\u003ee\u003c/strong\u003e), 11 Hz (\u003cstrong\u003ef\u003c/strong\u003e), and 12 Hz (\u003cstrong\u003eg\u003c/strong\u003e) AMF stimulation. *p ≤0.05. \u003cstrong\u003eh\u003c/strong\u003e, Power spectrum of MagTIES with 5 s AMF at 10 Hz 3 times with 5 s intervals. \u003cstrong\u003ei\u003c/strong\u003e, Averaged traces of power intensity at 20 Hz under the same condition as in (\u003cstrong\u003eh\u003c/strong\u003e). Orange, 20B/4M group (n = 4). Black, 20B/4H group (n = 6). \u003cstrong\u003ej\u003c/strong\u003e, Change of power intensity at 20 Hz, 22 Hz, and 24 Hz from 20B/4M and 20B/4H group by the same condition as (H). *p ≤0.05 \u003cstrong\u003ek\u003c/strong\u003e, Power spectrum of MagTIES with 1 s AMF at 10 Hz for 15 times with 1 s intervals. \u003cstrong\u003el\u003c/strong\u003e, Averaged traces of power intensity at 20 Hz by the same condition as (\u003cstrong\u003ek\u003c/strong\u003e). Orange, 20B/4M group (n = 5). Black, 20B/4H group (n = 4). \u003cstrong\u003em\u003c/strong\u003e, Change of power intensity at 20 Hz, 22 Hz, and 24 Hz from 20B/4M (n = 5) and 20B/4H group (n = 4) by the same condition as (\u003cstrong\u003ek\u003c/strong\u003e). *p ≤ 0.05\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3959025/v1/50d953a82946d9ca1cdb0859.png"},{"id":52440442,"identity":"5102dc30-c0ce-4473-80e4-d61987c4b443","added_by":"auto","created_at":"2024-03-11 16:56:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3708252,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3959025/v1/e5784f9d-4c39-4e51-8592-90d3b9bc6614.pdf"},{"id":52187461,"identity":"b6ecf7d6-f765-4aeb-9363-4ee55316ba47","added_by":"auto","created_at":"2024-03-07 18:52:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1272292,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementryInformation0208.docx","url":"https://assets-eu.researchsquare.com/files/rs-3959025/v1/ec5ed419517899c082c0e9fb.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nWe would like to disclose that a patent application related to the technologies presented in this manuscript has been filed. This disclosure is also noted in the manuscript. We assure that this does not affect the integrity of our research findings and our commitment to the ethical standards of scientific inquiry.","formattedTitle":"Wireless Brain Oscillation Control via Magnetoelectric Stimulation with Millisecond Precision","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRegulating brain activity through electrical stimulation is a pivotal technique in modern neuroscience research and clinical neurological therapy. It holds the key to unlocking treatments for a spectrum of neurological disorders that affect millions worldwide, ranging from Parkinson's disease and epilepsy to chronic pain. Deep Brain Stimulation (DBS), a prominent treatment using electrode implants, has relieved over 160,000 patients globally with various neurological disorders in the last 30 years\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Despite its clinical success, electrical implants for DBS face risks such as complications from surgery, infection, and potential damage due to micromotions during daily activities\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Consequently, the development of less invasive methods for electrical brain stimulation is essential.\u003c/p\u003e \u003cp\u003eIn pursuit of minimizing invasiveness, recent advances have shifted towards magnetic approaches\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, utilizing the ability of magnetic fields to penetrate the skull, bones, and tissues without interference, thus offering an ideal solution for remotely manipulating neuronal activity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Transcranial Magnetic Stimulation (TMS), the most prominent among these methods, employs strong magnetic fields more significant than 1.5 T to induce electrical currents non-invasively within the brain. However, TMS's spatial and depth precision is limited in its capacity to stimulate the deeper brain regions without affecting the surface areas\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, thereby restricting its scope of application. The limitations of those neuromodulation methods catalyzed the exploration of alternative magnetic-based neuromodulation strategies capable of reaching deeper neural targets with better spatial precision. In this context, magnetic nanoparticle-based neuromodulation emerges as a promising approach. These magnetic nanoparticles can be manipulated remotely with much fewer magnetic fields than TMS, offering a potentially transformative solution for deep brain stimulation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOver the last decade, various magnetic nanoparticle-based neuromodulation approaches have been invented for wirelessly stimulating deep brain neurons with minimal invasiveness, such as magnetothermal stimulation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, magnetomechanical stimulation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, magnetochemogenetics\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and magnetoelectric stimulation\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Among them, magnetoelectric stimulation is the only approach that doesn\u0026rsquo;t require expressing specific thermosensitive ion channels, mechanosensitive ion channels, or receptors at target neurons. This approach not only bypasses the complexities associated with gene delivery and overexpression of exogenous genes but also sidesteps the challenge of variable and poorly understood intrinsic mechanosensor expression, complicating the predictability of neuromodulation effects in diverse neuronal types. While magnetoelectric stimulation offers these advantages, it, too, faces its own set of challenges.\u003c/p\u003e \u003cp\u003eCurrent magnetoelectric nanomaterials for neuromodulation predominantly focus on magnetostriction-based approaches, utilizing superparamagnetic and piezoelectric materials\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The effectiveness of these methods largely depends on the coupling efficiency between the magnetic and piezoelectric elements. Core-shell architectures are frequently employed in these systems to optimize this interaction. Previous studies have explored various nanomaterial composites in spherical and disc shapes, such as CoFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-BaTiO\u003csub\u003e3\u003c/sub\u003e nanoparticles\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, CoFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-BiFeO\u003csub\u003e3\u003c/sub\u003e nanoparticles\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-CoFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-BaTiO\u003csub\u003e3\u003c/sub\u003e nanodiscs\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, demonstrating their potential in applications ranging from DBS\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, dissociation of Aβ\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e to tumor cell apoptosis induction\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For effective DBS, these technologies often require a significant static magnetic field, approximately 220 mT, coupled with an alternating magnetic field of more than 6 mT at frequencies beyond 140 Hz\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, these systems primarily offer second-scale temporal precision. This limitation underscores a significant need for advancements toward millisecond-scale precision to achieve more precise modulation of neuronal activity.\u003c/p\u003e \u003cp\u003eRecognizing these challenges in the current state of the art, we turn our attention to the untapped potential of Magnetite Nanodiscs (MNDs), which possess the capability to transduce magnetic fields into mechanical forces\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). However, the prospect of harnessing the mechanical force generated by MNDs for magnetoelectric stimulation has yet to be explored. We hypothesize that adapting MND-mediated magnetic-driven torque force for magnetoelectric stimulation could lower the magnetic field requirements and enable the upscaling of magnetic apparatus for broader applications. Meanwhile, BaTiO\u003csub\u003e3\u003c/sub\u003e nanoparticles (BTOs) are the commonly used piezoelectric nanoparticles with a high piezoelectric coefficient\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These have been demonstrated for ultrasound-based wireless stimulation on neuron-like SH-SY5Y cells18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). However, the potential of utilizing magnetic-driven torque forces to induce a dielectric effect in BTOs remains uncharted.\u003c/p\u003e \u003cp\u003eBuilding upon this concept, our study introduces an innovative magnetoelectrical neuromodulation technique named Magnetic-driven Torque Induced Electric Stimulation (MagTIES). This approach utilizes uniquely configured piezoelectric BTOs and MNDs. By applying these nanoparticles sequentially to cells or tissues and conjugating BTOs and MNDs by biotin-avidin linkage, we propose that torque forces induced in MNDs by an alternating magnetic field can stimulate the release of electric fields from BTOs, which was strategically positioned between cell membranes and MNDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We hypothesize that this electric field can trigger voltage-gated ion channels on cell membranes, inducing action potentials in neurons. This innovative approach reduces the required magnetic intensity and frequency compared to previous magnetoelectric stimulation methods, potentially offering a more precise, less invasive, and deeper-reaching neuromodulation technique.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of nanomaterials\u003c/h2\u003e \u003cp\u003eMagnetite nanodiscs (MNDs) were synthesized following a two-step solvothermal synthesis procedure\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In the first step, hematite nanodiscs (HNDs) were produced by solvothermal synthesis. In the second step, HNDs were reduced to MNDs while maintaining the morphology. The crystal structures of these nanodiscs were characterized using Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e, g). The XRD and VSM results indicate that HNDs are fully reduced to MNDs after a two-step synthesis process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, S1a). HNDs, which served as our control group, were not magnetized by external magnetic fields. Additionally, BaTiO\u003csub\u003e3\u003c/sub\u003e nanoparticles (BTO), sourced from US Research Nanomaterial, Inc., were characterized by TEM and XRD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, h). All nanomaterials were functionalized for cellular applications; BTOs were coated with PEG to facilitate attachment to cell membranes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and further functionalized with neutravidin for linkage with nanodiscs. Consistent with previous studies \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, both MNDs and HNDs were coated with PMAO. The PMAO-coated nanodiscs were conjugated with biotin for linkage with BTOs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMagTIES induced neuronal responses in vitro\u003c/h2\u003e \u003cp\u003eTo validate our hypothesis regarding the efficacy of MagTIES, we initially applied the functionalized BTOs to primary cultured hippocampal neurons. Subsequently, we introduced the functionalized MNDs or HNDs to these neurons. The biotinylated MNDs or HNDs were linked to the neutravidin-conjugated BTOs through biotin-neutravidin binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). To visually confirm the binding of BTOs and MNDs in the primary cultured cells, we employed F\u0026ouml;rster Resonance Energy Transfer (FRET). For this purpose, the functionalized 100 nm BTOs (BTO\u003csub\u003e100\u003c/sub\u003e) and 250 nm MNDs (MND\u003csub\u003e250\u003c/sub\u003e) were conjugated with Alexa-488 and Alexa-594, respectively. Following applying BTO\u003csub\u003e100\u003c/sub\u003e and MND\u003csub\u003e250\u003c/sub\u003e to the cultured neurons, illumination with blue light at 470 nm was used to excite the Alexa-488 on the BTO\u003csub\u003e100\u003c/sub\u003e. This resulted in the emission of green fluorescence from Alexa-488, which subsequently transferred energy to the nearby Alexa-594 on the MND\u003csub\u003e250\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). The FRET ratio was significantly increased after the application of MNDs, indicating successful binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, S1b). Moreover, the Scanning Electron Microscopy (SEM) image of a neuron in the BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e group shows the BTOs were attached to the cell membrane, and the MNDs were sitting on top of BTOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). Similar results can be observed in SEM image of neuron with BTO\u003csub\u003e100\u003c/sub\u003e and 250 nm HNDs (HND\u003csub\u003e250\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el)\u003c/p\u003e \u003cp\u003eWe then measured the magnetoelectric stimulated Ca\u003csup\u003e2+\u003c/sup\u003e responses in the cultured hippocampal neurons with a custom-made air-core coil in the upright fluorescence microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, S2). Upon applying the functionalized BTO\u003csub\u003e100\u003c/sub\u003e and MND\u003csub\u003e250\u003c/sub\u003e, magnetic field-induced Ca\u003csup\u003e2+\u003c/sup\u003e responses were observed during the application of slow and weak AMF of 50 mT at 10 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Notably, repeated AMF at this intensity and frequency elicited multiple Ca\u003csup\u003e2+\u003c/sup\u003e responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Such responses were not observed in the control group with BTO\u003csub\u003e100\u003c/sub\u003e and HND\u003csub\u003e250\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, f). The fluorescence responses were significantly higher in the BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh), indicating the specificity of the MagTIES-induced responses. Interestingly, if we rearranged the application sequence to place the MND\u003csub\u003e250\u003c/sub\u003e between BTO\u003csub\u003e100\u003c/sub\u003e and the cell membrane, the AMF-induced responses were significantly smaller (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, g-h). This result indicates that the arrangement of BTOs between MNDs and cell membranes is critical for inducing neuronal activity.\u003c/p\u003e \u003cp\u003eTo further elucidate the temporal precision of MagTIES, we employed Di-8-ANEPPS, a radiometric voltage-sensitive dye (VSD), to measure action potentials in cultured neurons at a temporal resolution of 1 kHz. Di-8-ANEPPS shifts its emission spectrum upon neuronal depolarization when excited at 470 nm, enabling the quantification of membrane potential changes by comparing green to red emission light (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei).\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Fluorescence imaging revealed that Di-8-ANEPPS predominantly localizes at the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej), ensuring the accuracy of potential change measurements. By applying short alternating magnetic field (AMF) pulses of 100 ms at 50 mT and 10 Hz, we observed MagTIES-induced neuronal spikes within milliseconds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). Each AMF cycle alternates the external magnetic field between two directions. This bidirectional alternation in a cycle can generate two distinct torque forces by the magnetite nanodiscs (MNDs). Consequently, we recorded one or two spikes per 10 Hz stimulation cycle over 100 ms periods, demonstrating the millisecond-scale temporal precision enabled by MagTIES.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMagTIES with nanoparticles in different sizes\u003c/h2\u003e \u003cp\u003eUnlike bulk BaTiO3, the piezoelectric coefficients of single BTOs have been shown to correlate with their size inversely\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Smaller BTOs, under 120 nm, possess higher piezoelectric coefficients, which have a piezoelectric coefficient (d\u003csub\u003e33\u003c/sub\u003e) more significant than 1500 pC/N\u003csup\u003e20\u003c/sup\u003e. BaTiO\u003csub\u003e3\u003c/sub\u003e nanoparticles larger than 300 nm had d\u003csub\u003e33\u003c/sub\u003e smaller than 300 pC/N\u003csup\u003e20\u003c/sup\u003e. In this context, we further investigated neuronal responses by combining MND\u003csub\u003e250\u003c/sub\u003e with BTOs of different sizes, including 100 nm (BTO\u003csub\u003e100\u003c/sub\u003e), 300 nm (BTO\u003csub\u003e300\u003c/sub\u003e), and 500 nm (BTO\u003csub\u003e500\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). We found that BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e groups elicited significantly stronger neuronal responses than BTO300/MND250 and BTO500/MND250 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f). These results indicate that stimulated response is inversely correlated to the size of BTOs.\u003c/p\u003e \u003cp\u003eConversely, the size of MNDs might also affect the efficacy of MagTIES. The torque force generated by MNDs is positively correlated to the size of nanodiscs. We hypothesized that smaller MNDs might induce less piezoelectric responses from BaTiO\u003csub\u003e3\u003c/sub\u003e nanoparticles (BTOs) and thereby affect the neuronal activity induced by MagTIES. To test this hypothesis, we compared the stimulated neuronal responses using MagTIES with BTO\u003csub\u003e100\u003c/sub\u003e combined with MND\u003csub\u003e250\u003c/sub\u003e, 220 nm MNDs (MND\u003csub\u003e220\u003c/sub\u003e), or 135 nm MNDs (MND\u003csub\u003e135\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-i). The cultured neurons activated in response to all combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-i). We found that the BTO100/MND250 group had a significantly more robust response than the BTO100/MND135 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). The BTO100/MND250 group had a slightly more robust response than the BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e220\u003c/sub\u003e group. However, these two groups have no significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). These results show that MagTIES with larger MNDs can induce more effective responses. In addition, the cell viability test shows that the cell death rate was meager in both BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e and BTO\u003csub\u003e100\u003c/sub\u003e/HND\u003csub\u003e250\u003c/sub\u003e groups after MagTIES (Fig. S3). These results collectively suggest the effectiveness and biosafety of MagTIES when employing BTO\u003csub\u003e100\u003c/sub\u003e combined with MND\u003csub\u003e250\u003c/sub\u003e; this combination was used in the following stages of this study.\u003c/p\u003e \u003cp\u003eA previous study demonstrated that applying MND\u003csub\u003e250\u003c/sub\u003e alone during AMF can activate neuronal activity by triggering the intrinsic mechanosensitive ion channel, TRPC, in cultured neurons\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In MagTIES, a layer of BTOs is positioned between the MNDs and cell membranes, which reduces the direct mechanical force transduced from MNDs to membranes. We found that applying the TRPC-specific antagonist, SKF96365, does not reduce MagTIES-induced neuronal activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, d). We further confirmed that MagTIES-induced Ca\u003csup\u003e2+\u003c/sup\u003e responses depended on voltage-gated channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-e) by using antagonist of voltage-gated Na\u003csup\u003e+\u003c/sup\u003e channels, tetrodotoxin (TTX), and the antagonist of voltage-gated Ca\u003csup\u003e2+\u003c/sup\u003e channels, mibefradil. These results indicate that TRPC is not critical for the MagTIES-induced activity of voltage-gated ion channels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMagTIES Induced Neuronal Activity In Vivo\u003c/h2\u003e \u003cp\u003eTo evaluate the \u003cem\u003ein vivo\u003c/em\u003e efficacy of MagTIES, particularly its ability to target deep brain regions, we selected the amygdala, a crucial area for emotion processing located deep within the brain\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. We utilized stereotactic injection to introduce a combination of BTO\u003csub\u003e100\u003c/sub\u003e and MND\u003csub\u003e250\u003c/sub\u003e into the amygdala of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Following a day of recovery, mice were exposed to MagTIES within an 11 cm coil, designed to generate an AMF tailored explicitly for our experiment. The mice were acclimated to the chamber, measuring 10 cm in diameter and 9 cm in height, for 30 minutes before stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). The stimulation protocol consisted of 10 times 30 s stimulation periods interspersed with 30 s rest intervals, with the AMF set at 50 mT and 10 Hz.\u003c/p\u003e \u003cp\u003eTo systematically evaluate the effect of MagTIES on neuronal activity, we varied the ratios and amounts of BTOs and MNDs injected. The experimental groups included varying ratios such as 40 \u0026micro;g BTOs with 4 \u0026micro;g MNDs (40B/4M), 20 \u0026micro;g BTOs with 4 \u0026micro;g MNDs (20B/4M), and 4 \u0026micro;g BTOs with 4 \u0026micro;g MNDs (4B/4M). We also examined different total amounts at the same ratio, including combinations like 20 \u0026micro;g BTOs with 20 \u0026micro;g MNDs (20B/20M) and 40 \u0026micro;g BTOs with 40 \u0026micro;g MNDs (40B/40M). Before craniotomy with stereotactic injection, functionalized BTOs and functionalized MNDs were characterized by FRET \u003cem\u003ein vitro\u003c/em\u003e to confirm the function of biotin-avidin linkage in each batch of materials (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-j). Intriguingly, we observed that the expression of c-Fos, an immediate early gene marker for neuronal activation, was significantly higher in the injected hemisphere compared to the contralateral side in the 40B/4M and 20B/4M groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, d). However, this trend was not as pronounced in the 4B/4M group. No significant differences were observed between the contralateral and ipsilateral sides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Interestingly, increasing the total amount of BTO and MND injections in the 20B/20M and 40B/40M groups did not significantly enhance c-Fos expression in the ipsilateral amygdala (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). These findings indicate that MagTIES with a specific amount of 20 \u0026micro;g BTOs and 4 \u0026micro;g MNDs injection can effectively induce neuronal activity \u003cem\u003ein vivo\u003c/em\u003e. In contrast, the control group with 20 \u0026micro;g BTOs and 4 \u0026micro;g HNDs injections (20B/4H) showed no significant changes in c-Fos expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, e), underscoring the specificity of the MagTIES-induced response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eModulating the neural oscillation by MagTIES\u003c/h2\u003e \u003cp\u003eBrain oscillations are crucial for various functions across different brain regions\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Neuromodulation technologies with high temporal precision, such as electrical DBS and optogenetics, can target and modulate these oscillations to specific frequencies\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. This capability is essential for manipulating neuronal circuitry by controlling neuronal activity with precise timing. However, tuning the frequency of brain oscillation via magnetic nanoparticle-based neuromodulation technologies has not been previously reported. Here, we utilized fiber photometry for recording real-time neuronal activity \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, circumventing interference from magnetic stimulation to the measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). AAV-hSyn-GCaMP7s-WPRE were unilaterally injected into the basolateral amygdala (BLA). After 3 to 7 weeks, the 20 \u0026micro;g BTOs and 4 \u0026micro;g MNDs (20B/4M) or 20 \u0026micro;g BTOs and 4 \u0026micro;g HNDs (20B/4H) were stereotaxic injected into the same location. The optical fiber with 400 \u0026micro;m diameter was implanted into the BLA. Post-surgical recovery, the Ca\u003csup\u003e2+\u003c/sup\u003e responses \u003cem\u003ein vivo\u003c/em\u003e were measured by fiber photometry in a magnetic apparatus with a diameter of 10 cm and a height of 19 cm (Fig. S5e-g).\u003c/p\u003e \u003cp\u003eFirst, we perform the MagTIES using 50 mT AMF at 10 Hz for 30 s. In the 20B/4M group, we observed a significant increase in fluorescence change (dF/F) compared to pre-stimulation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-h). In contrast, there was no notable fluorescence change in the control group with 20B/4H (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-h). In 20B/4M group, reducing the magnetic field intensity to 40 mT, 30 mT, and 20 mT still resulted in an observable trend of increased fluorescence, but without statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Interestingly, further analysis using Fast Fourier Transform (FFT) revealed increased brain oscillations at 20 Hz during 10 Hz AMF application in the 20B/4M group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej), a phenomenon not seen in the 20B/4H group. The power spectrum intensity at 20 Hz was significantly increased in 20B/4M injected mice when applying 50 mT AMF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek), which cannot be observed in 20B/4H injected mice. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). The increased power spectrum intensity trend was also notable when reducing the magnetic field intensity to 40 mT, 30 mT, and 20 mT, but without statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el).\u003c/p\u003e \u003cp\u003eDifferent AMF frequencies were used for MagTIES to determine whether the oscillation frequency corresponded to the MagTIES frequency. While we increased the frequency of AMF from 10 Hz to 11 Hz and 12 Hz, we can observe the apparent increment of power intensity at 20 Hz, 22 Hz, and 24 Hz, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, S6a). Align with our hypothesis, the oscillation of Ca\u003csup\u003e2+\u003c/sup\u003e responses was precisely two times the applied frequency of AMF. When using 10 Hz AMF to the 20B/4M group, the change of power intensity at 20 Hz was ~\u0026thinsp;7-fold more prominent than the baseline intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, e), which is significantly higher than the change of power intensity in 20B/4H group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, e). But the shift in power intensity at nearby frequencies doesn\u0026rsquo;t show the difference between the 20B/4M group and 20B/4H group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Similarly, by using MagTIES with 11 Hz, 50 mT AMF, The increase of power spectrum intensity at the 22 Hz was significantly larger in the 20B/4M group than 20B/4H group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, f). The power intensity was not changed at 20 H and 24 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). We can observe similar results using MagTIES with 12 Hz and 50 mT AMF. Only a change of power intensity at 24 Hz was increased in the 20B/4M group compared to the 20B/4H group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), which cannot be observed in other frequencies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eFinally, we observed that MagTIES induced responses even when the AMF stimulation period was reduced to 5 seconds or 1 second. Specifically, applying AMF for a 5-second stimulation period, interspersed with 5-second intervals for three cycles, led to a marked increase in power spectrum intensity, notably at 20 Hz during the stimulation periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh-i). The change in power intensity at 20 Hz, observed 5 seconds before and after stimulation, was significantly greater in the 20B/4M group than in the 20B/4H group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej, S6b). A similar pattern emerged when AMF was applied for 1-second periods, followed by 1-second resting periods for 15 cycles. A significant increase in power spectrum intensity at 20 Hz was noted during these 1-second stimulation periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek-l). The rise in power intensity at 20 Hz was also significantly more significant in the 20B/4M group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003em, S6c). These fiber photometry results indicate the unique capability of MagTIES to manipulate neural oscillations in the amygdala with a high degree of temporal and frequency specificity, marking a significant advancement in the field of neuromodulation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study is the first to demonstrate a non-magnetostrictive method for magnetoelectric stimulation at the nanoscale, utilizing two separate nanomaterials: BTOs and MNDs. This approach offers a more straightforward yet effective method for stimulating biological tissues and cells. Unlike core-shell magnetoelectric nanoparticles that rely on a tight interface between magnetostrictive and piezoelectric materials, our use of separate BTO and MND materials, linked via biotin-avidin, simplifies the synthesis process and broadens potential applications. Our findings demonstrate that magnetic-driven torque forces from MNDs effectively induce a dielectric field in BTOs, triggering neuronal activity. This approach has been successfully implemented \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, showcasing the potential of MagTIES in neuromodulation.\u003c/p\u003e \u003cp\u003eUsing voltage imaging and fiber photometry, we revealed that this approach can trigger neuronal activity within milliseconds in vitro and precisely modulate brain oscillations to specific frequencies \u003cem\u003ein vivo\u003c/em\u003e. Significantly, MagTIES marks the first instance of a magnetic nanoparticle-based technology achieving modulation of brain oscillations with millisecond-scale temporal precision, positioning it at the forefront of advancements in neuromodulation technology. Furthermore, we reveal a nanomaterial size-dependent response in the MagTIES approach, where smaller BTOs and larger MNDs produce more robust neuronal responses. This finding adds an essential dimension to the design considerations for optimizing MagTIES systems.\u003c/p\u003e \u003cp\u003eConsidering that the electric field decreases with the square of the distance from its source, the effectiveness of the electric field from BTOs in influencing membrane potential would diminish with increased distance from the cell membrane. Intriguingly, our findings highlight the critical importance of the spatial arrangement of BTOs and MNDs in the effectiveness of MagTIES. When BTOs were directly attached to the cell membrane in the BTO\u003csub\u003e100\u003c/sub\u003e/MND\u003csub\u003e250\u003c/sub\u003e group, the magnetically induced responses were significantly more pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Conversely, when MNDs were positioned between the BTOs and the cell membrane, the neuronal responses were substantially weaker. This underscores the importance of optimizing the spatial configuration of these nanomaterials for maximized neuromodulation efficacy.\u003c/p\u003e \u003cp\u003eThe varied expression levels of intrinsic mechanosensitive ion channels across different cell types and species have been a limiting factor in applying transgene-free magnetomechanical stimulation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Our findings indicate that the intrinsic mechanosensitive ion channel, TRPC, is not essential for MagTIES-induced neuronal activity (Fig. S3a, d). In addition, our approach has demonstrated the ability to induce brain oscillations \u003cem\u003ein vivo\u003c/em\u003e with alternating magnetic fields (AMF) below 50 mT, a threshold lower than the one needed for activating intrinsic TRPC\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In contrast to approaches that depend on mechanosensitive ion channels, electric stimulation is already widely accepted for triggering neuronal activity. While we cannot entirely discount the involvement of intrinsic mechanosensitive ion channels, the effectiveness of MagTIES, regardless of innate mechano-sensitivity in neurons, significantly broadens its applicability. This magnetoelectric stimulation approach enables neuronal stimulation without necessitating knowledge of their specific mechanosensitive ion channel expression profiles, thus offering a more universally applicable neuromodulation strategy.\u003c/p\u003e \u003cp\u003eRegarding biosafety and clinical relevance, both BTO and MNDs have previously been reported as biocompatible materials\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. BTO is one of the most biocompatible piezoelectric materials with the highest piezoelectric coefficient\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. On the other hand, MNDs are made of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, a biocompatible material similar to Ferumoxytol, an FDA-approved magnetic nanoparticle for clinical use\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. As a transgenic-free approach, MagTIES sidesteps the complexities and potential side effects associated with gene delivery methods\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Future studies are necessary to understand the long-term biosafety of these materials and their feasibility for clinical use. Exploring combinations of various piezoelectric and superparamagnetic materials could further advance the capabilities of MagTIES. Additionally, considering the scalability and adaptability of the magnetic apparatus used in MagTIES, this technology holds promise for a wide range of applications in both fundamental research and clinical therapies. In conclusion, MagTIES introduces an innovative, less invasive, and precise approach to neuromodulation, particularly in deep brain regions. Its ability to target specific frequencies with high temporal accuracy opens new avenues for neurological research and treatment, setting a foundation for future explorations in this exciting field.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank the National Science and Technology Council (NSTC)\u0026nbsp;for funding (NSTC 110-2636-B-A49-003, NSTC 111-2636-B-A49-008, NSTC 112-2636-B-A49-006). Schematics in Fig. 2 were created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003ePC. Formal Analysis: CC, PC. Funding acquisition: PC. Investigation: CC, LC, MH, CT, YT, GT, PC. Methodology: CC, LC, JX, PC. Project administration: PC. Supervision: PC. Validation: CC, LC, MH, CT, YT, GT. Visualization: CC, PC. Writing – original draft: PC. Writing – review \u0026amp; editing: CC, PC\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e PC and CC have filed a patent in Taiwan (application No. 112130503) and in the U.S. (application No. 18/540750) describing magnetic field-induced electrical stimulation of cells, which is related to this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLozano, A. 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Genotoxicity in mice following AAV gene delivery: A safety concern for human gene therapy? \u003cem\u003eMolecular Therapy\u003c/em\u003e vol. 24 198\u0026ndash;201 Preprint at https://doi.org/10.1038/mt.2016.17 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3959025/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3959025/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eElectrical deep brain stimulation (DBS) is a pivotal technology in treating neurological disorders and advancing neuroscience research. Minimizing the invasiveness of conventional DBS can reduce the risk of large hardware implants. Our study introduces a novel wireless magnetoelectric neuromodulation method termed Magnetic-driven Torque-Induced Electrical Stimulation (MagTIES). Diverging from traditional magnetostriction-based magnetoelectric stimulation approaches, we utilized the torque force from magnetic nanodiscs during alternating magnetic fields (AMF) to induce a piezoelectric effect on piezoelectric nanoparticles. This technique triggered neuronal activity \u003cem\u003ein vitro \u003c/em\u003eand \u003cem\u003ein vivo\u003c/em\u003e at millisecond-scale temporal precision using weak AMF at slow frequency. Importantly, it allows fine-tuning brain oscillations in deep brain areas through AMF frequency adjustments. MagTIES represents a significant advancement in neuromodulation, providing a minimally invasive, transgene-free approach for precise and wireless brain activity control, with vast potential for neurological therapies and neuroscience research.\u003c/p\u003e","manuscriptTitle":"Wireless Brain Oscillation Control via Magnetoelectric Stimulation with Millisecond Precision","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-07 18:52:04","doi":"10.21203/rs.3.rs-3959025/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8cb7bc69-2c3f-472b-b268-2a48a2d6fce7","owner":[],"postedDate":"March 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":29038506,"name":"Biological sciences/Neuroscience"},{"id":29038507,"name":"Biological sciences/Biotechnology/Nanobiotechnology"}],"tags":[],"updatedAt":"2024-03-07T18:52:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-07 18:52:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3959025","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3959025","identity":"rs-3959025","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}