A Mechanogenetic Toolkit for Interrogating Cell Signaling in Space and Time.

Tools capable of imaging and perturbing mechanical signaling pathways with fine spatiotemporal resolution have been elusive, despite their importance in diverse cellular processes. The challenge in developing a mechanogenetic toolkit (i.e., selective and quantitative activation of genetically encoded mechanoreceptors) stems from the fact that many mechanically activated processes are localized in space and time yet additionally require mechanical loading to become activated. To address this challenge, we synthesized magnetoplasmonic nanoparticles that can image, localize, and mechanically load targeted proteins with high spatiotemporal resolution. We demonstrate their utility by investigating the cell-surface activation of two mechanoreceptors: Notch and E-cadherin. By measuring cellular responses to a spectrum of spatial, chemical, temporal, and mechanical inputs at the single-molecule and single-cell levels, we reveal how spatial segregation and mechanical force cooperate to direct receptor activation dynamics. This generalizable technique can be used to control and understand diverse mechanosensitive processes in cell signaling. VIDEO ABSTRACT.

Nanoscale Magneto-mechanical-genetics of Deep Brain Neurons Reversing Motor Deficits in Parkinsonian Mice.

Here, we introduce the magneto-mechanical-genetic (MMG)-driven wireless deep brain stimulation (DBS) using magnetic nanostructures for therapeutic benefits in the mouse model of Parkinson's disease (PD). Electrical DBS of the subthalamic nucleus (STN) is an effective therapy for mitigating Parkinson's motor symptoms. However, its broader application is hampered by the requirement for implanted electrodes and the lack of anatomical and cellular specificity. Using the nanoscale magnetic force actuators (m-Torquer), which deliver torque force under rotating magnetic fields to activate pre-encoded Piezo1 ion channels on target neurons, our system enables wireless and STN-specific DBS without implants, addressing key unmet challenges in the DBS field. In both late- and early-stage PD mice, MMG-DBS significantly improved locomotor activity and motor balance by 2-fold compared to untreated PD mice. Moreover, MMG-DBS enabled sustained therapeutic effects. This approach provides a non-invasive and implant-free DBS with cellular targeting capability for the effective treatment of Parkinsonian symptoms.

Hydrogel Magnetomechanical Actuator Nanoparticles for Wireless Remote Control of Mechanosignaling In Vivo.

As a new enabling nanotechnology tool for wireless, target-specific, and long-distance stimulation of mechanoreceptors in vivo, here we present a hydrogel magnetomechanical actuator (h-MMA) nanoparticle. To allow both deep-tissue penetration of input signals and efficient force generation, h-MMA integrates a two-step transduction mechanism that converts magnetic anisotropic energy to thermal energy within its magnetic core (i.e., Zn0.4Fe2.6O4 nanoparticle cluster) and then to mechanical energy to induce the surrounding polymer (i.e., pNiPMAm) shell contraction, finally delivering forces to activate targeted mechanoreceptors. We show that h-MMAs enable on-demand modulation of Notch signaling in both fluorescence reporter cell lines and a xenograft mouse model, demonstrating its utility as a powerful in vivo perturbation approach for mechanobiology interrogation in a minimally invasive and untethered manner.

Magnetogenetics with Piezo1 Mechanosensitive Ion Channel for CRISPR Gene Editing.

Regulation of genetic activity in single cells and tissues is pivotal to determine key cellular functions in current biomedicine, yet the conventional biochemical activators lack spatiotemporal precision due to the diffusion-mediated slow kinetics and nonselectivity. Here, we describe a magnetogenetic method for target-specific activation of a clustered regularly interspaced short palindromic repeats (CRISPR) system for the regulation of intracellular proteins. We used magnetomechanical force generated by the magnetic nanostructure to activate pre-encoded Piezo1, the mechanosensitive ion channel, on the target cell. The activated Piezo1 further triggers the intracellular Ca2+ signaling pathway, inducing the pre-encoded genes to express genes of interest (GOIs), which is Cas9 protein for the CRISPR regulation of the target proteins. We demonstrated that this magnetogenetic CRISPR system successfully edits the target genome for both in vitro and pseudo-in vivo environments, providing a versatile magnetic platform for remote gene editing of animals with various size scales.

Non-contact long-range magnetic stimulation of mechanosensitive ion channels in freely moving animals.

Among physical stimulation modalities, magnetism has clear advantages, such as deep penetration and untethered interventions in biological subjects. However, some of the working principles and effectiveness of existing magnetic neurostimulation approaches have been challenged, leaving questions to be answered. Here we introduce m-Torquer, a magnetic toolkit that mimics magnetoreception in nature. It comprises a nanoscale magnetic torque actuator and a circular magnet array, which deliver piconewton-scale forces to cells over a working range of ~70 cm. With m-Torquer, stimulation of neurons expressing bona fide mechanosensitive ion channel Piezo1 enables consistent and reproducible neuromodulation in freely moving mice. With its long working distance and cellular targeting capability, m-Torquer provides versatility in its use, which can range from single cells to in vivo systems, with the potential application in large animals such as primates.

Magnetothermally Activated Nanometer-level Modular Functional Group Grafting of Nanoparticles.

Nanoparticles with multifunctionality and high colloidal stability are essential for biomedical applications. However, their use is often hindered by the formation of thick coating shells and/or nanoparticle agglomeration. Herein, we report a single nanoparticle coating strategy to form 1 nm polymeric shells with a variety of chemical functional groups and surface charges. Under exposure to alternating magnetic field, nanosecond thermal energy pulses trigger a polymerization in the region only a few nanometers from the magnetic nanoparticle (MNP) surface. Modular coatings containing functional groups, according to the respective choice of monomers, are possible. In addition, the surface charge can be tuned from negative through neutral to positive. We adopted a coating method for use in biomedical targeting studies where obtaining compact nanoparticles with the desired surface charge is critical. A single MNP with a zwitterionic charge can provide excellent colloidal stability and cell-specific targeting.

Magnetic Control of Axon Navigation in Reprogrammed Neurons.

While neural cell transplantation represents a promising therapy for neurodegenerative diseases, the formation of functional networks of transplanted cells with host neurons constitutes one of the challenging steps. Here, we introduce a magnetic guidance methodology that controls neurite growth signaling via magnetic nanoparticles (MNPs) conjugated with antibodies targeting the deleted in colorectal cancer (DCC) receptor (DCC-MNPs). Activation of the DCC receptors by clusterization and subsequent axonal growth of the induced neuronal (iN) cells was performed in a spatially controlled manner. In addition to the directionality of the magnetically controlled axon projection, axonal growth of the iN cells assisted the formation of functional connections with pre-existing primary neurons. Our results suggest magnetic guidance as a strategy for improving neuronal connectivity by spatially guiding the axonal projections of transplanted neural cells for synaptic interactions with the host tissue.

Small, Clickable, and Monovalent Magnetofluorescent Nanoparticles Enable Mechanogenetic Regulation of Receptors in a Crowded Live-Cell Microenvironment.

Multifunctional magnetic nanoparticles have shown great promise as next-generation imaging and perturbation probes for deciphering molecular and cellular processes. As a consequence of multicomponent integration into a single nanosystem, pre-existing nanoprobes are typically large and show limited access to biological targets present in a crowded microenvironment. Here, we apply organic-phase surface PEGylation, click chemistry, and charge-based valency discrimination principles to develop compact, modular, and monovalent magnetofluorescent nanoparticles (MFNs). We show that MFNs exhibit highly efficient labeling to target receptors present in cells with a dense and thick glycocalyx layer. We use these MFNs to interrogate the E-cadherin-mediated adherens junction formation and F-actin polymerization in a three-dimensional space, demonstrating the utility as modular and versatile mechanogenetic probes in the most demanding single-cell perturbation applications.

A magnetically powered nanomachine with a DNA clutch.

Machines found in nature and human-made machines share common components, such as an engine, and an output element, such as a rotor, linked by a clutch. This clutch, as seen in biological structures such as dynein, myosin or bacterial flagellar motors, allows for temporary disengagement of the moving parts from the running engine. However, such sophistication is still challenging to achieve in artificial nanomachines. Here we present a spherical rotary nanomotor with a reversible clutch system based on precise molecular recognition of built-in DNA strands. The clutch couples and decouples the engine from the machine's rotor in response to encoded inputs such as DNA or RNA. The nanomotor comprises a porous nanocage as a spherical rotor to confine the magnetic engine particle within the nanospace (∼0.004 µm3) of the cage. Thus, the entropically driven irreversible disintegration of the magnetic engine and the spherical rotor during the disengagement process is eliminated, and an exchange of microenvironmental inputs is possible through the nanopores. Our motor is only 200 nm in size and the clutch-mediated force transmission powered by an embedded ferromagnetic nanocrystal is high enough (∼15.5 pN at 50 mT) for the in vitro mechanical activation of Notch and integrin receptors, demonstrating its potential as nano-bio machinery.

In vivo magnetogenetics for cell-type-specific targeting and modulation of brain circuits.

Neuromodulation technologies are crucial for investigating neuronal connectivity and brain function. Magnetic neuromodulation offers wireless and remote deep brain stimulations that are lacking in optogenetic- and wired-electrode-based tools. However, due to the limited understanding of working principles and poorly designed magnetic operating systems, earlier magnetic approaches have yet to be utilized. Furthermore, despite its importance in neuroscience research, cell-type-specific magnetic neuromodulation has remained elusive. Here we present a nanomaterials-based magnetogenetic toolbox, in conjunction with Cre-loxP technology, to selectively activate genetically encoded Piezo1 ion channels in targeted neuronal populations via torque generated by the nanomagnetic actuators in vitro and in vivo. We demonstrate this cell-type-targeting magnetic approach for remote and spatiotemporal precise control of deep brain neural activity in multiple behavioural models, such as bidirectional feeding control, long-term neuromodulation for weight control in obese mice and wireless modulation of social behaviours in multiple mice in the same physical space. Our study demonstrates the potential of cell-type-specific magnetogenetics as an effective and reliable research tool for life sciences, especially in wireless, long-term and freely behaving animals.

Molecular Decrowding by Tissue Expansion Allows Precise Determination of the Spatial Distribution of Synaptic Proteins at a Nanometer Scale by exTEM.

To understand how the molecular machinery of synapses works, it is essential to determine an inventory of synaptic proteins at a subsynaptic resolution. Nevertheless, synaptic proteins are difficult to localize because of the low expression levels and limited access to immunostaining epitopes. Here, we report on the exTEM (epitope-exposed by expansion-transmission electron microscopy) method that enables the imaging of synaptic proteins in situ. This method combines TEM with nanoscale resolution and expandable tissue-hydrogel hybrids for enhanced immunolabeling with better epitope accessibility via molecular decrowding, allowing successful probing of the distribution of various synapse-organizing proteins. We propose that exTEM can be employed for studying the mechanisms underlying the regulation of synaptic architecture and function by providing nanoscale molecular distribution of synaptic proteins in situ. We also envision that exTEM is widely applicable for investigating protein nanostructures located in densely packed environments by immunostaining of commercially available antibodies at nanometer resolution.

Fast detection of SARS-CoV-2 RNA via the integration of plasmonic thermocycling and fluorescence detection in a portable device.

The diagnosis of severe acute respiratory syndrome 2 (SARS-CoV-2) infection by quantitative PCR with reverse transcription (RT-qPCR) typically involves bulky instrumentation in centralized laboratories and an assay time of 1-2 h. Here, we show that SARS-CoV-2 RNA can be detected in 17 min via a portable device integrating reverse transcription, fast thermocycling (via plasmonic heating through magneto-plasmonic nanoparticles) and in situ fluorescence detection following magnetic clearance of the nanoparticles. The device correctly classified all nasopharyngeal, oropharyngeal and sputum samples from 75 patients with COVID-19 and 75 healthy controls, with good concordance in fluorescence intensity with standard RT-qPCR (Pearson coefficients > 0.7 for the N1, N2 and RPP30 genes). Fast, portable and automated nucleic acid detection should facilitate testing at the point of care.

Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles.

Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to answer a host of important biological questions. This protocol describes a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. The key components of this tool are a magnetoplasmonic nanoparticle (MPN) actuator that delivers defined spatial and mechanical cues to receptors through target-specific one-to-one engagement and a micromagnetic tweezers (µMT) that remotely controls the magnitude of force exerted on a single MPN. In our approach, a SNAP-tagged cell-surface receptor of interest is conjugated with a single-stranded DNA oligonucleotide, which hybridizes to its complementary oligonucleotide on the MPN. This protocol consists of four major stages: (i) chemical synthesis of MPNs, (ii) conjugation with DNA and purification of monovalent MPNs, (iii) modular targeting of MPNs to cell-surface receptors, and (iv) control of spatial and mechanical properties of targeted mechanosensitive receptors in live cells by adjusting the µMT-to-MPN distance. Using benzylguanine (BG)-functionalized MPNs and model cell lines expressing either SNAP-tagged Notch or vascular endothelial cadherin (VE-cadherin), we provide stepwise instructions for mechanogenetic control of receptor clustering and for mechanical receptor activation. The ability of this method to differentially control spatial and mechanical inputs to targeted receptors makes it particularly useful for interrogating the differential contributions of each individual cue to cell signaling. The entire procedure takes up to 1 week.

Antioxidant and antiinflammatory activities of xanthorrhizol in hippocampal neurons and primary cultured microglia.

Xanthorrhizol, a natural sesquiterpenoid isolated from the rhizome of Curcuma xanthorrhiza Roxb (Zingiberaceae), has antibacterial activities and protective effects against cisplatin-induced hepatotoxicity. In this study, we investigated the activities of xanthorrhizol as an antioxidant or antiinflammatory agent using neuronal and microglial cells. Xanthorrhizol had potent neuroprotective effects on glutamate-induced neurotoxicity and reactive oxygen species (ROS) generation in the murine hippocampal HT22 cell line. Also, xanthorrhizol inhibited H(2)O(2)-induced lipid peroxidation in rat brain homogenates. The properties of xanthorrhizol as an antiinflammatory agent were investigated in microglial activation by lipopolysaccharide. It reduced the expression of cyclooxygenase-2 and the inducible nitric oxide synthase, which consequently resulted in the reduction of nitric oxide. The production of proinflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha in activated microglial cells, was reduced by xanthorrhizol. These results suggest that xanthorrhizol could be an effective candidate for the treatment of Alzheimer's disease- and other neurological disease-related ROS and inflammation.