Spatial Control Research Articles

Spatially Controlled Self-Assembly of Supramolecular Hydrogels Enabled by Light-Triggered Catalysis.

Spatial control over supramolecular self-assembly prevails in living system, yet remains difficult to replicate in synthetic scenarios. Here, on the basis of a hydrazone formation-mediated supramolecular hydrogelation system, access to patterning of supramolecular hydrogels is demonstrated via a light-triggered catalysis strategy. A photoacid generator that can produce protons in aqueous solutions upon irradiation is employed. The generated protons lead to a drop in pH of around three units (initial pH 7.0), effectively accelerating the formation and self-assembly of the hydrazone gelators. Because of the light-triggered catalysis, the hydrogelation samples in the presence of photoacid generator show lower critical gelation concentration, higher stiffness, and denser networks. Importantly, by performing selective irradiation using differently shaped masks, various spatially resolved supramolecular hydrogels following the shapes of the masks are fabricated. The concept of using light-triggered catalysis to realize spatial control over supramolecular self-assembly provides an alternative approach toward bottom-up fabrication of structured soft materials for various applications such as tissue engineering, single cell manipulation, and biosensing.

Nanobody-thioesterase chimeras to specifically target protein palmitoylation

The complexity of the cellular proteome is massively expanded by a repertoire of chemically distinct reversible post-translational modifications (PTMs) that control protein localisation, interactions, and function. The temporal and spatial control of these PTMs is central to organism physiology, and mis-regulation of PTMs is a hallmark of many diseases. Here we present an approach to manipulate PTMs on target proteins using nanobodies fused to enzymes that control these PTMs. Anti-GFP nanobodies fused to thioesterases (which depalmitoylate protein cysteines) depalmitoylate GFP tagged substrates. A chemogenetic approach to enhance nanobody affinity for its target enables temporal control of target depalmitoylation. Using a thioesterase fused to a nanobody directed against the Ca(v)1.2 beta subunit we reduce palmitoylation of the Ca(v)1.2 alpha subunit, modifying the channel’s voltage dependence and arrhythmia susceptibility in stem cell derived cardiac myocytes. We conclude that nanobody enzyme chimeras represent an approach to specifically manipulate PTMs, with applications in both the laboratory and the clinic.

Spatial Confinement of a Dual Activatable DNAzyme Sensor in the Cavity of a DNA Nanocage for Logic‐Gated Molecular Imaging

AbstractDespite intense interest in design of DNAzyme sensors for molecular detection and imaging in living cells, their intracellular applications are still hampered by limited spatial control and poor bio‐stability. Here we present controlled spatial confinement of a rationally designed, microRNA (miRNA)‐activatable DNAzyme sensor probe (mDz) within the cavity of DNA nanocage, enabling efficient intracellular delivery with improved bio‐stability for AND‐gate molecular imaging. The mDz that possesses inactive DNAzyme activity is designed by the introduction of a blocking DNA strand, while miR‐21 mediated strand displacement reaction allows for the formation of an intact DNAzyme structure for metal‐ion‐mediated catalytic reaction. Furthermore, the DNA nanocage serves as a nanocarrier for intracellular delivery of mDz, in which the cavity is accessible to the dual targets for logic‐gated molecular imaging, while the confinement effect can provide steric protection of mDz by obstructing nuclease from entering the cavity of the DNA nanocage, resulting in enhanced bio‐stability and improved molecular imaging precision. This strategy paves a way for the engineering of activatable DNA nanosensors with both self‐delivery and self‐protection capabilities to detect diverse intracellular targets.

Development of Transiently Strainable Benzocycloheptenes for Catalyst-Free, Visible-Light-Mediated [3 + 2]-Cycloadditions.

Dynamic photogeneration of ephemeral and reactive species is enabling for chemical reactions, providing spatial and temporal control. A previous study from our group established the ability of 6,7-dihydro-5H-benzo[7]annulene, benzocycloheptene (BC7), to convert photochemical energy into ring strain, enabling the rapid cycloaddition of alkyl azides with the reversibly formed and transient trans-isomer, affording versatile nonaromatic triazolines. Despite the conceptual advances of the previous study, some challenges remained: the fragility of the triazoline products, the low regioselectivity for the cycloaddition, a need for an iridium-based photosensitizer and organic-based solvents, and a lack of convenient linchpin functional group handles. Herein, we communicate the development of a second generation of BC7 molecules that overcome the issues of the first generation. A method to convert fragile triazoline products to stable triazoles was developed. The alkene component was polarized with a carbonyl group, dramatically improving the regioselectivity while simultaneously red-shifting the absorbance of the cycloalkene into the visible region, which was expected to facilitate direct excitation and eliminate the need for photocatalysts. However, experiments indicated that the cycloaddition involved passage through a triplet manifold, complicating the direct excitation strategy. This was successfully overcome by attaching a bromine atom directly to the alkene moiety, which accelerated singlet-to-triplet intersystem crossing by the heavy atom effect. Further exploration identified sites of substitution that can increase the water solubility and provide a handle for the loading of chemical tools and probes.

Spatial Control of CAR T Cell Activation Using Tumor-Homing Polymers.

CAR T cell therapies often lack specificity, leading to issues ranging from inadequate antigen targeting to off-tumor toxicities. To counter that lack of specificity, we expanded tumor targeting capabilities with universal CAR and spatially defined CAR T cell engagement with targets through a combination of synthetic biology and biomaterial approaches. We developed a novel framework, called "In situ Mobilization: Polymer Activated Cell Therapies" (IMPACT) for polymer-mediated, anatomical control of IF-THEN gated CAR T cells. With IMPACT, a regulated payload such as a BiTE or tumor-targeting CAR will only be expressed after engineered cells engage a tumor-localizing polymer ("IF" condition). In this first demonstration of IMPACT, we engineered CAR T cells to respond to fluorescein that is displayed by an injectable polymer that binds to and is retained in fibrin deposits in tumor microenvironments. This interaction then drives selective and conditional expression of a protein within tumors ("THEN" condition). Here, we develop the polymer and CAR T cell infrastructure of IMPACT and demonstrate tumor-localized CAR T cell activation in a murine tumor model after the intravenous administration of polymer and engineered T cells.

Spatial Confinement of a Dual Activatable DNAzyme Sensor in the Cavity of a DNA Nanocage for Logic-Gated Molecular Imaging.

Despite intense interest in design of DNAzyme sensors for molecular detection and imaging in living cells, their intracellular applications are still hampered by limited spatial control and poor bio-stability. Here we present controlled spatial confinement of a rationally designed, microRNA (miRNA)-activatable DNAzyme sensor probe (mDz) within the cavity of DNA nanocage, enabling efficient intracellular delivery with improved bio-stability for AND-gate molecular imaging. The mDz that possesses inactive DNAzyme activity is designed by the introduction of a blocking DNA strand, while miR-21 mediated strand displacement reaction allows for the formation of an intact DNAzyme structure for metal-ion-mediated catalytic reaction. Furthermore, the DNA nanocage serves as a nanocarrier for intracellular delivery of mDz, in which the cavity is accessible to the dual targets for logic-gated molecular imaging, while the confinement effect can provide steric protection of mDz by obstructing nuclease from entering the cavity of the DNA nanocage, resulting in enhanced bio-stability and improved molecular imaging precision. This strategy paves a way for the engineering of activatable DNA nanosensors with both self-delivery and self-protection capabilities to detect diverse intracellular targets.

Biomolecular condensates: A new lens on cancer biology.

Cells are compartmentalized into different organelles to ensure precise spatial temporal control and efficient operation of cellular processes. Membraneless organelles, also known as biomolecular condensates, are emerging as previously underappreciated ways of organizing cellular functions. Condensates allow local concentration of protein, RNA, or DNA molecules with shared functions, thus facilitating spatiotemporal control of biochemical reactions spanning a range of cellular processes. Studies discussed herein have shown that aberrant formation of condensates is associated with various diseases such as cancers. Here, we summarize how condensates mechanistically contribute to malignancy-related cellular processes, including genomic instability, epigenetic rewiring, oncogenic transcriptional activation, and signaling. An improved understanding of condensate formation and dissolution will enable development of new cancer therapies. Finally, we address the remaining challenges in the field and suggest future efforts to better integrate condensates into cancer research.

Optogenetic control of receptor-mediated growth cone dynamics in neurons.

Development of neuronal connections is spatially and temporally controlled by extracellular cues which often activate their cognate cell surface receptors and elicit localized cellular responses. Here, we demonstrate the use of an optogenetic tool to activate receptor signaling locally to induce actin-mediated growth cone remodeling in neurons. Based on the light-induced interaction between Cryptochrome 2 (CRY2) and CIB1, we generated a bicistronic vector to co-expresses CRY2 fused to the intracellular domain of a guidance receptor and a membrane-anchored CIB1. When expressed in primary neurons, activation of the growth inhibitory PlexA4 receptor induced growth cone collapse, while activation of the growth stimulating TrkA receptor increased growth cone size. Moreover, local activation of either receptor not only elicited the predicted response in light-activated growth cones but also an opposite response in neighboring no-light-exposed growth cones of the same neuron. Finally, this tool was used to reorient growth cones toward or away from the site of light activation and to stimulate local actin polymerization for branch initiation along axonal shafts. These studies demonstrate the use of an optogenetic tool for precise spatial and temporal control of receptor signaling in neurons and support its future application in investigating cellular mechanisms of neuronal development and plasticity.

Immobilization of BMP-2 in porous hydrogels to spatially regulate osteogenesis.

Sustained release of bone morphogenetic protein 2 (BMP-2) is used to enhance bone regeneration, but immobilizing BMP-2 in three-dimensional scaffolds could enable spatial regulation of stem cell differentiation and bone formation. Here, we fabricate porous granular hydrogels presenting BMP-2 on the surface to regulate stem cell growth and differentiation. Immobilization of BMP-2 and cell-adhesive ligands is achieved by surface-specific functionalization of microgels, which are jammed to form microporous hydrogels. Varying surface ligand density regulated spreading, proliferation and differentiation of cells. In addition, modulating the distribution of cell-adhesive ligands and BMP-2 allowed spatial control over cell adhesion and osteogenic differentiation.

Frequency response mechanism of surface smoothing and material removal on fused silica

To meet the stringent requirements for sub-nanometer surface roughness and full-spectrum spatial frequency error control in high-precision optical systems, this study systematically investigates the effects of varying Cerium oxide (CeO2) abrasive particle sizes and pitch pad hardness on the surface quality of fused silica glass during computer-controlled optical surfacing (CCOS). This study integrates experimental characterization with mathematical modeling to analyze the mechanisms by which abrasive particle size and polishing tool hardness influence material removal rates, contact pressure distribution, and surface roughness. The results indicate that smaller CeO2 particles effectively reduce high-spatial frequency roughness, making them suitable for precise micro-surface finishing, while larger particles exhibit higher material removal efficiency, making them more suitable for macro-surface machining. Hard pitch pads induce greater indentation depth and concentrated pressure, significantly improving mid-spatial frequency surface errors, while softer pitch pads are more suitable for overall surface smoothing. Experimental findings further validate the feasibility and effectiveness of the proposed optimization strategies in achieving ultra-precision processing at picometer scales across the full spectrum of frequency bands. This study provides a theoretical basis and practical guidance for selecting process parameters in the manufacturing of high-precision optical components.

Regio-Selective Mechanical Enhancement of Polymer-Grafted Nanoparticle Composites via Light-Mediated Crosslinking.

Polymer-brush-grafted nanoparticles (PGNPs) that can be covalently crosslinked post-processing enable the fabrication of mechanically robust and chemically stable polymer nanocomposites with high inorganic filler content. Modifying PGNP brushes to append UV-activated crosslinkers along the polymer chains would permit a modular crosslinking strategy applicable to a diverse range of nanocomposite compositions. Further, light-activated crosslinking reactions enable spatial control of crosslink density to program intentionally inhomogeneous mechanical responses. Here, a method of synthesizing composites using UV-crosslinkable brush-coated nanoparticles (referred to as UV-XNPs) is introduced that can be applied to various monomer compositions by incorporating photoinitiators into the polymer brushes. UV crosslinking of processed UV-XNP structures can increase their tensile modulus up to 15-fold without any noticeable alteration to their appearance or shape. By using photomasks to alter UV intensity across a sample, intentionally designed inhomogeneities in crosslink density result in predetermined anisotropic shape changes under strain. This unique capability of UV-XNP materials is applied to stiffness-patterned flexible electronic substrates that prevent the delamination of rigid components under deformation. The potential of UV-XNPs as functional, soft device components is further demonstrated by wearable devices that can be modified post-fabrication to customize their performance, permitting the ability to add functionality to existing device architectures.

Xolography for Biomedical Applications: Dual-Color Light-Sheet Printing of Hydrogels With Local Control Over Shape and Stiffness.

Current challenges in tissue engineering include creation of extracellular environments that support and interact with cells using biochemical, mechanical, and structural cues. Spatial control over these cues is currently limited due to a lack of suitable fabrication techniques. This study introduces Xolography, an emerging dual-color light-sheet volumetric printing technology, to achieve control over structural and mechanical features for hydrogel-based photoresins at micro- to macroscale while printing within minutes. A water-soluble photoswitch photoinitiator system and a library of naturally-derived, synthetic, and thermoresponsive hydrogels for Xolography are proposed. Centimeter-scale, 3D constructs with positive features of 20µm and negative features of ≈100µm are fabricated with control over mechanical properties (compressive moduli 0.2kPa-6.5MPa). Notably, switching from binary to grayscaled light projection enables spatial control over stiffness (0.2-16kPa). As a proof of concept, grayscaled Xolography is leveraged with thermoresponsive hydrogels to introduce reversible anisotropic shape changes beyond isometric shrinkage. Xolography of viable cell aggregates is finally demonstrated, laying the foundation for cell-laden printing of dynamic, cell-instructive environments with tunable structural and mechanical cues in a fast one-step process. Overall, these innovations unlock unique possibilities of Xolography across multiple biomedical applications.

Spatioselective Imaging of Noncoding RNAs in Mitochondria via an Organelle-Specific DNA Assembly Strategy.

Precise imaging of noncoding RNAs (ncRNAs) in specific organelles allows decoding of their functions at subcellular level but lacks advanced tools. Here we present a DNA-based nanobiotechnology for spatially selective imaging of ncRNA (e.g., microRNA (miRNA)) in mitochondria via an organelle-specific DNA assembly strategy. The target miRNA-initiated assembly of DNA hairpins is inhibited by the block of toehold-mediated strand displacement reaction but can be exclusively activated by a mitochondria-encoded ribosomal RNA (rRNA) for hybridization chain reaction, enabling spatial control over miRNA imaging. We demonstrate that the conditionally controlled DNA assembly technology allows for minimization of nonspecific activation and thus improves the spatial precision of miRNA detection. In addition, the strategy is adaptable to visualizing other ncRNAs such as long noncoding RNAs in mitochondria, highlighting the universality of the approach. Overall, this work provides a useful tool for spatially selective imaging of ncRNAs and investigating the functions of organelle-located RNA.

Nanoscale Cellular Traction Force Quantification: CRISPR-Cas12a Supercharged DNA Tension Sensors in Nanoclustered Ligand Patterns.

High-throughput measurement of cellular traction forces at the nanoscale remains a significant challenge in mechanobiology, limiting our understanding of how cells interact with their microenvironment. Here, we present a novel technique for fabricating protein nanopatterns in standard multiwell microplate formats (96/384-wells), enabling the high-throughput quantification of cellular forces using DNA tension gauge tethers (TGTs) amplified by CRISPR-Cas12a. Our method employs sparse colloidal lithography to create nanopatterned surfaces with feature sizes ranging from sub 100 to 800 nm on transparent, planar, and fully PEGylated substrates. These surfaces allow for the orthogonal immobilization of two different proteins or biomolecules using click-chemistry, providing precise spatial control over cellular signaling cues. We demonstrate the robustness and versatility of this platform through imaging techniques, including total internal reflection fluorescence microscopy, confocal laser scanning microscopy, and high-throughput imaging. Applying this technology, we measured the early stage mechanical forces exerted by 3T3 fibroblasts across different nanoscale features, detecting forces ranging from 12 to 56 pN. By integrating the Mechano-Cas12a Assisted Tension Sensor (MCATS) system, we achieved rapid and high-throughput quantification of cellular traction forces, analyzing over 2 million cells within minutes. Our findings reveal that nanoscale clustering of integrin ligands significantly influences the mechanical responses of cells. This platform offers a powerful tool for mechanobiology research, facilitating the study of cellular forces and mechanotransduction pathways in a high-throughput manner compatible with standard cell culture systems.

Integrating microfluidics, hydrogels, and 3D bioprinting for personalized vessel-on-a-chip platforms.

Thrombosis, a major cause of morbidity and mortality worldwide, presents a complex challenge in cardiovascular medicine due to the intricacy of clotting mechanisms in living organisms. Traditional research approaches, including clinical studies and animal models, often yield conflicting results due to the inability to control variables in these complex systems, highlighting the need for more precise investigative tools. This review explores the evolution of in vitro thrombosis models, from conventional polydimethylsiloxane (PDMS)-based microfluidic devices to advanced hydrogel-based systems and cutting-edge 3D bioprinted vascular constructs. We discuss how these emerging technologies, particularly vessel-on-a-chip platforms, are enabling researchers to control previously unmanageable factors, thereby offering unprecedented opportunities to pinpoint specific clotting mechanisms. While PDMS-based devices offer optical transparency and fabrication ease, their inherent limitations, including non-physiological rigidity and surface properties, have driven the development of hydrogel-based systems that better mimic the extracellular matrix of blood vessels. The integration of microfluidics with biomimetic materials and tissue engineering approaches has led to the development of sophisticated models capable of simulating patient-specific vascular geometries, flow dynamics, and cellular interactions under highly controlled conditions. The advent of 3D bioprinting further enables the creation of complex, multi-layered vascular structures with precise spatial control over geometry and cellular composition. Despite significant progress, challenges remain in achieving long-term stability, incorporating immune components, and translating these models to clinical applications. By providing a comprehensive overview of current advancements and future prospects, this review aims to stimulate further innovation in thrombosis research and accelerate the development of more effective, personalized approaches to thrombosis prevention and treatment.

Strain-Induced Photochemical Opening of Ferrocene[6]cycloparaphenylene: Uncaging of Fe2+ with Green Light.

We present the synthesis, structural analysis, and remarkable reactivity of the first carbon nanohoop that fully incorporates ferrocene in the macrocyclic backbone. The high strain imposed on the ferrocene by the curved nanohoop structure enables unprecedented photochemical reactivity of this otherwise photochemically inert metallocene complex. Visible light activation triggers a ring-opening of the nanohoop structure, fully dissociating the Fe-cyclopentadienyl bonds in the presence of 1,10-phenanthroline. This process uncages Fe2+ ions captured in the form of [Fe(phen)3]2+ complex in high chemical yield and can operate efficiently in a water-rich solvent with green light excitation. The measured quantum yields of [Fe(phen)3]2+ formation show that embedding ferrocene into a strained nanohoop boosts its photoreactivity by 3 orders of magnitude compared to an unstrained ferrocene macrocycle or ferrocene itself. Our data suggest that the dissociation occurs by intercepting the photoexcited triplet state of the nanohoop by a nucleophilic solvent or external ligand. The strategy portrayed in this work proposes that new, tunable reactivity of analogous metallamacrocycles can be achieved with spatial and temporal control, which will aid and abet the development of responsive materials for metal ion delivery and supramolecular, organometallic, or polymer chemistry.

A nanoparticle-based wireless deep brain stimulation system that reverses Parkinson's disease.

Deep brain stimulation technology enables the neural modulation with precise spatial control but requires permanent implantation of conduits. Here, we describe a photothermal wireless deep brain stimulation nanosystem capable of eliminating α-synuclein aggregates and restoring degenerated dopamine neurons in the substantia nigra to treat Parkinson's disease. This nanosystem (ATB NPs) consists of gold nanoshell, an antibody against the heat-sensitive transient receptor potential vanilloid family member 1 (TRPV1), and β-synuclein (β-syn) peptides with a near infrared-responsive linker. ATB NPs by stereotactic injection target dopamine neurons expressing TRPV1 receptors in the substantia nigra. Upon pulsed near-infrared irradiation, ATB NPs, serving as nanoantennae, convert the light into heat, leading to calcium ion influx, depolarization, and action potentials in dopamine neurons through TRPV1 receptors. Simultaneously, β-synuclein peptides released from ATB NPs cooperate with chaperone-mediated autophagy initiated by heat shock protein, HSC70, to effectively eliminate α-synuclein fibrils in neurons. These orchestrated actions restored pathological dopamine neurons and locomotor behaviors of Parkinson's disease.

A review of 3D bioprinting for organoids

Abstract Current two-dimensional (2D) cell models for effective drug screening suffer from significant limitations imposed by the lack of realism in the physiological environment. Three-dimensional (3D) organoids models hold immense potential in mimicking the key functions of human organs by overcoming the limitations of traditional 2D cell models. However, current techniques for preparation of 3D organoids models had limitations in reproducibility, scalability, and the ability to closely replicate the complex microenvironment found in vivo. Additionally, traditional 3D cell culture systems often involve lengthy and labor-intensive processes that hinder high-throughput applications necessary for a large-scale drug screening. Advancements in 3D bioprinting technologies offer promising solutions to these challenges by enabling precise spatial control over cell placement and material composition, thereby facilitating the creation of more physiologically relevant organoids than current techniques. This review provides a comprehensive summary of recent advances in 3D bioprinting technologies for creating organoids models, which begins with an introduction to different types of 3D bioprinting techniques (especially focus on volumetric bioprinting (VBP) technique), followed by an overview of bioinks utilized for organoids bioprinting. Moreover, we also introduce the applications of 3D bioprinting organoids in disease models, drug efficiency evaluation and regenerative medicine. Finally, the challenges and possible strategies for the development and clinical translation of 3D bioprinting organoids are concluded.

Cytoplasmic Regulation of the Poly(A) Tail Length as a Potential Therapeutic Target.

Virtually all mRNAs acquire a poly(A) tail co-transcriptionally, but its length is dynamically regulated in the cytoplasm in a transcript-specific manner. The length of the poly(A) tail plays a crucial role in determining mRNA translation, stability, and localization. This dynamic regulation of poly(A) tail length is widely used to create post-transcriptional gene expression programs, allowing for precise temporal and spatial control. Dysregulation of poly(A) tail length has been linked to various diseases, including cancers, inflammatory and cardiovascular disorders, and neurological syndromes. Cytoplasmic poly(A) tail length is maintained by a dynamic equilibrium between cis-acting elements and cognate factors that promote deadenylation or polyadenylation, enabling rapid gene expression reprogramming in response to internal and external cellular cues. While cytoplasmic deadenylation and its pathophysiological implications have been extensively studied, cytoplasmic polyadenylation and its therapeutic potential remain less explored. This review discusses the distribution, regulation, and mechanisms of Cytoplasmic Polyadenylation Element-Binding Proteins (CPEBs), highlighting their dual roles in either promoting or repressing gene expression depending on cellular context. We also explore their involvement in diseases such as tumor progression and metastasis, along with their potential as targets for novel therapeutic strategies.

Optical control of spin waves in hybrid magnonic-plasmonic structures.

Magnonics, which harnesses the unique properties of spin waves, offers promising advancements in data processing due to its broad frequency range, nonlinear dynamics, and scalability for on-chip integration. Effective information encoding in magnonic systems requires precise spatial and temporal control of spin waves. Here, we demonstrate the rapid optical control of spin-wave transport in hybrid magnonic-plasmonic structures. By using thermoplasmonic heating in yttrium iron garnet films integrated with gold nanodisk arrays, we achieve a suppression of spin-wave signals by 20 dB using single laser pulses lasting just a few hundred nanoseconds. Our results reveal a strong correlation between plasmonic light absorption and spin-wave manipulation, as supported by micromagnetic simulations that emphasize the crucial role of magnonic refraction. This study establishes thermoplasmonics as a powerful tool for controlling spin-wave propagation, bridging the fields of magnonics and plasmonics, and paving the way for the development of multifunctional hybrid magnonic devices.