Piezo1-Targeted Magnetoacoustic Nanobubbles Rescue Alzheimer’s Pathology by Electromechanical Neuromodulation and Amyloid‑β Clearance

Fangxuan Chu; Xiaoqing Zhou; Ren Ma; Ruixu Liu; Xu Liu; Kai Zhu; Yuheng Wang; Xin Wang; Ying Li; Shunqi Zhang; Tao Yin; Zhipeng Liu

doi:10.1021/acsnano.5c13702

. 2025 Dec 16;20(2):1962–1982. doi: 10.1021/acsnano.5c13702

Piezo1-Targeted Magnetoacoustic Nanobubbles Rescue Alzheimer’s Pathology by Electromechanical Neuromodulation and Amyloid‑β Clearance

Fangxuan Chu ¹, Xiaoqing Zhou ¹, Ren Ma ¹, Ruixu Liu ¹, Xu Liu ¹, Kai Zhu ¹, Yuheng Wang ¹, Xin Wang ¹, Ying Li ¹, Shunqi Zhang ¹, Tao Yin ¹, Zhipeng Liu ^1,^*

PMCID: PMC12825367 PMID: 41399980

Abstract

Amyloid-β (Aβ) is considered a core pathological feature of Alzheimer’s disease (AD), and its clearance efficiency is highly dependent on the function of the Piezo1 channel in microglia. However, the activity of Piezo1 is impaired under the pathological conditions of AD, and existing pharmacological strategies struggle to achieve precise targeted intervention in deep brain regions. To address these concerns, our research proposes a synergistic therapeutic paradigm leveraging transcranial magneto-acoustic stimulation (TMAS) to actuate microglial-Piezo1-targeted magnetic nanobubbles (PT-MNBs) for AD treatment. TMAS noninvasively focuses physical energy into deep brain lesion areas through a magnetoacoustic coupling field and drives PT-MNBs to generate responsive mechanical and electrical stimulation signals. PT-MNBs achieve microglia-specific anchoring through surface-modified phosphatidylserine, while conjugated anti-Piezo1 antibodies precisely deliver mechano-electrical stimulation signals to antibody-functionalized Piezo1 ion channels in microglial populations. This synchronously activates the mechanical- and voltage- sensitive domains of Piezo1 to recruit microglia to areas of inflammation and increase Aβ clearance, ameliorating synaptic plasticity impairment and ultimately reversing the pathological progression of AD. This dual-action mechanism achieves spatially precise manipulation of cellular mechanical and electrical activity in deep brain regions of AD mice and enhances Piezo1 function through precise energy delivery, enhancing their ability to clear Aβ plaques, opening an avenue for a noninvasive, deep-targeted physical stimulation-mediated nanoparticle synergistic therapy for AD.

Keywords: transcranial magnetoacoustic stimulation, magnetic nanobubbles, Piezo1, microglia, Alzheimer’s disease

Introduction

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder and the leading cause of dementia worldwide, affecting millions of individuals and imposing enormous socioeconomic burdens on healthcare systems. The pathological progression of AD is closely associated with the abnormal deposition of β-amyloid (Aβ), which aggregates to impair neuronal activity, triggering a series of neuroinflammatory cascades and ultimately leading to synaptic impairment and neuronal death. , Although Aβ accumulation begins decades before clinical symptoms emerge, the inability of the brain to clear these toxic aggregates is recognized as a critical driver of AD progression. Microglia, the macrophage-like immune cells of the central nervous system, serve as indispensable regulators of AD. These cells are crucial for monitoring the brain microenvironment through the dynamic extension and retraction of processes and maintaining neural homeostasis through the phagocytosis of abnormal proteins and the regulation of neuroinflammatory responses. Upon encountering Aβ plaques, microglia actively recognize and phagocytose these deposits while establishing a leakproof mechanical barrier that prevents the outward spread of amyloid fibrils, thus alleviating Aβ-triggered neurotoxicity. , Notably, the mechanical stiffness of Aβ plaques (>1 MPa), which markedly differs from that of the soft brain parenchyma (∼200–500 Pa), is recognized as a critical factor in regulating microglial phagocytic activity through mechanosensitive pathways. This responsiveness to mechanical stimuli implies the presence of specific mechanosensors in microglia.

As premier mechanotransducers, Piezo1 ion channels exhibit exceptional specificity and sensitivity in converting extracellular mechanical forces into intracellular signaling cascades. Piezo1 senses extracellular mechanical stimuli to induce Ca² ⁺ influx to mediate the mechanotransduction of microglia. , Through its intrinsic mechanosensory capacity, this channel enables microglia to sense the stiffness of Aβ fibrils, thereby triggering protective responses against Aβ plaques and ultimately preventing the spread of Aβ pathology. , Interestingly, owing to its mechanical sensitivity, Piezo1 acts as a mediator of ultrasonic neuroregulation to modulate cellular signaling and animal behavior. , Piezo1 also exhibits voltage sensitivity, and its conformation can be altered by localized electric fields through activation of its voltage-sensing domain. For example, the voltage-sensitive properties of Piezo1 can modulate the electrical environment to regulate immunomodulation of macrophage polarization. Given the role of Piezo1 in regulating microglial responses to Aβ pathology and its sensitivity to both mechanical and electrical stimuli, the development of strategies capable of precisely and noninvasively activating the Piezo1 channel in microglia within deep brain regions holds significant importance for restoring their Aβ clearance function and mitigating AD progression.

Traditional pharmacological agonists struggle to cross the blood–brain barrier and achieve lesion-targeted delivery, while single-modal ultrasound or electrical stimulation approaches face challenges in terms of cellular specificity or spatial resolution. , Transcranial magnetoacoustic stimulation (TMAS), an emerging noninvasive neuromodulation technique with high focusing and spatial resolution in deep brain regions, integrates the magnetic fields and ultrasonic fields to generate localized mechanical forces and magnetoacoustically coupled electric fields, thereby enabling regulation of Piezo1 through mechano-electrical coupling effects. To further increase the stimulation specificity and activation efficacy of Piezo1 in microglia, the integration of TMAS with Piezo1-targeted magnetic nanobubbles (PT-MNBs) enables dual precise regulation of microglial Piezo1. The PT-MNBs consist of a superparamagnetic Fe₃O₄-modified liposomal shell encapsulating a perfluoropropane (C₃F₈) gas core. The gas-filled core endows the PT-MNBs with ultrasound-responsive characteristics that significantly amplify the localized acoustic field effects, while their magnetic properties further strengthen the magnetoacoustic coupling electric signals, allowing them to generate enhanced mechanical and electrical stimulation signals near cells. PT-MNBs specifically anchor to microglia via surface modification with phosphatidylserine (PS), , whereas the conjugated anti-Piezo1 antibodies deliver mechano-electrical stimulation signals precisely to antibody-functionalized Piezo1 ion channels. Thus, the combination of TMAS and PT-MNBs holds promise for achieving triple synergistic effects within the AD brain. (1) PT-MNBs respond to TMAS to enhance the local acoustic field and magnetoacoustically coupled electric field and precisely focus the energy near microglia. (2) PT-MNBs respond to TMAS to precisely deliver mechano-electrical stimulation signals to antibody-functionalized Piezo1 ion channels, thereby activating Piezo1 channels and transducing mechanical and electrical stimuli into intracellular signals. (3) Piezo1 channel activation and subsequent extracellular Ca² ⁺ influx recruit microglia to inflammatory sites, inducing microglial aggregation, phagocytosis, and compression of Aβ plaques, ultimately ameliorating impairments in synaptic plasticity and suppressing the inflammatory cascade in AD model mice. In conclusion, we present a therapeutic paradigm involving TMAS-driven PT-MNBs that modulates microglial function through dual mechanical- and electrical regulation and promotes Piezo1 function through precise energy delivery, thereby harnessing its dual functionality as both a promoter of neuromodulation and a therapeutic target to clear Aβ deposits and ameliorate AD pathology.

Results

Characterization of PT-MNBs

In our first experiments, we designed magnetic nanobubbles (MNBs) consisted of a superparamagnetic Fe₃O₄-modified liposome shell and a perfluoropropane (C₃F₈) gas core, which endows them with excellent acoustic and magnetic properties, and further functionalized them with anti-Piezo1antibodies and PS to obtain microglia Piezo1-targeted magnetic nanobubbles (PT-MNBs), allowing PT-MNBs to specifically bind/target and modulate microglial Piezo1 (Figure A). The PS mediate precise anchoring to microglia because of their well-documented ability to target M/MΦs. , PS specifically targets microglia through a mechanism mediated by the “eat-me” signal recognition system during apoptosis process. By simulating apoptotic cell signals, it achieves precise targeting through the intrinsic PS receptor recognition system of microglia. , Transmission electron microscopy revealed that the PT-MNBs appeared as round vesicles (Figure B), and the average diameter and zeta potential of the PT-MNBs were 127.84 ± 10.28 nm and −25.78 ± 0.90 mV, respectively (Figure C, Supplementary Figure 1A,B). Several techniques were used to verify the successful preparation of PT-MNBs. Scanning electron microscopy and energy dispersive spectrometry (EDS) mapping revealed the symmetrical distributions of C, Fe, P, O and N in the PT-MNBs (Figure D,E). The functional groups in the PT-MNBs were confirmed by FTIR spectroscopy (Figure F), the peaks in the ranges of 1000–1100 cm^–1 and 540–650 cm^–1 were attributed to the stretching oscillations of P = O and Fe–O bonds, respectively. X-ray photoelectron spectroscopy (XPS) revealed characteristic P 2p (approximately 133 eV) and Fe 2p (approximately 713 eV) peaks in PT-MNBs, indicating the successful integration of PS and Fe₃O₄ into the PT-MNBs (Figure G). In addition, PT-MNBs also contained several different functional groups, including C = O/N–O, C–N and O = C–O, according to the survey, C 1s, O 1s, and N 1s spectra (Figure G–L), which matched the chemical structure of the primary materials used in their manufacture. Thermogravimetric analysis (TGA) revealed that the PS and anti-Piezo1 antibodies accounted for 18% and 64% of the total weight of the PT-MNBs, respectively (Figure M). In addition, the remanence and coercive forces of the PT-MNBs were almost zero, indicating that there was no residual magnetization after exclusion of the extraneous magnetic force (Figure N), and magnetic attraction experiments further verified the magnetic properties of the PT-MNBs (Supplementary Figure 1C). Echogenicity analysis confirmed that the prepared PT-MNBs had an excellent response to ultrasonic vibration (Supplementary Figure 1D). The PT-MNBs exhibited excellent cell precision and targeting due to their nanoscale size and surface functionalization with antibodies and PS, and had superior mechanical and magnetic sensitivity as gas-filled protein structures loaded with superparamagnetic Fe₃O₄. To verify the ability of PT-MNBs to target microglia, BV2 cells were incubated with Cy5.5-labeled PT-MNBs and bare-MNBs (without PS modification). Confocal microscopy revealed stronger Cy5.5 red fluorescence signals on the surface of cells incubated with PT-MNBs than were observed in the bare-MNBs group (Figure O). Furthermore, flow cytometry analysis revealed a significantly greater mean fluorescence intensity in PT-MNBs-treated cells than in those treated with bare-MNBs (Figure P). These results indicate that PS modification endows PT-MNBs with the ability to target microglia, which is consistent with the well-documented role of PS as an “eat-me” signal that can be recognized by specific receptors on the surface of microglia. , We also observed that PT-MNBs did not exert cytotoxic effects on neurons cocultured with microglia in vitro (Figure Q).

TMAS-Responsive PT-MNBs for Precise Neurostimulation

TMAS is a noninvasive stimulation system established with transcranial ultrasound stimulation and magnetic field (Figure A). This system could generate the mechanical force of ultrasound and a magneto-acoustic electric field, which is capable of acting on mechanical and voltage-sensitive ion channels and noninvasively modulates neural activity in specific deep brain regions. We next investigated the ability of PT-MNBs to respond to TMAS in mice (Figure B). TMAS generated a sound pressure of 0.5 MPa at a depth of 25 mm for intracranial stimulation in mice that was consistent with the results of the simulation calculation in the TMAS+ PT-MNBs-treated brains of mice (Figure C,D). The simulated electric field strength was approximately 0.1 V/m in the TMAS+PT-MNBs-treated brains of mice, which was consistent with the measured electric field intensity (Figure E–G). The simulated current density generated in PT-MNBs under TMAS exposure was approximately 0.1 A/m² in the brain of mice (Supplementary Figure 1E), and the signals were able to reach the hippocampus in mice. The presence of PT-MNBs increased the accuracy of TMAS in targeting specific brain regions and cells, avoiding scatter-induced activation of nontarget brain regions and cells. To verify whether TMAS combined with PT-MNBs can temporarily open the tight connection of the BBBenabling PT-MNBs to enter the brainwe conducted Evans blue (EB) dye to verify the permeability of BBB. EB extravasation into the brain clearly increased only after treatment with both TMAS and PT-MNBs; however, EB extravasation into the brain was reduced 60 min after TMAS and PT-MNBs (Supplementary Figure 1F). These results indicate that the BBB opening mediated by TMAS+PT-MNBs treatment was transient and not permanent. This was further illustrated by IVIS imaging of the brain, which revealed that the PT-MNBs entered the brain under the action of TMAS (Supplementary Figure 1G). In addition, major organs including the heart, lungs, liver, spleen, and kidneys, were collected for ex vivo imaging at 24 h following TMAS+PT-MNBs administration. The results revealed fluorescence signals in the liver and kidneys, suggesting that PT-MNBs were primarily metabolized and cleared via the hepatic and renal pathways without significant accumulation or adverse effects in other organs (Supplementary Figure 1H). Notably, the fluorescence signals in the liver and kidneys of the TMAS treated-group were not significantly greater than those in the control group without TMAS treatment. This finding demonstrated that our TMAS treatment did not cause abnormal accumulation of PT-MNBs in metabolic organs or pose additional toxicity risks (Supplementary Figure 1H). The brains (hippocampal DG and CA1 areas) and the main organs (heart, lung, liver, spleen, and kidney) revealed no hemorrhage, erythrocyte extravasation or tissue damage after TMAS+PT-MNBs treatment (Supplementary Figure 1I,J)). Together, these findings verified the safety of this method

Given the role of Piezo1 in regulating microglial responses to Aβ pathology and its dual sensitivity to both mechanical and electrical stimulation, we first monitored the effects of TMAS + PT-MNBs on the Piezo1 ion channel in microglia. Prior to TMAS, the extracellular Ca²⁺ level was greater than the intracellular Ca²⁺ level, and Piezo1 channels, to which PT-MNBs attach through receptor–ligand linkages on the cell membrane, were closed. We hypothesized that TMAS led to the magnetoacoustic response of PT-MNBs and the activation of Piezo1 ion channels, followed by the subsequent influx of extracellular Ca²⁺ into cells, enabling Piezo1 ion channels to function as electromechanical sensors that receive stronger magnetoacoustic effects from TMAS and to convert mechanical and electrical stimuli from TMAS-actuated PT-MNBs into intracellular signals (Figure H). We first verified the effect of TMAS-actuated PT-MNBs on microglial Piezo1 channels by using whole-cell recordings to monitor Piezo1-mediated currents. PT-MNBs were added to the microglial culture medium prior to TMAS stimulation, and we found that PT-MNBs dramatically induced transient currents under the action of TMAS, exerting a stronger effect than TMAS alone (Figure I,J). Importantly, these currents were efficiently inhibited in microglia by blocking the Piezo1 channel with si-Piezo1 (Figure I,J). Our results showed that when stimulation of TMAS + PT-MNBs was stopped, the current could return to the baseline level and no persistent current was observed in the whole cell patch-clamp experiment (Figure I,J). These results also suggest that Piezo1 is opened in a functional rather than a destructive manner. The opening of Piezo1 ion channels, as nonselective ion channels, allows Ca²⁺ influx into cells, leading to changes in the intensity of intracellular Ca²⁺ signals. , To investigate whether TMAS-actuated PT-MNBs regulate Ca²⁺ activity in response to Piezo1 activation in microglia, Ca²⁺ imaging of microglia during TMAS combined with PT-MNBs treatment was conducted, and Piezo1-dependent Ca²⁺ influx was greater in TMAS+PT-MNBs-treated microglia than in TMAS-treated microglia (Figure K,L; Supplementary Figure 1K). Similarly, these evoked Ca²⁺ responses were dramatically reduced in microglia transfected with si-Piezo1 (Figure K,L). Collectively, these findings demonstrate that TMAS-actuated PT-MNBs act on microglia to a significant extent by activating Piezo1 ion channels, which translate coupling stimulation into downstream electrochemical signals.

TMAS-Actuated PT-MNBs Alleviate Spatial Cognitive Impairment and Synaptic Plasticity Deficits in 5xFAD Mice

5xFAD mice show AD-like pathologies in a stage-dependent manner: Aβ deposition at 1.5–2 months of age; microglial proliferation and neuroinflammation at 3 months of age; and progressive cognitive and memory dysfunction at 4 months of age. Therefore, we subsequently investigated whether TMAS+PT-MNBs could affect the spatial cognitive function of 5xFAD mice. After the injection of PT-MNBs (0.03 mL/kg, IV), TMAS was applied to the bilateral hippocampus for 5 min. The stimulation parameters for TMAS were as follows: burst repetition frequency of 1 Hz; 10 ms bursts; total duration of 120 s; average peak pressure of 0.5 MPa; magnetic field strength of 0.3 T. After receiving 6 weeks of TMAS+PT-MNBs treatment, the Morris water maze (MWM) test was performed to evaluate spatial learning and memory, which are highly correlated with hippocampal function (Supplementary Figure 2A). All groups had comparable swimming speeds during the initial training and spatial exploration phases (Supplementary Figure 2B), indicating that there was no noticeable deficit in athletic performance across the groups, but the escape latency of the AD + TMAS and AD + TMAS + PT-MNBs groups was shorter than that of the AD group. In addition, the escape latency of the AD + TMAS + PT-MNBs group was shorter than that of the AD + TMAS group, especially on the last day of training and during the exploration phase (Supplementary Figure 2C), suggesting that TMAS exerted stronger neuromodulatory effects under the action of PT-MNBs. Similar results were obtained for cognitive scores based on swimming paths (Supplementary Figure 2D,E)). The quantified analysis of the trajectories of the mice revealed that the mice in the AD group had greater difficulty finding the platform than did the mice in the AD + TMAS group, whereas the mice in the AD + TMAS + PT-MNBs group crossed the platform position more often and stayed in the correct quadrant longer than did those in the AD and AD + TMAS groups(Supplementary Figure 2F,G)). The novel object recognition (NOR) test and Y-maze test are used to assess short-term object recognition memory and spatial working memory, respectively , Statistical analysis revealed that PT-MNBs further increased the recognition index (RI) and percentage of spontaneous alternations in AD model mice in response to TMAS stimulation (Supplementary Figure 2H–M)), indicating that TMAS was able to exert stronger neuromodulatory effects under the action of PT-MNBs and alleviate cognitive impairment in AD model mice

Next, we determined the effects of TMAS-actuated PT-MNBs on hippocampal long-term potentiation (LTP) and depotentiation (DEP), which constitute the mechanistic basis of learning and memory. Following TMAS, LTP after theta burst stimulation was increased and DEP after low-frequency stimulation was decreased in 5xFAD mice, but the field excitatory postsynaptic potential (fEPSP) slope was greater and the DEP slope was lower in the AD + TMAS + PT-MNBs group than in the AD + TMAS group (Figure A–E), indicating that TMAS was able to increase synaptic plasticity more markedly in 5xFAD mice under the action of PT-MNBs. As the dendritic spines of neurons are the basic units for synaptic formation, we further quantified the abundance of the dendritic spines and synapses via Golgi-Cox staining and transmission electron microscopy (TEM), respectively. The densities of dendritic spines and synapses in the hippocampus region were increased by TMAS + PT-MNBs treatment (Figure F–K). In particular, TMAS-driven PT-MNBs further increased the density of motile functional (thin, stubby, and mushroom-like) spines without affecting the density of immature filamentous spines (Figure I). Moreover, TMAS-actuated PT-MNBs also increased the levels of neuronal cytoskeleton-associated proteins (MAP2) and synaptic plasticity-associated proteins (SYP, PSD-95 and NR2B) in the hippocampi of 5xFAD mice (Figure L–N, Supplementary Figure 3). Thus, we found that TMAS + PT-MNBs treatment is a simple and effective method for activating neural signaling and eliciting reproducible synaptic responses in 5xFAD mice. The pattern of these synaptic responses changed less when TMAS alone was applied to the hippocampus, indicating that PT-MNBs can successfully enhance the neuromodulatory effect of TMAS in 5xFAD mice.

TMAS-actuated PT-MNBs improved the synaptic function of hippocampal neurons in 5xFAD mice. A Implantation of electrodes for electrophysiological recordings. B fEPSP curves before and after theta burst stimulation. (n = 8). C Changes in fEPSP slope from the PP to the DG. D,E The average fEPSP slope from the last 15 min of the corresponding recordings shown in Figure C (n = 8). F Micrographs of dendritic spines from hippocampal neurons and different types of dendritic spines. Scale bar, 250 μm. G,H Representative images of dendritic spines and a graph of the corresponding data (n = 8). I The proportions of different types of neuronal dendritic spines (n = 8). J,K TEM images of hippocampal neuron synaptic structures and a graph of the corresponding data (n = 5). Synapses: dashed boxes. Scale bar, 200 nm. L,M Representative immunofluorescence images and fluorescence intensity of NeuN and MAP2 in the brain (n = 5). Scale bar, 50 μm. N Representative Western blot bands of SYP, PSD95 and NR 2B in the hippocampus (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 vs the AD group. #p < 0.05 vs the AD + TMAS group.

TMAS-Actuated PT-MNBs Activate Microglial Piezo1 to Mediate Aβ Plaque Clearance in 5xFAD Mice

Following our observation that TMAS-actuated PT-MNBs improved spatial cognition and increased synaptic plasticity in 5xFAD mice, we next investigated the potential biophysical mechanisms underlying this effect. Microglia are the endogenous immune and phagocytic cells of the brain and regulate multiple pathological processes in AD, and PS can target microglia to recognize and phagocytose pathogens in areas of inflammation. , Our previous research revealed that Piezo1, a mechanical and voltage-sensitive ion channel with a tripartite propeller-shaped morphology, , acts as an intrinsic modulator of ultrasound- and TMAS-mediated neuromodulation in the brain. We hypothesized that PT-MNBs act not only as magnetic force actuators to increase the stimulation effects and precision of TMAS but also that the surface functionalization of PT-MNBs with Piezo1-targeting antibodies and PS allows for cell-specific targeting and modulation of microglial Piezo1, enabling microglial Piezo1 ion channels to serve as electromechanical transducers and Aβ sensors. Therefore, we first analyzed the expression of microglial Piezo1, which is strongly associated with ultrasonic mechanical force and the magnetoacoustic electric field during TMAS stimulation. Consistent with our hypothesis, Piezo1 expression was increased in hippocampal microglia in the AD + TMAS group, and Piezo1 was robustly upregulated in 5xFAD mice exposed to both PT-MNBs and TMAS compared with that in mice exposed to TMAS alone (Figure A,B). These findings suggest that PT-MNBs act as magnetic force actuators to further enhance the mechanical and electrical effects of TMAS on Piezo1 due to the presence of Piezo1-targeting antibodies. Piezo1 is a mechanical and voltage-sensitive ion channel that is triggered to sense physical forces, including mechanical and electromechanical stimuli, allowing calcium ions to flow inward. , To investigate whether TMAS-actuated PT-MNBs modulate microglial Ca²⁺ activity in response to Piezo1 activation, we used fiber-optic photometry to monitor changes in calcium signaling in microglia expressing GCaMP6f in 5xFAD mice. Hippocampal microglia of 5xFAD mice were engineered to express GCaMP6f via viral transduction. Three weeks later, PT-MNBs were injected, and TMAS was applied to the brain at the following parameters: burst repetition frequency of 1 Hz, 10 ms bursts, total duration of 120 s, acoustic pressure of 0.5 MPa and electric field intensity of 0.1 V/m (Figure C). The results revealed a consistent increase in GCaMP6f fluorescence in response to TMAS stimulation in the presence of PT-MNBs (Figure D–F). TMAS treatment also elicited a Ca²⁺ response in the brains of 5xFAD mice, but the peak amplitude was substantially increased following TMAS+PT-MNBs treatment at the same parameters (Figure D–F). Remarkably, after TMAS-actuated PT-MNBs activated microglial Piezo1 and triggered calcium influx, the microglia predominantly accumulated around Aβ plaques and increased the Iba1/Aβ ratio (Figure G,H). TMAS+ PT-MNBs had a greater effect than did TMAS alone, suggesting that in response to TMAS, PT-MNBs recruited more microglia to cluster around Aβ plaques and reduced Aβ deposition via Piezo1 in the brains of 5xFAD mice. Furthermore, we also observed strong colocalization of microglia with Aβ and CD68 (a marker of phagocytic microglia), and the levels of the microglial phagocytosis-related receptors TREM2, P2Y6 and GPR34 were also increased in the hippocampal and cortical tissues of the AD + TMAS + PT-MNBs group (Figure I,J). Furthermore, similar results were found in BV2 cells in vitro, as TMAS-actuated PT-MNBs activated Piezo1 to a significantly greater extent, accelerating the migration of BV2 cells and BV2 cell-mediated Aβ phagocytosis (Supplementary Figure 4A–D))

TMAS-actuated PT-MNBs activated Piezo1 and promoted the phagocytosis of Aβ plaques by microglia. A Representative images of Iba1 and Piezo1 in the hippocampi of 5xFAD mice. Scale bar, 50 μm. B Fluorescence intensity of Piezo1 (n = 5). C Schematic showing CX3CR1-GCaMp6f virus delivery and fiber implantation in the hippocampus. D Representative calcium traces elicited by TMAS+PT-MNBs stimulation. E,F Mean GCaMP6s fluorescence intensity and heatmap of the hippocampus of 5xFAD mice after treatment with different stimuli (n = 4). G Representative images of Aβ and Iba1 in the hippocampus of 5xFAD mice. Scale bar, 25 μm. H Quantification of overlapping Iba1 and Aβ plaques of different sizes (n = 5). I Representative images and colocalization analysis of Iba1 (green), CD68 (red) and Aβ (acid blue) in the hippocampus of 5xFAD mice. Scale bar, 25 μm. J mRNA levels of TREM2, P2Y6 and GPR34 in the hippocampus and cortex (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 vs the AD group. #p < 0.05 vs the AD + TMAS group.

PT-MNBs, as magnetic force actuators of TMAS, further localized and amplified the ultrasonic and magnetoacoustic effects of TMAS through bubble oscillations and superparamagnetism, and the surface of the PT-MNBs was functionalized with anti-Piezo1 antibodies and PS. PT-MNBs specifically regulated microglial function by aggregating acoustic and electric field energy generated by TMAS near microglia and opening Piezo1 ion channels, accelerating microglial migration and phagocytotic activity to induce Aβ plaque clearance, demonstrating the specificity and efficacy of TMAS+PT-MNBs in the treatment of AD.

TMAS-Actuated PT-MNBs Alleviate Microglia-Associated Neuroinflammation in 5xFAD Mice

Neuroinflammation is a central player in the pathogenesis of AD, and microglia are critical effectors of the neuroinflammatory response in the brain. Overactivation of the pro-inflammatory microglial subset, characterized by high levels of TNF-α, IL-6 and CD86, promotes the seed-like spread of Aβ, whereas the anti-inflammatory subset, characterized by increased CD206 and IL-10, inhibits the transfer of Aβ between neurons and reduces the spread of Aβ in the CNS. , Although there is considerable interest in how physical signals modulate immune cell function, few studies have focused on how mechanical or electrical signals regulate microglial function. Under TMAS-actuated PT-MNBs stimulation, the enhanced synergistic interaction of mechanical and electrical signals further activated microglial Piezo1 (Figure A,B), and Piezo1 increased the sensitivity of microglia to mechanical and electrical stimulation. ,,, Therefore, we next investigated whether mechanical and electrical signals generated by TMAS-actuated PT-MNBs could modulate microglial immune function in the brains of 5xFAD mice upon activation of Piezo1. Our data revealed that the expression of CD86 was strongly suppressed, whereas the fluorescence intensity of an CD206 was increased in the hippocampus and cortex of 5xFAD mice after TMAS+PT-MNBs stimulation (Figure A–D); moreover, TMAS+PT-MNBs treatment strongly inhibited microglial somatic hypertrophy and the adoption of an amoeboid morphology, in addition to decreasing the size of microglia and causing them to exhibit a rounder shape, similar to that of resting microglia (Figure A–D). Immunohistochemistry revealed that the overactivated inflammatory microglia was significantly attenuated after TMAS+PT-MNBs stimulation (Figure E), and the complexity of microglial branches 16- and 20 μm from the nucleus was significantly greater in the AD+TMAS+PT-MNBs group than in the AD+TMAS group (Figure F,G). These results indicate that TMAS+PT-MNBs stimulation can increase branching complexity and reduce the overactivation of microglia and that PT-MNBs clearly enhance the effect of TMAS (Figure E–G). Furthermore, the release of proinflammatory cytokines, including TNF-α, IL-1β and IL-6, was strongly suppressed, whereas the release of anti-inflammatory cytokines, including IL-4, IL-10 and IL-13, was increased in the brains of 5xFAD mice after TMAS+PT-MNBs stimulation (Figure H–J, Supplementary Figure 5A). BV-2 cells as mature microglia and treated them with Aβ to induce an in vitro inflammatory model of AD. The results revealed that TMAS+PT-MNBs stimulation modulated microglial polarization and reduced the secretion of inflammatory cytokines in BV-2 cells (Supplementary Figure 5B–H)). Importantly, we detected both a significant colocalization of microglia with TREM2 and Aβ and an increase in the levels of disease-related microglial (DAM) markers (TREM2 and APOE) in the hippocampal tissues of the AD + TMAS + PT-MNBs group compared with those in the AD group(Supplementary Figure 5I,J)). In brief, these results indicate that the therapeutic effect of TMAS+PT-MNBs treatment is at least partially mediated by the redirection of microglial activation toward the beneficial DAM phenotype, which not only suppresses inflammatory responses in the brain but also actively promotes microglia to enter a reparative state capable of phagocytosing and clearing Aβ

TMAS-actuated PT-MNBs regulated microglial polarization and suppressed the release of inflammatory factors. A,B The images of Iba1 (red) and CD68 (green) of the mice in each group. Scale bar, 50 μm. C,D Representative images of Iba1 (red) and CD206 (green) expression in the hippocampus and cortex of the mice in each group. Scale bar, 50 μm. E Representative immunohistochemical staining of microglia in the hippocampus. Scale bar, 100 μm. F Magnified image showing microglial morphology in the hippocampal region. G Number of microglia branches in the hippocampus (n = 5 mice). H Representative Western blot bands of inflammatory cytokines in the hippocampus. I,J Relative mRNA levels of inflammatory cytokines in the hippocampus and cortex of the mice in each group (n = 5 mice). *p < 0.05, **p < 0.01, ***p < 0.001 vs the AD group. #p < 0.05 vs the AD + TMAS group.

Pyroptosis is an inflammation-induced form of cell death, and the NLRP3 inflammasome accelerates microglial secretion of inflammatory factors and triggers pyroptosis, resulting in impaired microglial phagocytosis and clearance in AD. Piezo1/NLRP3 can mediate pyroptosis in macrophages. Therefore, we further investigated how TMAS-actuated PT-MNBs relieve neuroinflammation upon activation of Piezo1. Our results revealed that the levels of pyroptosis proteins, including NLRP3, GSDMD, ASC, Caspase1-p20, IL-1β and IL-18, were decreased after TMAS+PT-MNBs stimulation, and the fluorescence intensity of ASC on microglia was also attenuated after TMAS+PT-MNBs stimulation (Supplementary Figure 6A–C)). Our previous study demonstrated that Piezo1 can activate microglial autophagy, thereby promoting Aβ clearance and maintaining brain homeostasis On the other hand, microglial autophagy inhibits the expression of NLRP3 inflammasome components and the secretion of mature IL-1β and IL-18 through the phagocytosis of Aβ. The levels of autophagy-related proteins, including LC3, Beclin-1 and p62, were significantly changed (Supplementary Figure 6D–G)), and the numbers of autophagosomes and lysosomes were increased in the hippocampus after TMAS+PT-MNBs treatment(Supplementary Figure 6H). Taken together, these results may prove to some extent that TMAS+PT-MNBs stimulation can induce microglial autophagy to inhibit pyroptosis through the phagocytosis of harmful substances such as Aβ, thereby attenuating neuroinflammation in 5xFAD mice.

The Effects of TMAS-Actuated PT-MNBs Involving Piezo1 Activation

To clarify the mechanism by which TMAS + PT-MNBs stimulation alters cognitive function, we assessed gene changes at the transcriptome level in the hippocampus via RNA-sequencing analysis (Figure A). Compared with the AD group, the TMAS+PT-MNBs-treated group presented 2781 diverse genes (DEGs), among which 1321 were upregulated and 1460 were downregulated (Figure B,C). Notably, TMAS+PT-MNBs treatment significantly upregulated Piezo1 levels in the hippocampi of 5xFAD mice (Figure D). The GO enrichment analysis further showed that these DEGs were associated with the following biological processes: cognition, synaptic plasticity, calcium ion transmembrane transport, microglial cell activation, phagocytosis, the cellular response to Aβ, the inflammatory response and autophagy (Figure E–G), which was consistent with our findings. We further analyzed the RNA-sequencing data and screened the up- and downregulated genes highlighted in the hierarchical cluster analysis. With the upregulation of Piezo1 expression, genes associated with cognition, learning and memory and synapses were upregulated, and these genes participated in the TMAS+PT-MNBs treatment-induced changes in cognitive function in 5xFAD mice (Figure H). After TMAS+PT-MNBs stimulation, the levels of proinflammatory and anti-inflammatory-related genes were downregulated and upregulated, respectively (Figure I), illustrating the ability of TMAS-actuated PT-MNBs treatment to reduce neuroinflammation and regulate the immune microenvironment of the brain. Under the action of TMAS+PT-MNBs, autophagy- and pyroptosis-related genes were also up- and downregulated, respectively (Figure J,K). These results suggest that the effect of TMAS+PT-MNB treatment involves the activation of Piezo1, which in turn triggers a series of biological processes of 5xFAD mice.

Therapeutic effects of TMAS-actuated PT-MNBs in 5xFAD mice involve the activation of Piezo1 (n = 4). A Schematic of the RNA sequencing analysis. B Changes in gene expression in the hippocampus of 5xFAD mice after TMAS+PT-MNBs stimulation. C Heatmap showing DEGs in 5xFAD mice after TMAS+PT-MNBs stimulation. D RT-qPCR and RNA sequencing were used to analyze the expression of Piezo1 (n = 5). E–G GO enrichment analysis of the biological processes associated with the DEGs after TMAS+PT-MNBs stimulation. H–K Heatmap showing the changes in the expression of genes related to Piezo1; cognition; learning and memory; synapse; proinflammatory and anti-inflammatory; autophagy; phagocytosis; and pyroptosis-related genes after TMAS+PT-MNBs stimulation. Genes with a log2-fold change >0 and p < 0.05 were considered differentially expressed.

Piezo1 Knockdown Inhibits the Neuromodulatory Effect of TMAS+PT-MNBs Stimulation in 5xFAD Mice

To understand whether Piezo1 is a crucial modulator of the regulatory effect of TMAS+PT-MNB treatment and whether Piezo1 is a target, either directly or indirectly, of TMAS and PT-MNBs in the brains of 5xFAD mice, we knocked down Piezo1 by stereotaxically injecting an adeno-associated virus (AAV) into the hippocampus of 5xFAD mice 2 weeks before TMAS+PT-MNBs treatment (Figure A). A control vector, AAV-shCtrl, was injected into control animals in the same manner. Behavioral and in vivo electrophysiological tests were then performed after TMAS+PT-MNBs stimulation. We confirmed that, compared with AAV-shCtrl-treated 5xFAD mice, AAV-shPiezo1-treated 5xFAD mice required more to search for the hidden platform in the MWM test (Figure B) and exhibited decreases in the RI and percentage of spontaneous alternations (Supplementary Figure 7A–E)). Furthermore, TMAS+PT-MNBs-induced synaptic plasticity of the hippocampal perforant pathway (PP)-DG circuit was strongly attenuated in AAV-shPiezo1-treated mice compared with that in AAV-shCtrl-treated mice Figure C, Supplementary Figure 7F,G); moreover, the spine density and number of neuronal synapses were reduced and the cytoskeleton structure was disrupted (Figure D–G), indicating that Piezo1 knockdown inhibited the improvement in spatial cognition and increase in synaptic plasticity in 5xFAD mice caused by TMAS+PT-MNBs stimulation. Immunofluorescence and quantitative real-time PCR analysis revealed that AAV-shPiezo1 administration suppressed the increase in Piezo1 expression in microglia induced by TMAS+PT-MNBs stimulation (Figure H,I). Interestingly, even after TMAS+PT-MNBs treatment, the Aβ level was further elevated in the brains of the AAV-shPiezo1-treated mice compared with those of the AAV-shCtrl-treated mice (Figure J-L). The aggregation and microglia-mediated phagocytosis of Aβ were diminished, resulting in the proinflammatory polarization of microglia and increased secretion of inflammatory factors (Supplementary Figure 7H–K)). The numbers of autophagosomes and lysosomes in the hippocampus were decreased in the AAV-shPiezo1-treated mice(Supplementary Figure 7L), and the expression levels of an autophagy marker (LC3) and a pyroptosis marker (ASC) in microglia were decreased and increased, respectively, in the AAV-shPiezo1-treated mice (Supplementary Figure 7M,N)), indicating that Piezo1 knockdown inhibited the restoration of microglial function caused by TMAS+PT-MNBs stimulation, including the inhibition of Aβ phagocytosis and clearance and the activation of neuroinflammation in the brain. These results provide further confirmation that microglial Piezo1 is an intrinsic mediator of the neuromodulatory effect of TMAS+PT-MNBs treatment and a target of TMAS and PT-MNBs in the brains of 5xFAD mice

Hippocampal Piezo1 knockdown inhibited the effect of TMAS+PT-MNBs stimulation in 5xFAD mice. A Schematic illustration of the experimental procedure. B Escape latencies of the MWM test in the training and exploratory phases (n = 8). C Changes in LTP and DEP slopes (n = 8). D,E Representative images of dendritic spines and statistical analysis of the density of hippocampal neurons (n = 8). Scale bar, 10 μm. F Representative immunofluorescence images of NeuN and MAP2 expression in the brain. Scale bar, 50 μm. G TEM images of hippocampal neuron synaptic structures. Dashed boxes: synapses. Scale bar, 200 μm. H Images of Iba1 (red) and Piezo1 (green) in the hippocampus of 5xFAD mice. Scale bar, 50 μm. I mRNA levels of Piezo1 in the hippocampus of 5xFAD mice (n = 5). J Images of Aβ (green) and Iba1 (red) in the hippocampus of 5xFAD mice. Scale bar, 25 μm. K Quantification of Iba1 and Aβ overlap in plaques of different sizes (n = 5). L Immunohistochemical staining of Aβ in the brains of 5xFAD mice. Scale bar, 20 μm. ^& p < 0.05, ^&& p < 0.01 vs the AAV-shCtrl group.

Discussion

AD is a complex brain disease with neuropathological characteristics that include Aβ accumulation and microglial clearance dysfunction. , Our study establishes a synergistic therapeutic paradigm involving TMAS-driven PT-MNBs, which precisely restore Piezo1 channel function through noninvasive bimodal stimulation, thereby modulating microglial function and alleviating AD pathology. Compared with conventional Piezo1 agonists (e.g., Yoda1), which have certain limitations, including a short half-life and poor brain permeability, this paradigm offers three principal innovations. (1) The energy of TMAS is focused and amplified by PT-MNBs, achieving spatiotemporally controlled noninvasive stimulation of deep hippocampal regions with <2 mm spatial resolution. (2) PT-MNBs are dually functionalized with phosphatidylserine (PS) and anti-Piezo1 antibodies, enabling them to achieve microglia-specific anchoring and the precise delivery of mechano-electrical signals to Piezo1, which synchronously activates its ion channel domains. (3) Piezo1 activation drives Ca² ⁺ influx and reprograms microglia into a neuroprotective phagocytic state, increasing Aβ clearance and alleviating neuroinflammation and neuronal damage in 5xFAD mice (Supplementary Figure 8). Piezo1 knockdown significantly reduced TMAS-driven PT-MNBs-induced synaptic plasticity responses, neuronal long-term potentiation (LTP) and spatial cognition in 5xFAD mice. This paradigm converts nanomaterial-mediated physical energy into cell- type-specific biological effects for “precision energy medicine”. Additionally, the ease of the MNBs’ surface modification allows cell targeting by attaching ligands or other targeting molecules, which could be applied in fields beyond neurostimulation, particularly oncology (for tumor ablation) and immunology (for immune cell activation).

TMAS has emerged as an approach for the noninvasive physical regulation of the nervous system. ,− The physical basis for TMAS is related to the magnetoacoustic coupling action of conductive tissue. Applying focused ultrasound irradiation to tissues in a stable magnetic field causes the vibration of conductive particles inside the tissue. These vibrating particles are also susceptible to the Lorentz force in the magnetic field, and a locally induced electric field is formed in the direction of the vector produced by the stable magnetic field and the acoustic field in the ultrasonic focusing region. ,, We previously reported that TMAS can regulate activity in the mouse motor cortex, eliciting an EMG amplitude (2.73 ± 0.32 mV); TMAS can also increase the neuronal expression of c-Fos, CaMKII and CREB to modulate synaptic plasticity and neural rhythmic oscillations with high spatial resolution and focus in the hippocampi of mice. ,, However, the heterogeneity of different brain regions and the presence of the skull may lead to insufficient mechanical or electrical sensitivity in certain areas of the brain, restricting the efficiency of energy delivery to Aβ-enriched regions. , To address this, we introduced PT-MNBs as energy transduction amplifiers to achieve deep and accurate neurostimulation upon the application of TMAS. PT-MNBs are unique gas-rich magnetic protein nanostructures with excellent acoustic and magnetic properties. The perfluoropropane (C₃F₈) gas core of the PT-MNBs generates a stable cavitation effect under ultrasonic fields, increasing local mechanical forces at the nanobubble–cell interface. In addition to its proven biocompatibility and clinical approval as an ultrasound contrast agent (e.g., Sonazoid), C₃F₈ has a low diffusion coefficient that enhances its in vivo stability and extends its circulation half-life. The Fe₃O₄ nanoparticles on their shells further increase the electric field strength of TMAS through magnetoacoustic coupling. Owing to functionalization with both anti-Piezo1 antibodies and PS, the PT-MNBs establish nanoscale energy-focused centers at the microglia–Aβ plaque interface, endowing target cells with sensitivity to the mechanical and electrical stimulation of TMAS to effectively regulate cellular functions. Moreover, since only cells near the PT-MNBs would be stimulated by TMAS, the therapeutic efficacy could be improved and the off-target effects of TMAS could be reduced even if the focal spot is not easily controllable.

Microglia are crucial phagocytic and immune cells that are involved in the development of AD pathology; specifically, the inability of microglia to clear harmful Aβ deposits and regulate inflammation is considered important factors in AD pathology. , Piezo1, an ion channel sensitive to both mechanical and electrical stimuli, serves not only as a biosensor of Aβ plaque stiffness but also as a crucial effector that regulates microglia-mediated Aβ clearance. , The unique structure of this channel enables it to respond to diverse physical stimuli (mechanical, electrical, and magnetic), efficiently converting extracellular physical signals into intracellular molecular cascades. ,,− We have demonstrated that Piezo1 functions as both a TMAS energy transducer and therapeutic target, particularly in terms of restoring microglial Aβ clearance capacity. TMAS-driven PT-MNBs generate spatiotemporally synchronized mechanical oscillations and localized electric fields, leveraging anti-Piezo1 antibody-mediated anchoring to achieve efficient Piezo1 channel gating. Activated Piezo1 triggers Ca²⁺ influx, which enhances the mechanosensation of Aβ stiffness, thereby orchestrating microglial recruitment, compaction, and phagocytosis of Aβ plaques. This successfully overcomes the problem of cellular heterogeneity and achieves both mechanoelectrical activation of microglial Piezo1 and Aβ plaque clearance, providing spatiotemporally precise and cell type-selective neuromodulation. Moreover, TMAS-actuated PT-MNBs promote microglial autophagy and inhibit NLRP3 inflammasome activation, rebalancing microglial responses from proinflammatory to phagocytic states and reshaping the immune microenvironment of the brain in 5xFAD mice. This consequently improves synaptic plasticity and spatial cognitive function by significantly enhancing hippocampal neurogenesis, axonal growth, and LTP. These findings demonstrate that, as surface-functionalized magnetic force actuators, PT-MNBs can increase the effectiveness and specificity of TMAS in regulating neural activity in AD model mice and that Piezo1 is the main mediator of this process. In addition, the Piezo1-mediated currents enabled through TMAS combined PT-MNBs treatment could be significantly attenuated by suppressing Piezo1 ion channel activity; however, a small amount of Ca²⁺ influx was still detected, suggesting that the intrinsic properties of the MNBs, including their mechanical vibrations and magnetic signatures, may also induce changes in Ca²⁺ influx through other sensitive ion channels under the action of TMAS, albeit to a lesser extent. Therefore, additional evidence, such as calcium imaging with different sensitive channel blockers, is needed to elucidate the contributions of other sensitive ion channels. MNBs are unique gas-rich magnetic protein nanostructures with excellent acoustic and magnetic properties that can activate Piezo1 under the action of TMAS (Supplementary Figure 9A). However, this effect was weak, spatially diffuse and not effectively focused on the Piezo1 channel. The antibody-guided energy focusing of PT-MNBs, which significantly enhances the efficiency of Piezo1 activation (vs unmodified MNBs, Supplementary Figure 9A,B), enables synergistic TMAS mechano-electrical stimulation and is essential for restoring microglial Aβ clearance. While we identified Piezo1 as the mechanical- and voltage-sensitive ion channel involved in the TMAS-mediated response of PT-MNBs in the brain, we speculate that Piezo1 channels may also amplify the effect of this treatment modality by cooperating with other ion channels since the neuromodulatory effects of TMAS+PT-MNB treatment were not completely eliminated in the AAV-shPiezo1 group; for example, TRPP1/2, TRPC1 or TRPV4 may contribute to the Piezo1-mediated neuromodulatory effects of TMAS+PT-MNBs treatment by enhancing Piezo1 function. , Therefore, elucidating the effects of TMAS on other ion channels will further expand its application for neuromodulation in the future.

In our study, we observed no side effects of TMAS actuated PT-MNBs on neurons or microglia in vitro, in brain tissue or in other major organs (Supplementary Figure 1I,J)), indicating that the PT-MNBs were well tolerated and did not cause apparent cell damage. Additionally, mechanical and thermal damage may be reduced by performing TMAS at a low intensity to prevent inertial cavitation and persistent overheating. For this reason, we limited the application of TMAS to a low ultrasound intensity, i.e., 0.5 MPa, which is also within the range of parameters valid for most previously reported neuromodulations in the brain (<0.6 MPa) − The electric field strength generated by TMAS in the brains of mice was approximately 100 mV/m, which is a subthreshold stimulus and within the range of effective electrical stimulation strengths for in vivo studies (10–338 mV/m). − Despite the size difference between nanomaterials (diameter ∼100 nm) and ion channels (diameter ∼1 nm), nanomaterials can modulate ion channel activity through local energy fields (mechanical, electromagnetic and thermal) without the need for direct size matching; we will also subsequently investigate the effect of PT-MNBs of different sizes on magnetoacoustic energy transfer to Piezo1 to determine the optimal size of PT-MNBs in response to a physical field. In addition, the ease of MNBs surface modification could enable cellular targeting by attaching ligands or other targeting molecules for the treatment of Parkinson’s disease or brain tumors or even in applications beyond neurostimulation. Overall, the TMAS-driven PT-MNBs platform establishes a transformative paradigm for bridging physical energy fields with cellular mechanoelectrical responses, and the integration of the field responsiveness of PT-MNBs with the noninvasive deep brain stimulation capabilities of TMAS offers many opportunities and advantages for precision neuromodulation for AD treatment.

Study Limitations

While this study demonstrates the promising therapeutic potential of TMAS-driven PT-MNBs for ameliorating AD pathologies, several limitations should be acknowledged. First, the precise biophysical transduction mechanism linking TMAS-driven PT-MNBs-induced electromechanical stimulation to Piezo1 channel activation warrants further elucidation. Although we have established a strong correlation, directly visualizing the nanoscale mechanical deformation of the cell membrane or the specific conformational changes in the Piezo1 protein in real-time remains technically challenging. A related challenge is the inherent difficulty in decoupling the contributions of the individual physical fields (acoustic and magnetic). The coupled nature of multimodal stimulation means that simply switching off one field may alter the overall biophysical environment. Therefore, more complex experimental designs or theoretical models are needed to define the unique parameters of each field responsible for channel gating. Second, our mechanistic exploration, while underscoring the critical role of microglial Piezo1, does not fully rule out the potential contribution of other cell types (e.g., astrocytes and neurons) or alternative mechano-/electrosensitive pathways that might be comodulated by our intervention and contribute to the observed neuroprotective outcomes. Third, the overall targeting efficiency and heterogeneous distribution of PT-MNBs within the complex AD brain microenvironment present considerable challenges. While our results confirm increased overall brain accumulation with TMAS, the precise fraction of the injected dose that effectively reaches and enters the intended microglia in different brain regions, especially in areas with varying degrees of pathology, remains to be precisely quantified and optimized. Finally, the clinical application of TMAS parameters requires further investigation. Our current study was conducted on a mouse model with a relatively thin skull, and the penetration depth and focusing efficiency of the magnetoacoustic energy in a larger, human brain need to be thoroughly evaluated through sophisticated computational modeling and large-animal studies. Addressing these limitations in future work will be crucial for advancing this strategy toward clinical application.

Conclusion

AD is a complex brain disease with neuropathological characteristics that include Aβ accumulation and microglial clearance dysfunction. Our study establishes a synergistic therapeutic paradigm involving TMAS-driven PT-MNBs, which precisely restore Piezo1 channel function through noninvasive bimodal stimulation, thereby modulating microglial function and alleviating AD pathology. This paradigm offers three principal innovations. (1) The energy of TMAS is focused and amplified by PT-MNBs, achieving spatiotemporally controlled noninvasive stimulation of deep hippocampal regions with <2 mm spatial resolution. (2) PT-MNBs are dually functionalized with phosphatidylserine (PS) and anti-Piezo1 antibodies, enabling them to achieve microglia-specific anchoring and the precise delivery of mechano-electrical signals to Piezo1, which synchronously activates its ion channel domains. (3) Piezo1 activation drives Ca² ⁺ influx and reprograms microglia into a neuroprotective phagocytic state, increasing Aβ clearance and alleviating neuroinflammation and neuronal damage in 5xFAD mice. Piezo1 knockdown significantly reduced TMAS-driven PT-MNBs-induced synaptic plasticity responses, neuronal LTP and spatial cognition in 5xFAD mice. This paradigm converts nanomaterial-mediated physical energy into cell- type-specific biological effects, realizing “precision energy medicine” by which the integration of neuromodulation techniques enables spatiotemporally precise regulation of deep brain regions, offering broad therapeutic potential for neurological and psychiatric disorders.

Methods

Preparation and Characterization of PT-MNBs

2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), and 1, 2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) were mixed in EtOH at a ratio of 50%:40%:5%:5% to prepare a lipid solution. The solution was mixed with high-purity water (1 m) containing dispersed superparamagnetic iron oxide via bath sonication (60 °C, 5 min), after which the organic solvent was removed via dialysis, yielding dispersions of PS-coated superparamagnetic iron oxide particles. The dispersions were mixed with glycerol and 1, 2-propylene glycol at a ratio of 80%:10%:10% in 3-ml glass vials, mixed with perfluoropropane (C₃F₈) in glass vials and mechanically oscillated for 45 s to obtain biotinylate magnetic nanobubbles coated with PS (MNBs). Finally, the biotinylated MNBs were used to prepare PT-MNBs via avidin–biotin coupling. Briefly, 30 μg of avidin was added to dispersions containing 10⁸ MNBs, followed by incubation with 3 μg of biotinylated anti-Piezo1 antibodies for 30 min. All the incubations were carried out at room temperature (25 °C). To obtain Cy5.5-labeled PT-MNBs, 8 μL of Cy5.5 (Thermo Fisher Scientific, US) was mixed with the dispersions, and rotary evaporation (60 °C, 40 min) was performed. Then, Cy5.5-labeled PT-MNBs were prepared in the same way as PT-MNBs in the dark.

The surface topography and structural features of the PT-MNBs were characterized via TEM (TF20, FEI, US). The size and zeta potential of the PT-MNBs were measured on a Zetasizer Nano ZS instrument (NanoBrook 90plus PALS, Brookhaven, US). Energy dispersive spectrometry mapping (TF20, US) revealed that the PT-MNBs had a homogeneous elemental distribution. XPS was applied to evaluate the functional groups of the PT-MNBs using a photoelectron spectrometer (Thermo Scientific Nexsa G, K-Alpha, US). FTIR spectra were obtained using a Vertex 70 spectrophotometer (Bruker Optics). TGA was performed with a thermogravimetric analyzer (TG 209 F3 Tarsus, Netzsch, Germany). The magnetic hysteresis curves of the PT-MNBs were obtained using a vibrating sample magnetometer (PPMS-9T, Quantum Design, US). The magnetic response of the PT-MNBs was verified with a static magnet. Echo analysis (Ultrasound System, Esaote, Italy) was used to evaluate the acoustic response of the PT-MNBs.

Characterization of the Acoustic Field and Magnetoacoustic Field in the Mouse Brain

The TMAS system used in this study was an integrated setup where the ultrasound and magnetic field components were electronically synchronized and physically coapplied to function as a unified device. The TMAS system consisted of 2 function generators (TFG6920A, Shuying, China; AFG3252, Tektronix, United States), an RF amplifier (GA2500, RITEC, United States), a 0.5 MHz focused US transducer (FP-1M, IOA-AC, China; Supplementary Figure 10) and an oscilloscope (MSO4104, Tektronix, United States). The acoustic intensity was measured with a calibrated hydrophone (NH-1000, PA, UK). A permanent magnet generated a static magnetic field for TMAS, and the strength of this field was tested using a Gauss meter (Lakeshore, US). The magnetic field at the target site was approximately 0.3 T. We employed a 0.5 MHz focused US transducer (FP-1M, IOA-AC, China), featuring a center frequency of 0.5 MHz and a focal spot size of ∼ 1.04 mm (Supplementary Figure 10), allowing the accurate stimulation of the bilateral hippocampus in mice. Two miniature electrode plates were implanted in the hippocampus of the mice and connected to amplifiers, filters and oscilloscopes to measure the magnetoacousticelectric field intensity in the brain under the action of the TMAS.

3D Finite Element Simulations

Multiphysics simulations were executed using the three-dimensional finite element analysis software COMSOL Multiphysics (v6.0). Based on computational acoustics methods, the acoustic field generated by the focused ultrasound transducer was simulated via the acoustic simulation module. Using the Hall voltage formulation, a numerical calculation module for Hall voltage was integrated into COMSOL to model the magneto-acoustic coupling electric field and current density. The parameters used in the simulation, including ultrasonic transducer specifications, magnetostatic field parameters, properties of PT-MNBs, and the acoustic and electrical properties of the realistic mouse brain model, were set to optimally match actual small animal experimental conditions.

Cell Culture and Stimulation

The cells were cultivated in a sterile incubator at 37 °C, in 5%CO2 and at a constant pH (pH: 7.2–7.4). BV2 microglia and HT-22 hippocampal neurons were grown in DMEM supplemented with 10% FBS and 1% PS. The medium was replaced every 2 days to ensure nutrient availability and the cells were passaged for 2 or 3 days to ensure adequate space for growth. The cells were cultured in confocal dishes, and PT-MNBs were carefully added to and mixed with the medium prior to stimulation. The stimulation parameters for TMAS were as follows: burst repetition frequency of 1-Hz, 10 ms bursts, total duration of 120 s, peak positive acoustic pressure of 0.5 MPa, and duration of 2 min. The use of these parameters allowed TMAS to be applied in short bursts, thus reducing thermal effects. To construct an in vitro model of AD, cells were exposed to HiLyte FluorTM 555-labeled Aβ at a concentration of 500 nM (AS-60480–01, AnaSpec, Inc.) or Aβ protein fragments at a concentration of 300 nM (A834109, Macklin Biochemical Co., Ltd.) for 12 h and then subjected to TMAS.

Determination of the Targeting Ability and Cytotoxicity of PT-MNBs In Vitro

BV2 cells were cultured with 100 μL of Cy5.5-labeled PT-MNBs and (Thermo Fisher Scientific, US) for 90 min, stained with Hoechst 33342 (Beyotime, China), and visualized with a laser confocal scanning microscope (LSM710, Germany). The cytotoxicity of PT-MNBs to cultured hippocampal neuronal cells was assessed using MTT assays. Neurons were treated with PT-MNBs in LEDTect 96-well plates, 0.4 mg/mL MTT was added to the medium, and the mixture was mixed for 6 h, and the absorbance was read at 570 nm.

Transfection with SiRNA

Piezo1-specific siRNA (si-Piezo1) can specifically inhibit the expression of the Piezo1 through the RNA interference mechanism. si-Piezo1 and negative control siRNAs were purchased from GenePharma and dissolved in 5 × siRNA buffer. BV2 microglia were transfected with 50 nM si-Piezo1 via Lipofectamine RNAiMAX (Thermo Fisher Scientific). The cells were collected 24 h post transfection and used for the indicated experiments.

Live-Cell Calcium Imaging

The intracellular Ca²⁺ dynamics of BV2 microglia were monitored by imaging Ca²⁺ influx during TMAS+PT-MNBs treatment. Briefly, the medium was changed to Ca²⁺ imaging buffer supplemented with 2.5 μM Fura-2 (Invitrogen) and 0.05% Pluronic F-127 (Invitrogen), after which the cells were cultured at 37 °C for 30 min in the dark The cells were then rinsed with Ca²⁺ imaging buffer to remove the residual dye prior to TMAS, and PT-MNBs were added to the medium prior to TMAS. Intracellular images of Fura-2 were taken at fixed time intervals by a Nikon Eclipse Ti2-E microscope at an excitation wavelength of 340–380 nm. An intracellular calcium ion probe (BBcellProbe F03, China) was also used for Ca²⁺ imaging, and images were obtained using confocal microscopy at an excitation wavelength of 505 nm.

Whole-Cell Patch-Clamp Experiment

Piezo1 currents were recorded using the whole-cell patch-clamp technique. Patch pipet electrodes were fabricated from borosilicate glass capillaries using a micropipette puller (Model P-97, Sutter Instrument, USA). The recording electrodes (3–5 MΩ) were filled with an internal solution containing (in mM): 133 CsCl, 5 EGTA, 10 HEPES, 1 CaCl₂, 1 MgCl₂, 4 MgATP, and 0.4 Na₂GTP (the pH was adjusted to 7.3 with CsOH). The extracellular solution contained (in mM): 10 glucose, 127 NaCl, 3 KCl, 1 MgCl₂, 10 HEPES, and 2.5 CaCl₂ (the pH was adjusted to 7.3 with NaOH). Cells were plated in culture dishes and incubated with PT-MNBs for 30 min prior to recording. After the whole-cell configuration was achieved, a standardized 5 min period was allowed for intracellular stabilization. TMAS stimulation was applied while the membrane current was continuously acquired with the voltage clamped at – 50 mV, ensuring that there was no significant lag between the stimulation and the acquisition of the electrophysiological response. Data were sampled at 10 kHz and low-pass filtered at 2 kHz using an Axopatch 200B amplifier (Axon Instruments) and a data collection system (DigiData 1322A, Axon Instruments). For data analysis, the acquired current traces were processed using Clampfit software, where the peak current amplitude during stimulation was quantified and normalized to the cell membrane capacitance to yield the current density for statistical comparison.

Transwell Assay

The cells were plated in a Costar Transwell plate and grown for 12 h at 37 °C in an incubator. Briefly, the cells in each group were fixed with 4% PFA for 20 min and dyed with 0.5% crystal violet for 10 min. The cells in the upper chamber were considered nonmigrating cells and were removed. Images were acquired on a Leica microscope (Wetzlar, Germany).

Animal Care

5xFAD mice show AD-like pathologies in a stage-dependent manner, with Aβ deposition at 1.5–2 months of age, with microglial proliferation and neuroinflammation at 3 months of age, followed by progressive cognitive decline at 4 months of age. 5xFAD transgenic mice and wild-type mice were obtained from Beijing HFK BioTechnology Co. Ltd. (China). 5xFAD mice overexpress the human APP and PSEN1 proteins under the control of the Thy1.2 promoter with 5 AD-associated mutations: the Swedish (K670N/M671L), Florida (I716 V) and London (V717I) mutations in APP and the M146L and L286 V mutations in PSEN1. The accumulation of these five FAD mutations affects mainly the production of Aβ, causing the mice to express high levels of Aβ₄₂. All the mice were housed at an appropriate temperature (23–25 °C) and humidity (50–60%) at the Chinese Academy of Medical Sciences and Peking Union Medical College. All the mice were provided adequate food and water. All experimental protocols were performed in accordance with the regulations of the Animal Care and Ethics Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College (SYXK: 2025–0002). In addition, every effort was made to reduce harm to the mice and to minimize the number of animals used in the study.

In Vivo Stimulation of TMAS

During stimulation, the mice were anaesthetized with 1% isoflurane gas. Sterile medical ultrasound gel was applied between the ultrasound transducer and the skull to ensure efficient energy delivery. Each mouse was gently restrained, and PT-MNBs (0.03 mL/kg, IV) were injected into the lateral tail vein using a 30G needle, ensuring a slow and constant administration rate. After the coordinates corresponding to the bilateral hippocampus (AP: 2.0 mm, ML: ± 1.4 mm, DV: 1.5 mm) were located, and TMAS (burst repetition frequency of 1 Hz, 10 ms bursts, total duration of 120 s, average peak pressure of 0.5 MPa, magnetic field strength of 0.3 T) was applied to the bilateral hippocampus for 5 min (Figure B). A single transducer was used to sequentially stimulate the hippocampus on each side. The mice were treated for 6 weeks and then used in follow-up experiments. EB extravasation was measured to monitor BBB permeability in the region of interest. The Cy5.5 fluorescence intensity in the brain regions into which Cy5.5-labeled PT-MNBs were injected was assessed at excitation and emission wavelengths of 710 and 780 nm, respectively.

Stereotactic Virus Injection

Since 5xFAD mice show full-blown Aβ deposition, microglial dysfunction and spatial cognitive impairment at 4 months of age, stereotactic virus injection can be performed in these mice at this age. Four-month-old 5xFAD mice were anaesthetized with 30% urethane (0.4 mL/kg, i.p.) and then immobilized in a stereotaxic instrument (SN-3, Narishige, Japan). A total of 1 μL of AAV9-shPiezo1 or AAV9-shCtrl (BrainVTA Co. Ltd., China) was injected into the bilateral hippocampus (AP: 2.0 mm, ML: ± 1.4 mm, DV: 1.5 mm) of 5xFAD mice at a rate of 0.05 μL/min 14 days before TMAS. The microinjector was kept in place in the brain for 10 min to enable the virus to spread and then carefully retracted.

In Vivo Electrophysiological Recordings

In vivo electrophysiological recordings were performed to record synaptic transmission between neurons in the PP–DG circuit. Twenty-4 h after the last treatment, the mice were subjected to the standard procedure for in vivo electrophysiological recordings (Supplementary Figure 2N). Briefly, the mice were anaesthetized with 30% urethane (0.4 mL/kg, i.p.), and body temperature was maintained via a heating pad. Stimulating and recording electrodes were positioned in the PP (AP: 3.8 mm, ML: 3 mm, DV: 1.5 mm) and DG (AP: 2.0 mm, ML: 1.4 mm, DV: 1.5 mm) of the hippocampus. First, the response of the PP to a single stimulus at an intensity (0.3–0.5 mA, 0.2 ms, 0.03 Hz) that elicited 70% of the maximum response was captured every 30 s for 30 min as the baseline response. Then, high-frequency theta burst stimulation (TBS; 30 trains of 12 pulses, 200 at 5 Hz) and low-frequency stimulation (LFS; 1 Hz, 15 min) were applied to evoke LTP and DEP, respectively. All the data were monitored every 60 s for 1 h. Clampfit 10.0 software (Molecular Devices, Sunnyvale, CA, USA) was used for initial data processing. After the electrophysiological experiment, the mice were immediately sacrificed, and the brains were collected and processed for subsequent analyses.

In Vivo Fiber Photometry Recording

For microinjection and optical fiber implantation, the mice were anaesthetized and fixed in a stereotaxic instrument. A total of 2.5 μL of rAAV-CX3CR1-GCaMP6f-WPRE-polyA (BrainVTA Co. Ltd., China) was delivered into the hippocampal DG area at a rate of 30–50 nL/min with a microsyringe (Hamilton, 33GA, Switzerland). For in vivo fiber photometry recording, unilateral optic fibers (RWD Life Science, O.D.: 1.25 mm, core: 200 μm, NA: 0.39) were then implanted into the hippocampal DG region and secured with dental cement.

For optical fiber recording, 3 weeks after implantation and recovery, the mice were subjected to TMAS+ PT-MNB treatment. The fluorescence intensity was recorded using an optical fiber recording system (RWD Life Science, China) at an excitation wavelength of 470 nm.

NOR Test

The NOR test was conducted using a 35 × 35 × 25 cm open field apparatus. First, the training phase began, and the mice were allowed to move freely for 5 min in a box containing two similar objects. After 2 h, Test 1 was administered, and the mice continued to explore both objects for 5 min following the replacement of one familiar object with a novel one. After 24 h, in Test 2, the novel object was displaced with another novel object, and the same experimental procedures were performed as in Test 1. The RI was determined using the following equation: Time_novel/(Time_novel + Time_old)*100% and Visits_novel/(Visits_novel + Visits_old)*100%.

Y-Maze Test

The working memory of the mice after treatment was detected using the Y-maze test. The Y-maze comprised a horizontal maze with three arms (35 × 8 × 18 cm). After being placed at the end of one arm of the Y-maze, the mice were able to move for 10 min. The total number of arm entries and the order in which the arms were entered were recorded. The spontaneous alternation percentage was determined using the following equation: (number of sequential visits to the three distinct arms/total number of visits to arm entries-2) × 100%.

MWM Test

The MWM test was conducted in a round pool (diameter: 100 cm, height: 70 cm) filled with water dyed white with nontoxic TiO₂. The temperature of the water was between 23 and 25 °C, and the water depth was 40 cm. The MWM test consisted of the initial training, spatial exploration test, reversal training and reversal exploration test phases. The mice were put into the water from the four different quadrants (I–IV) and underwent training trials (4 trials per day), during which they were allowed to search for the hidden platform for 60 s. During this time, if the mice successfully found the platform, the time was recorded as the escape latency. If the hidden platform was not found, the mouse was led to the platform, and the escape latency was recorded as 60 s. The escape latency, swimming speed and swimming path in each trial were recorded. Cognitive performance was assessed from the swimming paths according to the following criteria, with a higher score indicating better cognitive function: thigmotaxis = 1, random = 2, corrected = 3 and direct = 4. The cognitive score was calculated as = (the total score in the 4 trials/16) × 100%. During the spatial exploration phase, the platform was removed from quadrant I, and the mice were released into the pool from the platform opposite to the one that previously contained the platform (quadrant III) and allowed to swim for 60 s. In the reversal training phase, the learning flexibility of the mice was tested by placing the platform in the middle of quadrant III. The mice underwent training trials during the reversal training phase, and the reversal exploration phase was carried out after the final trial. The testing method was the same as that in the initial training phase. the assessment was carried out.

Golgi-Cox Staining

Dynamic changes in the dendritic spines of neurons were analyzed using Golgi staining. Dissected brain tissue was immersed in Golgi-Cox solution for 14 days at 23–26 °C in the dark. Coronal sections of brain tissue (150 μm thick) were prepared using a vibrating microtome (VT1000S, Leica, Germany). The sections were incubated with 6% Na₂CO₃ (20 min), 70% EtOH (10 min), 90% EtOH (15 min), 100% EtOH (20 min) and xylene (20 min). The brain sections were embedded in neutral resin, mounted on slides and coverslipped. The morphology and density of the dendritic spines were assessed via a light microscope (DM3000, Leica, Germany) and ImageJ software. To determine the number of dendritic spines, straight branches with a clear resolution and a length greater than 10 μm were counted. In addition, we calculated the proportions of four types of dendritic spines, including filopodia (long spines without a head), thin spines (long spines with a small head and a diameter not longer than 0.6 μm), mushroom spines (long spines with a large head and a diameter longer than 0.6 μm) and stubby spines (short spines without a head).

TEM

The mice were deeply anaesthetized and underwent cardiac perfusion with precooled 0.9% NaCl and 4%PFA (Solarbio, China). Hippocampal tissue was extracted, isolated, cut into 1–3 mm³ blocks and immersed in 2.5% glutaraldehyde solution overnight. The blocks were then immersed in 1% OsO4 and 1% aqueous uranyl acetate for 2 h. After dehydration with ethanol solutions, the tissue blocks were embedded in 812/Araldite and sectioned on an ultramicrotome. The sections were placed on copper grids, after which the neuronal ultrastructure, autophagosomes and lysosomes in the region of the hippocampus were observed at 100 kV with a JEOL 1400 transmission electron microscope (JEOL USA, Peabody, MA).

Histological Staining and Analysis

The mice were transcardially perfused with precooled PBS and 4% PFA under anesthesia, and the isolated tissues were placed in 4% PFA and fixed for 48 h. The tissues were then immersed in paraffin, and the sections were subjected to H&E staining for histological examination.

For immunofluorescence staining, brain tissue containing the hippocampus was cut into 35 μm coronal sections. The sections were blocked for 60 min in blocking buffer (0.5% Triton X-100 with 10% NGS) at room temperature, and then immersed in primary antibody at 4 °C for 12 h. On the second day, the brain sections were washed in PBS and stained with a conjugated fluorescent secondary antibody (A-11029 or A-27040, 1:1000, Thermo Fisher Scientific) for 1 h in the dark. The primary antibodies used included antibodies against NeuN (#24307, CST, diluted 1:500), MAP2 (PA1–10005, Invitrogen, diluted 1:1000), SYP (ab32127, Abcam, diluted 1:1000), PSD-95 (ab238135, Abcam, diluted 1:1000), NR-2B (ab65783, Abcam, diluted 1:1000), Iba-1 (019–19741, 016–26721, Wako Chemicals, diluted 1:500), Piezo1 (NBP2–75617, Novus Biologicals, diluted 1:500), Aβ (#14974, CST, diluted 1:1000), CD68 (ab53444, Abcam, diluted 1:500), TREM2 (#91068, CST, diluted 1:500), CD86 (ab213044, Abcam, diluted 1:500), CD206 (ab8918, Abcam, diluted 1:500), LC3 (83506, CST, diluted 1:1000), and ASC (#67824, CST, diluted 1:500).

For immunohistochemical staining, brain sections were cultured at 4 °C for 12 h with anti-Iba1 (ab5076, Abcam, 1:500) and anti-Aβ (#9888, CST, 1:1200) antibodies. An HRP-conjugated IgG antibody (ab205718, Abcam, 1:500) was used as a secondary antibody. Cell visualization and data analysis were conducted via microscopy (Olympus, FV1000, Japan) and ImageJ software.

Western Blotting

Mouse hippocampal and cortical tissues were dissected, incubated in 150 μL of RIPA buffer with 1% PMSF for 15 min for lysis, and centrifuged (12000 × g, 15 min) in a precooled centrifuge, after which the supernatant was obtained. The protein concentration was subsequently measured using a BCA protein assay kits (Beyotime Biotechnology, China). Samples containing equal quantities of protein (30 μg) were separated via 8–12% SDS-PAGE, transferred to PVDF membranes, and then blocked with 5% skim milk for 1 h, followed by incubation with primary antibodies. The next day, the PVDF membranes were washed and incubated with secondary antibodies. After the membranes were washed with TBST for 4 × 10 min, the protein bands were imaged using a chemiluminescence system (Tanon 5200, China). The primary antibodies used were against IL-4 (PA5–25165, Thermo Fisher, 1:1000), SYP (ab32127, Abcam, 1:1000), IL-1β (ab254360, Abcam, 1:1000), NR-2B (ab65783, Abcam, 1:1000), TNF-α (#11948, CST, 1:1000), PSD-95 (ab238135, Abcam, 1:1000), IL-6 (#12912S, CST, 1:1000), IL-10 (ab189392, Abcam, 1:1000), IL-13 (#85677, CST, 1:1000), and GAPDH (ab8245, Abcam, 1:1000). The band densities were quantified and analyzed using ImageJ software.

Real-Time Quantitative PCR (RT–qPCR)

Total RNA was isolated from mouse hippocampal and cortical tissues with TRIzol solution (Invitrogen, USA). The RNA concentration was determined with a NanoDrop spectrophotometer (Thermo Fisher Scientific, US), and then the RNA was reverse transcribed into cDNA using PrimeScript RT Master Mix (Vazyme, R323). RT-qPCR was conducted with SYBR Green I mixture (Vazyme, Q712) on a 7500 Fast Real-time PCR instrument (Applied Biosystems, USA). The sequences of the primers used were as follows: TNF-α, 5′-TGCCTATGTCTCAGCCTCTTC-3′ and 5′-GGTCTGGGCCATAGAACTGA-3′; IL-1β, 5′-ATTGTGGCTGTGGAGAAG-3′ and 5′-TTGTGAGGTGCTGATGTA-3′; IL-6, 5′-TGTGCAATGGCAATTCTGAT-3′ and 5′- GGTACTCCAGAAGACCAGAGGA-3′; IL-4, 5′-GGTCTCAACCCCCAGCTAGT-3′ and 5′-GCCGATGATCTCTCTCAAGTGAT-3′; IL-10, 5′-TGTGTCAGCCCTCAGAGTAC-3′ and 5′-CACTGACACTTCGCACAA-3′; IL-13, 5′-CAATTGCAATGCCATCTACAGGAC-3′ and 5′-CGAAACAGTTGCTTTGTGTAGCTGA-3′; Piezo1, 5′-TCATCATCCTTAACCACATGGTG-3′ and 5′-TGAAGACGATAGCTGTCATCCA-3′; TREM2, 5′-CTGATCACAGCCCTGTCCCAA-3′ and 5′-CGTCTCCCCCAGTGCTTCAA-3′; GPR34, 5′-CTTCAGGAAAGCTTCAACTC-3′ and 5′-GTAACTATCAGGAGGAGAGC-3′; P2Y6, 5′-GTGAGGATTTCAAGCGACTGC-3′ and 5′-TCCCCTCTGGCGTAGTTATAGA-3′, and GAPDH, 5′- AGGTCGGTGTGAACGGATTTG-3′ and 5′-TGTAGACCATGTAGTTGAGGTCA-3′. APOE qPCR primer pair from Beyotime Biotechnology (QM01222S). mRNA expression levels were quantified using the 2-ΔΔCT method.

Enzyme-Linked Immunosorbent Assay (ELISA)

The cell supernatant was collected and prepared for subsequent experimental analysis. The levels of TNF-α, IL-1β, IL-6, IL-10 and IL-4 in the cell supernatant were measured following the manufacturer’s instructions by using a mouse TNF-α ELISA kit (Cusabio, CSB-E04741m), mouse IL-1β ELISA kit (Cusabio, CSB-E08054m), mouse IL-6 ELISA kit (Cusabio, CSB-E04639m), mouse IL-10 ELISA kit (Elabscience, E-EL-M0046c) and mouse IL-4 ELISA kit (MultiSciences, EK204HS-48).

RNA Sequencing Analysis

RNA was isolated from mouse hippocampal tissue using TRIzol, and RNA integrity was assessed using the Agilent 2100 Bioanalyzer System. High quality samples were used for library preparation. After the quality of the library was checked, the libraries were sequenced on an Illumina NovaSeq 6000 platform (Illumina, USA), and 150 bp paired-end reads were generated. HISAT2 v2.0.5 was used to construct the reference genome and compare the paired-end clean reads to the reference genome. Differential expression analysis was performed using DESeq2 software (1.20.0). For differential gene expression analysis, the Benjamini and Hochberg method was used to adjust the P values. Genes with an adjusted P value ≤ 0.05, which was considered significant, and a |log2FoldChange|≥0.0 according to DESeq2 were considered differentially expressed. Fragments per kilobase million (FPKM) values were also used to quantify gene expression levels. GO enrichment analysis was conducted to evaluate the major biological functions of the DEGs. The DEGs were subjected to GO enrichment analysis using ClusterProfiler software (3.8.1). Terms with a P value ≤ 0.05 were regarded as significantly enriched. GSEA of the GO data was performed using the GSEA tool (http://www. broadinstitute.org/gsea/index.jsp). Heatmaps, volcano plots, bar plots and bubble plots were generated using the pheatmap, ggplot2, ggrepel and GOplot packages in R.

Statistical Analysis

All the data are expressed as means ± SEMs. SPSS 26.0 and GraphPad Prism 8 were used for statistical analysis and graph preparation. The significance of differences was assessed using one- or two-way analysis of variance (ANOVA) followed by the least significant difference (LSD) multiple comparison test. If the interaction effect was significant, Bonferroni’s post hoc correction was performed. The threshold for statistical significance was established at a p-value of 0.05. The value of “n” represents biological replicatesspecifically, the number of independent mice per experimental group. For the neuron-related analyses, we quantified 3–5 nonoverlapping visual fields per brain region from each replicate, with the final data reflecting the mean of these technical replicates per mouse. All analyses were performed using semiautomated or automated software to minimize subjective bias and were conducted by two independent researchers in a blinded fashion to ensure consistency.

Supplementary Material

nn5c13702_si_001.pdf^{(6.5MB, pdf)}

Acknowledgments

This study was supported by the National Natural Science Foundation of China (823B2028, 81927806, 52077223, and 32330059); CAMS Innovation Fund for Medical Sciences (2021-I2M-1-058 and 2022-12M-2-003); the Natural Science Foundation of Tianjin (24JCYBJC00570); State Key Laboratory of Advanced Medical Materials and Devices Research Grant (24ZXZSSS00110); Fundamental Research Funds for the Central Universities of Peking Union Medical College (3332025197); and the Postdoctoral Fellowship Program of CPSF (GZC20251897 and 2025M772880). We thank BioRender for support of the graphical abstract, Figures 1A; 2A, B, F, H; 3A; 4C; 6A; and 7A and Supplementary Figures 2A, H, K, N and 8.

Glossary

Abbreviations

ADA: Alzheimer’s disease
TMAS: Transcranial magneto-acoustic stimulation
Aβ: β-Amyloid
PT-MNBs: Piezo1-targeted magnetic nanobubbles
PS: Phosphatidylserine
MNBs: Magnetic nanobubbles
C3F8: Perfluoropropane
EDS: Energy dispersive spectrometry
XPS: X-ray photoelectron spectroscopy
TGA: Thermogravimetric analysis
EB: Evans blue
MWM: Morris water maze
NOR: Novel object recognition
RI: Recognition index
LTP: Long-term potentiation
DEP: Depotentiation
fEPSP: Field excitatory postsynaptic potential
SYP: Presynaptic vesicle membrane protein
PSD-95: Postsynaptic scaffold protein
NR 2B: N-Methyl-d-aspartate receptor 2B
LFS: Low-frequency stimulation
TBS: Theta burst stimulation
PP: Perforant pathway
DG: Dentate gyrus
GCaMP6f: Genetically encoded calcium indicator
GO: Gene ontology
DEGs: Diverse genes
AAV: Adeno-associated virus
EMG: Electromyography

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.5c13702.

Characterizations of PT-MNBs (zeta potential, magnetic properties, echogenicity analysis and biodistribution in main organs); behavioral results (MWM test, NOR test, and Y-maze test) and the expression of synaptic-related proteins; data on Piezo1-mediated phagocytosis and microglial inflammatory responses in vitro; schematic of the mechanism of TMAS-driven PT-MNBs; comparison of the effects of PT-MNBs and MNBs; characteristics of the 0.5 MHz focused ultrasound transducer (PDF)

Fangxuan Chu: performed the experiments and wrote the manuscript; Xiaoqing Zhou, Ren Ma, and Ruixu Liu: conceived the experiments, conducted the experiments, and discussed the data; Xu Liu, Kai Zhu, and Yuheng Wang: provided technical guidance and analyzed the data; Xin Wang, Ying Li, Shunqi Zhang, and Tao Yin: analyzed the data and drew the figures; Zhipeng Liu: directed the study, designed the study, and revised the paper.

The authors declare no competing financial interest.

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PERMALINK

Piezo1-Targeted Magnetoacoustic Nanobubbles Rescue Alzheimer’s Pathology by Electromechanical Neuromodulation and Amyloid‑β Clearance

Fangxuan Chu

Xiaoqing Zhou

Ren Ma

Ruixu Liu

Xu Liu

Kai Zhu

Yuheng Wang

Xin Wang

Ying Li

Shunqi Zhang

Tao Yin

Zhipeng Liu

Abstract

Introduction

Results

Characterization of PT-MNBs

1.

TMAS-Responsive PT-MNBs for Precise Neurostimulation

2.

TMAS-Actuated PT-MNBs Alleviate Spatial Cognitive Impairment and Synaptic Plasticity Deficits in 5xFAD Mice

3.

TMAS-Actuated PT-MNBs Activate Microglial Piezo1 to Mediate Aβ Plaque Clearance in 5xFAD Mice

4.

TMAS-Actuated PT-MNBs Alleviate Microglia-Associated Neuroinflammation in 5xFAD Mice

5.

The Effects of TMAS-Actuated PT-MNBs Involving Piezo1 Activation

6.

Piezo1 Knockdown Inhibits the Neuromodulatory Effect of TMAS+PT-MNBs Stimulation in 5xFAD Mice

7.

Discussion

Study Limitations

Conclusion

Methods

Preparation and Characterization of PT-MNBs

Characterization of the Acoustic Field and Magnetoacoustic Field in the Mouse Brain

3D Finite Element Simulations

Cell Culture and Stimulation

Determination of the Targeting Ability and Cytotoxicity of PT-MNBs In Vitro

Transfection with SiRNA

Live-Cell Calcium Imaging

Whole-Cell Patch-Clamp Experiment

Transwell Assay

Animal Care

In Vivo Stimulation of TMAS

Stereotactic Virus Injection

In Vivo Electrophysiological Recordings

In Vivo Fiber Photometry Recording

NOR Test

Y-Maze Test

MWM Test

Golgi-Cox Staining

TEM

Histological Staining and Analysis

Western Blotting

Real-Time Quantitative PCR (RT–qPCR)

Enzyme-Linked Immunosorbent Assay (ELISA)

RNA Sequencing Analysis

Statistical Analysis

Supplementary Material

Acknowledgments

Glossary

Abbreviations

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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