Nature Nanotechnology

Microtubules are central components of cytoskeletal transport systems and have been widely repurposed as active elements in motor-driven nanodevices. However, site-specific functionalization of stabilized microtubules remains a fundamental challenge, as the tubulin lattice presents chemically indistinguishable binding sites along its length. Here we report a strategy for selective end-functionalization of stabilized microtubules using DNA origami nanostructures. By coupling DNA origami to Fab fragments targeting acetylated -tubulin Lys40 within the microtubule lumen, and exploiting steric exclusion of the origami from the lattice interior, binding is confined to accessible sites at microtubule ends and lattice defects. Using a six-helix bundle origami as a minimal construct, we demonstrate selective tip labelling of gliding microtubules without perturbing kinesin-driven motility. The same structures additionally mark lattice defects, enabling dynamic visualization of defect sites during transport. Furthermore, we show that tip-bound origami can hybridize with complementary DNA strands to capture cargo from surfaces in motion, establishing programmable, end-specific loading. This approach introduces a generalizable route to spatially controlled functionalization of cytoskeletal filaments, enabling new capabilities in molecular transport, nanoscale assembly, and the study of microtubule integrity and repair.

Causal manipulation of vagal gut-brain pathways empowers studies of metabolism and interoception. However, the anatomy and cytoarchitecture of vagal circuits pose challenges to deployment of optical or electrical stimulation probes. We present a wireless modulation of vagal circuits via magnetite nanodiscs (MNDs) targeted to specific nodose ganglia neurons via genetically delivered anchoring moieties. Under slow-varying magnetic fields, membrane-bound MNDs transduce mechanical torques that trigger depolarization mediated by endogenous mechanoreceptors in sensory neurons. When targeted to neurons expressing oxytocin or glucagon-like peptide 1 receptors in the left nodose ganglia, MND stimulation activates downstream hindbrain satiety circuits and reduces food intake. These findings establish MND-mediated stimulation as a targeted, implant-free platform for modulating gut-brain neural circuits and beyond.

The global success of mRNA vaccines has underscored the pivotal role of lipid nanoparticles (LNPs), yet how subtle chemical variations in ionizable lipids and their formulation parameters orchestrate complex immune landscapes remains largely elusive. Here, we report a novel ionizable lipid, N4Z, and demonstrate that its distinct chemical signature selectively intensifies early innate immune programs compared to its structural analogue, N4Y. Single-cell transcriptomic profiling at the injection site reveals that N4Z-based LNPs uniquely prime inflammatory and type I interferon-related transcriptional programs, accompanied by a rapid influx of B and CD4+ T cells. Beyond lipid chemistry, we show that formulation-level tuning, that is independent of the ionizable lipid structure, can reshape the systemic biodistribution from hepatic dominance toward lymphoid tissues. This optimization substantially enhances macrophage-associated antigen expression, which in turn amplifies polyfunctional CD4+ T cell responses, T follicular helper cell differentiation, and germinal center reactions. Our findings establish that the coordinated interplay between lipid engineering and formulation design provides a programmable platform for precision mRNA vaccination, achieving superior protective efficacy and neutralizing activity over clinically validated benchmarks.

Negatively charged DNA nanostructures, such as tetrahedral nanocages, are internalized by cells despite the electrostatic repulsion from the anionic cell membrane, and, paradoxically, cancer cells, which carry intrinsically higher negative charge due to overexpression of sialic acids on their cell surface, show markedly higher uptake than normal cells. This contradiction exposes a fundamental gap in our understanding of how these anionic nanostructures overcome this repulsion. Using chemical modulation of cell-surface sialylation in RPE1 cells to create three groups with altered sialylation levels, together with inhibitor-based dissection of endocytic pathways, we demonstrate that an increase in cell surface sialylation governs the uptake of DNA tetrahedra not through electrostatics but by structurally remodeling the cell membrane via rearrangement of the GM1 lipid raft microdomain, recruiting caveolae-mediated endocytosis as an additional pathway alongside clathrin-mediated endocytosis, thereby increasing the intake of the nanostructure. These findings reframe tumor hyper-sialylation as a determinant of the uptake of anionic nanostructures, such as DNA tetrahedra, and as a targetable parameter for rational optimization of DNA-based nanotherapeutics against cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/722926v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@10eede7org.highwire.dtl.DTLVardef@124dd56org.highwire.dtl.DTLVardef@13f5355org.highwire.dtl.DTLVardef@780ecf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transmembrane signaling mediated by cytokine receptors orchestrates key cellular processes such as proliferation, differentiation, and immune responses. While numerous high-resolution structures of cytokine receptor ectodomains are available, the structural organization of the largely disordered intracellular domain (ICD) has remained unclear. Here, we interrogate the axial organization of cytokine receptor signaling complexes at the plasma membrane by metal-induced energy transfer (MIET). For this purpose, we leveraged biofunctionalized nanodot arrays (bNDAs) to capture cell surface receptors at a defined distance from the substrate. Readout by fluorescence lifetime imaging microscopy enabled quantifying axial distances of proteins in the plasma membrane of cells at both ensemble and single-molecule levels with a resolution of [~]1 nm. Using the prototypic, biomedically relevant class I cytokine receptor GP130 as a model system, we uncover by MIET that the ICD extends randomly into the cytosol in the resting state, but surprisingly undergoes an axial compaction upon signal activation. These results demonstrate the potential of bNDA-supported MIET for resolving the axial architecture of signaling complexes within the cellular context.

The glycocalyx is a major regulator of membrane recognition, yet its specific influence on extracellular vesicles (EVs) cellular uptake remains poorly defined. We established a genetic glycoengineering platform to systematically investigate how the major glycan classes on small EVs (sEVs) modulate cell interactions and functional cargo delivery. Using an isogenic panel of HEK293F lines lacking distinct glycan biosynthetic pathways, we find that removing glycosaminoglycans ({Delta}GAG-sEVs) yields a strong increase in cellular uptake and delivery of diverse cargos, including DNA oligonucleotides, siRNA, proteins, and plasmid DNA. Glycan-modified recipient cells show that sEV-cell communication and internalization is jointly governed by glycan features on both membranes. {Delta}GAG-sEVs strongly improve gene delivery and expression in recipient cells and in a physiologically relevant human airway epithelial model. These findings establish glycan structures as tunable regulators of sEV uptake and position {Delta}GAG-sEVs as potent vehicles for improved drug delivery and gene therapy.

Neuroelectronic interfaces hold great promise to restore functions in neurological disorders or motor dysfunctions, but current devices struggle to integrate seamlessly within living tissues. Here we report a transformative approach to create bionic neurons that autonomously build integrated fluorescent fibrils and demonstrate their role as neuromodulators. Using a combination of cell biology, ultrastructural, imaging and nanospectroscopical approaches, we deciphered the unique biosynthetic pathway employed by the cells to self-fabricate these nanoelectronics and uncover their hybrid structure. Importantly, patch clamp recordings revealed their neuromodulatory potential, through the perturbation of membrane electrical properties and the early rising phase of the action potential. Deciphering how basic molecular elements self-organize into complex architectures within biological environments could unlock the ability to engineer natural electroactive systems directly inside living organisms. This capability could be used to create conductive pathways between arbitrarily defined neurons, microcircuits, or nervous system regions, effectively writing connections into living brains.

The ability to encode and reliably read nanoscale information is increasingly important for multiplexed biomolecular detection and super-resolution imaging. DNA origami provides a uniquely programmable platform for arranging structural and functional elements with nanometer precision, enabling the creation of identifiable nanoscale patterns. In this context, DNA origami-based barcodes that incorporate gold nanoparticles (AuNPs) to encode either origami geometry or the identity of specific biological targets within defined nanoparticle patterns have been paired with transmission electron microscopy imaging for decoding. However, surface-bond AuNPs may detach during handling, purification, or biological incubation, leading to misidentification or decoding errors in barcode analysis. Here we report a rational design for the controlled encapsulation of AuNPs within DNA origami tubes to enhance nanoparticle retention and structural integrity. We engineered curvature-inducing modifications in a flat rectangular DNA origami scaffold to promote inward folding and confinement of AuNPs. These barcodes can be further functionalized on the outer surface with bioactive aptamers and/or fluorescence dyes, enabling targeted interactions with cells and optical readout. Programable dimerization further expands multiplexing capacity. This design provides a robust framework for structurally stable origami barcodes and advances the development of high-resolution, multiplexed labeling and diagnostic platforms. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/725969v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@686c1aorg.highwire.dtl.DTLVardef@1914c4eorg.highwire.dtl.DTLVardef@28ad47org.highwire.dtl.DTLVardef@8847ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Macrophage small extracellular vesicles (sEVs) carry phenotype-linked cargo and bioactivity for immunomodulation and regeneration, but therapeutic translation is limited by low secretion and poor control of function. We introduce a music-activated piezoelectric nanofiber substrate (PES) that converted audible sound into programmable electrical stimulation to enhance sEV biogenesis while tuning macrophage polarization. Adjusting acoustic parameters increased sEV yield, while musically inspired "assemblies" biased macrophage phenotypes: dissonant, low-frequency stimuli promoted M1-like inflammation, whereas consonant, higher-frequency stimuli favored M2-like, regenerative states. These shifts produced distinct sEV cargo and bioactivities. We rationally designed customized music stimulus that maximized both vesicle production and M2 bias, yielding sEVs exhibited regeneration potentials. This work establishes a programmable acoustic-piezoelectric strategy to scale macrophage sEV production while tailoring their therapeutic potency.

Immunotherapy has transformed cancer treatment, yet cell-based therapies remain complex and costly, and immune checkpoint blockade (ICB) agents often suffer from limited stability and poor T-cell selectivity. Here, we develop an engineered dendritic cell-derived small extracellular vesicle (DC-sEV) nanoplatform for combinatorial immunotherapy via in situ T-cell activation and checkpoint reprogramming. DC-sEVs preserve intrinsic dendritic-cell immunobiology, enabling antigen presentation and potent T-cell activation. We further integrate high-efficiency cargo loading and membrane functionalization to selectively deliver ICB payloads to T cells, achieving dual reprogramming that sustains effector function and amplifies antitumor immunity. This approach reduced cancer cell viability to 44.05% in vitro and produced 82.12% tumor growth inhibition in vivo, establishing DC-sEVs as a targeted, scalable cell-free immunotherapy platform. HIGHLIGHTSO_LIDC-sEVs preserve antigen presentation, T-cell activation, and lymph node targeting C_LIO_LIChirality-assisted loading with pH-responsive functionalization enables efficient cytosolic delivery while maintaining membrane bioactivity C_LIO_LIEngineered DC-sEVs combine in situ T-cell priming and PD-1 silencing to enhance effector function C_LIO_LIIn situ T-cell reprogramming drives potent antitumor efficacy and favorable tumor microenvironment remodeling C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/717283v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@e3b05org.highwire.dtl.DTLVardef@44febcorg.highwire.dtl.DTLVardef@1b00479org.highwire.dtl.DTLVardef@f5db2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Delivering biomolecules into pollen tubes that deliver sperm cells for plant fertilization remains technically challenging due to thick cell walls and rapid polarized growth, hindering reproductive engineering. DNA nanotechnology offers a promising alternative over current delivery methods due to their biocompatibility, programmable design, low cytotoxicity, and stimulus-responsive properties, yet their application in plants remains underexplored. Here, we provide the first demonstration of tetrahedral DNA nanostructures (TDNs) as nanocarriers for active, endocytosis-mediated uptake into Arabidopsis pollen tubes, enabling spermidine delivery that shortens pollen tube elongation through actin reorganization and ROS modulation. TDN-treated pollen tubes grew through the Arabidopsis stigma and style, underwent capacitation, and maintained attraction to ovules in a semi-in-vivo assay, preserving reproductive fitness. Furthermore, we demonstrate that functionalization of TDNs with nuclear localization signal peptide significantly enhances nuclear localization. Collectively, these findings establish DNA nanostructures as effective nanocarriers for targeted biomolecule delivery and precise pollen tube modulation, advancing crop reproductive engineering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/710033v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@a09c01org.highwire.dtl.DTLVardef@623943org.highwire.dtl.DTLVardef@9d954corg.highwire.dtl.DTLVardef@1b4c35b_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Efficient and controllable delivery of genome-editing proteins remains a central challenge for therapeutic translation of gene-editing technologies. Extracellular vesicles (EVs) offer an attractive non-viral delivery modality due to their biocompatibility and large capacity for cytosolic cargo delivery. Yet, rational strategies to achieve controlled and programmable protein loading are still lacking. Here, we present NEO-TOP-EVs, an EV biogenesis-guided engineering platform that systematically integrates key features of three design principles inspired by vesicle formation: 1) PI(4,5)P2-mediated plasma membrane targeting, 2) ESCRT-dependent membrane scission, and 3) self-assembly-driven cargo clustering for enabling efficient encapsulation of genome-editing ribonucleoproteins. Together, the NEO design increased cargo incorporation and enhanced functional delivery of gene editing modalities under particle-normalized conditions. Using NEO-TOP-EVs, we achieve efficient delivery of Cas9 and adenine base editor ribonucleoproteins without nucleic acid templates. In an in vitro proof-of-concept, delivery of an adenine base editor targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) induces efficient splice-site disruption, resulting in reduced PCSK9 expression and enhanced LDL receptor activity. Proof-of-concept in vivo experiments provide preliminary evidence of functional Cre protein delivery to the liver. Together, these findings establish NEO-TOP-EVs as a modular platform for protein-based genome editing, demonstrating how biogenesis-informed EV engineering yields functional protein delivery at levels relevant to therapeutic development.

In vivo genome editing with CRISPR-Cas9 ribonucleoproteins (RNPs) holds substantial therapeutic promise, yet rapid bloodstream clearance and the absence of delivery systems capable of systemic tumor targeting have hindered its clinical translation. Herein, a supramolecular ternary complex platform is reported in which Cas9/sgRNA RNPs are co-assembled with tannic acid (TA) and phenylboronic acid (PBA)-conjugated polymers through sequential self-assembly, producing [~]30 nm core-shell ternary complexes that protect RNPs from enzymatic degradation and dissociate selectively at endosomal pH. Upon intravenous administration in subcutaneous tumor-bearing mice, these ternary complexes exhibit prolonged blood circulation and preferential tumor accumulation, achieving 37.2% gene editing at tumor sites compared with only 1.5% for free RNPs. The platform successfully knocks out previously undruggable oncogenes including mutant KRAS and polo-like kinase 1 (PLK1), markedly suppressing tumor growth in vivo. By integrating sequential supramolecular self-assembly with stimuli-responsive cargo release, this strategy establishes a generalizable framework for systemically administered in vivo CRISPR therapeutics.

Extracellular vesicles (EVs) are promising vehicles for nucleic acid delivery, yet efficient delivery of circular RNA (circRNA) remains challenging due to inefficient loading and limited intracellular expression. Here, we establish an EV-based platform that enables efficient circRNA delivery via in situ biogenesis and sorting. By optimizing intracellular circularization and translation through vector design, we markedly enhance circRNA expression. By combining this with Snu13-mediated EV sorting and enhanced vesicle biogenesis, we achieve efficient packaging of circRNA without compromising vesicle integrity. This integrated strategy enables robust and sustained protein expression following EV-based circRNA delivery. By leveraging this platform, we demonstrate a dendritic cell-targeting circRNA vaccine that elicits strong antigen-specific CD8+ T cell responses and antitumor efficacy. We further show that systemic delivery of BNP-encoding circRNA attenuates doxorubicin-induced myocardial fibrosis. Together, this work establishes a generalizable platform for circRNA therapeutics by overcoming key barriers in circRNA expression and EV-mediated delivery. TeaserEngineered EVs enable efficient circRNA delivery for sustained protein expression and therapy.

The cytosolic delivery of therapeutic proteins remains one of the most persistent challenges in modern drug delivery. Here, we report the discovery and characterization of an encapsulin-based protein nanocage, QtEnc, with unexpected permeability properties and the ability to internalize cargo proteins in vitro, fundamentally departing from existing protein nanocage cargo loading paradigms. This permeability enables simple, rapid, and single-step post-assembly cargo loading, accommodating cargos as large as 482 kDa, and allowing multiplexed cargo co-encapsulation with tunable ratios. Leveraging this property, we develop a modular QtEnc-based NanoCarrier (QtEncNC) with a pH-responsive cargo detachment module and an endosomal escape module, enabling low pH-triggered cargo release from assembled shells and subsequent endosomal escape for cytosolic delivery. Using a cytotoxic protein, BLF1, as a proof-of-concept QtEncNC payload, we demonstrate efficient cytosolic protein delivery in HeLa cells. These findings establish QtEncNC as a versatile and modular platform for cytosolic protein delivery with broad biomedical potential.

Self-assembled DNA nanostructures show great promise as functional devices, highly configurable materials, and in nanorobotics. Magnetic control can provide a powerful actuation mechanism in a broad range of contexts, since it affords a high-level of external control, it is biocompatible, and orthogonal to chemical or electrical stimuli. Here we demonstrate magnetic molecular nanoactuators by leveraging the unique site-specificity of DNA origami to assemble highly anisotropic magnetic nanocubes on high-aspect ratio DNA origami bundles. We traced and controlled 100s of our DNA origami nanorotors at the single-rotor level and demonstrated their programmable magnetic clamping and controlled rotation under uniform and rotating magnetic fields. By varying the population and inter-particle spacing of the nanocubes, magnetic torque values in the order of 10-100 pN nm are achieved at field strengths < 10 mT. Monte Carlo simulations reveal that assembly of nanocubes on DNA origami rotors leads to collective magnetic properties, with numerically estimated torque values in good agreement with the experiments. Our magnetic nanorotors offer a foundation for biocompatible nanorobotics, as well as high-throughput magnetic force and torque tweezers.

The nanofabrication of functional protein-based surfaces is challenging due to the chemical complexity of proteins and their unpredictable behavior at the solid-liquid interface. Many proteins of interest -such as antibodies or large enzymatic complexes - lack strong and dynamic protein-protein and protein-surface interactions necessary to drive self-assembly of stable arrays with high surface coverage. Additionally, adsorption-induced conformational changes at the solid-liquid interface could lead to a loss of activity and increase the risk of undesirable interfacial processes. Here we introduce SAKe, a kelch-like designer protein, as a versatile platform to address these challenges. Ancestral sequence reconstruction led to high thermal stability, and the high symmetry allowed modularity of the proteins core. Rational engineering of the bottom side allowed SAKe to form large (up to 5 micrometers in length), well-defined and pH-dependent two-dimensional assemblies while maintaining structural integrity, which is key for further development of functional materials. SAKe self-assembly was investigated through in-liquid atomic force microscopy on muscovite mica. High resolution imaging confirmed the integrity of the SAKe protein upon adsorption on the solid-liquid interface. These results showcase the SAKe protein as a platform for the further engineering of functional protein-based two-dimensional materials.

Nucleic acid nanostructures provide programmable architectures for molecular delivery but remain limited by rapid nuclease degradation, poor in vivo persistence and inefficient intracellular cargo release. Here we report a mirror-image L-DNA nanocube as a biologically persistent and modular therapeutic delivery platform. The nanocube self-assembles from synthetic L-DNA oligonucleotides into a structurally defined architecture that exhibits substantially enhanced resistance to enzymatic degradation and prolonged stability under physiological conditions compared with the corresponding D-DNA nanostructure. Surface functionalization with folic acid enables selective tumour targeting in vitro and in vivo. The L-DNA nanocube supports the delivery of chemically distinct therapeutic cargos, including doxorubicin, a bortezomib prodrug and MCL1-targeting small interfering RNA (siRNA). In tumour-bearing mice, L-DNA nanocube-mediated delivery improves therapeutic efficacy while reducing systemic toxicity relative to free drug and D-DNA nanocube controls. For siRNA delivery, we engineer a pH-responsive release mechanism that promotes endosomal escape and cytosolic cargo localization, as visualized by cryo-electron tomography, resulting in efficient gene silencing. Together, these results establish mirror-image nucleic acid nanostructures as a class of biologically functional nanomaterials for programmable intracellular therapeutic delivery.

Oligonucleotide therapeutics hold transformative potential, yet their clinical translation is hindered by delivery barriers, including rapid renal/hepatic clearance and poor organ specificity. Bottlebrush polymers conjugates have emerged as a promising vector to address these limitations, but conventional architectures with uniform backbones can only achieve an unmodifiable, rigid biodistribution profile. Here, we report a library of sequence-defined "digital" bottlebrush polymers, precisely engineered with controlled placements of chemical motifs that modify physiochemical properties - including lipids, cholesterol, and cationic groups - along a polyphosphodiester backbone. Systematic evaluation of the digital bottlebrush polymer library reveals distinct structure-property relationships and enables organ-biased systemic delivery to several traditionally difficult-to-reach tissues, including muscle and skin. In a mouse model of rheumatoid arthritis, a single dose of a spleen-homing polymer-conjugated antisense oligonucleotide targeting TNF- achieves potent knockdown and drives full functional recovery. These findings establish a versatile design framework for tailoring bottlebrush polymers to specific therapeutic applications.

Optical barcodes for pooled high-throughput screening must support large libraries while remaining decodable in a single imaging step. Existing approaches often trade design control for manufacturability: deterministic barcodes often require per-code redesign of particle fabrication, whereas stochastic combinatorial barcodes are difficult to generate as predefined batches. Here we introduce a chemically programmable barcoding architecture that decouples particle fabrication from barcode assignment. Using a contact-free multilaminar flow lithography platform with all-around three-dimensional sheathing, we continuously fabricate a universal hydrogel scaffold containing five spatially segregated DNA-addressable domains at rates >106 particles/h. Chosen barcode identities are subsequently written on demand onto the same template batch by domain-selective DNA hybridization. Single-domain measurements resolved 64 candidate optical states, indicating an experimentally informed theoretical upper bound of 645 {approx} 1.1 x 109 barcodes. We further implemented a predefined 59,049-code library by split-pool labeling, achieving an 88% recovery of decoded beads at a stringent posterior threshold (>0.95). After 11 days, >7,800 beads were correctly re-identified at >0.95 accuracy in matched fields of view. This strategy provides a highly scalable, chemically programmable route to build large, user-defined optical barcode libraries with single-image optical readout and longitudinal traceability.