Neuromodulation is crucial for the understanding of brain circuits and treatment of neurological diseases. This work demonstrates a new photoacoustic nanoparticlebased neural stimulation technique. Synthesized nanoparticles transduce nearinfrared light to ultrasound locally at the neuronal membrane and evoke neural activation in vitro and in vivo. Through targeting the mechanosensitive ion channel TRPV4, the modified nanotransducers achieve neural activation with enhanced specificity. Together, photoacoustic nanotransducers offer opportunities for nongenetic neuromodulation with deep tissue penetration.

Neural interfaces using biocompatible scaffolds provide crucial properties for the functional repair of nerve injuries and neurodegenerative diseases, including cell adhesion, structural support, and mass transport. Neural stimulation has also been found to be effective in promoting neural regeneration. This work provides a new strategy to integrate photoacoustic (PA) neural stimulation into hydrogel scaffolds using a nanocomposite hydrogel approach. Specifically, polyethylene glycol (PEG)-functionalized carbon nanotubes (CNT), highly efficient photoacoustic agents, are 2 embedded into silk fibroin to form biocompatible and soft photoacoustic materials. We show that these photoacoustic functional scaffolds enable non-genetic activation of neurons with a spatial precision defined by the area of light illumination, promoting neuron regeneration. These CNT/silk scaffolds offered reliable and repeatable photoacoustic neural stimulation. 94% of photoacoustic stimulated neurons exhibit a fluorescence change larger than 10% in calcium imaging in the light illuminated area. The on-demand photoacoustic stimulation increased neurite outgrowth by 1.74fold in a dorsal root ganglion model, when compared to the unstimulated group. We also confirmed that photoacoustic neural stimulation promoted neurite outgrowth by impacting the brain-derived neurotrophic factor (BDNF) pathway. As a multifunctional neural scaffold, CNT/silk scaffolds demonstrated non-genetic PA neural stimulation functions and promoted neurite outgrowth, providing a new method for non-pharmacological neural regeneration.

Neuromodulation at high spatial resolution poses great significance in advancing fundamental knowledge in the field of neuroscience and offering novel clinical treatments. Here, we developed a tapered fiber optoacoustic emitter (TFOE) generating an ultrasound field with a high spatial precision of 39.6 µm, enabling optoacoustic activation of single neurons or subcellular structures, such as axons and dendrites. Temporally, a single acoustic pulse of sub-microsecond converted by the TFOE from a single laser pulse of 3 ns is shown as the shortest acoustic stimuli so far for successful neuron activation. The precise ultrasound generated by the TFOE enabled the integration of the optoacoustic stimulation with highly stable patch-clamp recording on single neurons. Direct measurements of the electrical response of single neurons to acoustic stimulation, which is difficult for conventional ultrasound stimulation, have been demonstrated. By coupling TFOE with ex vivo brain slice electrophysiology, we unveil cell-type-specific responses of excitatory and inhibitory neurons to acoustic stimulation. These results demonstrate that TFOE is a non-genetic single-cell and sub-cellular modulation technology, which could shed new insights into the mechanism of ultrasound neurostimulation.

Graphical abstract A fiber-based optoacoustic emitter is developed, serving as the most miniaturized ultrasound point source so far, with sub-millimeter confinement. Controllable frequencies are achieved and further induce cell membrane sonoporation with frequency dependent efficiency. By solving the problem of compromise between sub-MHz frequency and sub-millimeter precision via breaking the diffraction limit, the FOE shows a great potential in region-specific cell modulation.

High precision neuromodulation is a powerful tool to decipher neurocircuits and treat neurological diseases. Current non-invasive neuromodulation methods offer limited precision at the millimeter level. Here, we report optically-generated focused ultrasound (OFUS) for non-invasive brain stimulation with ultrahigh precision. OFUS is generated by a soft optoacoustic pad (SOAP) fabricated through embedding candle soot nanoparticles in a curved polydimethylsiloxane film. SOAP generates a transcranial ultrasound focus at 15 MHz with an ultrahigh lateral resolution of 83 µm, which is two orders of magnitude smaller than that of conventional transcranial-focused ultrasound (tFUS). Here, we show effective OFUS neurostimulation in vitro with a single ultrasound cycle. We demonstrate submillimeter transcranial stimulation of the mouse motor cortex in vivo. An acoustic energy of 0.6 mJ/cm2, four orders of magnitude less than that of tFUS, is sufficient for successful OFUS neurostimulation. OFUS offers new capabilities for neuroscience studies and disease treatments by delivering a focus with ultrahigh precision non-invasively.

Highly precise neuromodulation with a high efficacy poses great importance in neuroscience. Here we developed a candle soot fiber optoacoustic emitter (CSFOE), capable of generating a high pressure of over 10 MPa with a central frequency of 12.8 MHz, enabling highly efficient neuromodulation in vitro. The design of the fiber optoacoustic emitter, including the choice of the material and the thickness of the layered structure, was optimized in both simulations and experiments. The optoacoustic conversion efficiency of the optimized CSFOE was found to be 10 times higher than the other carbon-based fiber optoacoustic emitters. Driven by a single laser, the CSFOE can perform dual-site optoacoustic activation of neurons, confirmed by calcium (Ca2+) imaging. Our work opens potential avenues for more complex and programmed control in neural circuits using a simple design for multisite neuromodulation in vivo.

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Neuromodulation is an invaluable approach for study of neural circuits and clinical treatment of neurological diseases. Here, we report semiconducting polymer nanoparticles based photoacoustic nanotransducers (PANs) for neural stimulation. Our PANs strongly absorb light in the near-infrared second window and generate localized acoustic waves. PANs can also be surfacemodified to selectively bind onto neurons. PAN-mediated activation of primary neurons in vitro is achieved with ten 3-nanosecond laser pulses at 1030 nm over a 3 millisecond duration. In vivo neural modulation of mouse brain activities and motor activities is demonstrated by PANs directly injected into brain cortex. With millisecond-scale temporal resolution, sub-millimeter spatial resolution and negligible heat deposition, PAN stimulation is a new non-genetic method for precise control of neuronal activities, opening potentials in non-invasive brain modulation.. then utilized the magneto-thermal effect of the paramagnetic nanoparticles to activate these channels 18 . In these studies, significant local temperature rise, exceeding the thermal threshold of the ion channels, e.g. 43 ˚C in the case of TRPV 1, was observed, thus raising concerns over safety of thermally activated brain stimulation. The Khizroev group used the magneto-electric nanoparticles under an applied magnetic field to perturb the voltage-sensitive ion channels for neuron modulation 19 . Notably, these magnetic stimuli-based techniques deliver a spatial precision relying on the confinement of the magnetic field, which is on the millimeter to centimeter scale.New technologies and concepts are still sought to achieve non-invasive, genetic free and precise neural stimulation.Here, we report the development and application of photoacoustic nanotransducers (PANs) to enable non-genetic neural stimulation in cultured primary neurons and in live brain (Figure 1a).Our PANs, based on synthesized semiconducting polymer nanoparticles, efficiently generate localized ultrasound by an optoacoustic process upon absorption of nanosecond pulsed light in the NIR-II window (1000 nm to 1700 nm) (Figure 1b). The NIR-II light has the capability of centimeter-deep tissue penetration 20,21 , which is beyond the reach of visible light currently used in optogenetics. We further modified the PAN surface for non-specific binding to neuronal membrane and specific targeting of mechanosensitive ion channels, respectively. We showed that upon excitation at 1030 nm PANs on the neuronal membrane successfully activated rat cortical neurons, confirmed by real time fluorescence imaging of GCaMP. We then demonstrated in vivo motor cortex activation and invoked subsequent motor responses through PANs directly injected into a mouse living brain. Importantly, the heat generated by the ns laser pulses is confined inside the PAN, resulting in a transient temperature rise during the photoacoustic process, evident by COMSOL simulations. Collectively, our finding shows photoacoustic nanotransducers as a new Collectively, we have ...