Nature Nanotechnology

DNA nanotechnology offers precise, biocompatible structures with strong potential for targeted drug delivery, yet current discovery approaches rely on testing individual designs, limiting exploration of structural diversity. Here, we introduce an evolutionary selection strategy that screens large libraries of DNA nanostructures, each folded from a single-stranded structure genome compatible with amplification and sequencing. Cellular internalization is used as the selection pressure: libraries are incubated with mammalian cells, internalized structures are recovered from lysates, and the process is iterated across multiple rounds in HEK293T and RAW264.7 cells. High-throughput sequencing of recovered structure genomes reveals cell-type-specific enrichment patterns, enabling the identification of individual nanostructures with preferential uptake. Selected candidates were synthesized and evaluated as purified structures, confirming differential internalization by quantitative flow cytometry and microscopy. Turning DNA nanostructure discovery into a selection-based process, could enable high-throughput exploration of structural diversity and provide an alternative route to identify nanostructures with cell-specific uptake properties for biomedical applications.

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.

Chirality in two-dimensional nanomaterials provides a powerful lever to control biological interfaces, yet the structural origins of nanoscale chirality remain poorly understood. Here, we systematically investigate chiral ligand modulated graphene quantum dots (GQDs) and reveal how ligand stereochemistry and edge chemistry modulate the formation of diverse chiral or achiral nanostructures. Spectroscopy, microscopy, and density functional theory with ring-puckering analysis identified six structural motifs, twisted-, twisted-boat, saddle-shaped, hybrid, unbuckled, and random. Among these, twisted-, twisted-boat, and saddle-shaped GQDs exhibited genuine nanoscale structural chirality, while unbuckled, hybrid, and random conformations lacked organized distortion. Importantly, structural chirality governed passive permeation into biological membrane (e.g. lipid membrane of extracellular vesicles), whereas achiral variants relied mainly on hydrophobic interactions. In contrast, active transport across biological membrane (e.g. endocytosis) is insensitive to nanoscale structural chirality but strongly influenced by chiral ligand identity and transporter recognition. Collectively, these results establish chiral ligand conjugation as a modular route to program both chiral and achiral motifs in graphene nanostructures and highlight nanoscale structural chirality as a design principle for engineering bio-nano interactions.

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.

We previously developed synthetic RNA-protein (sRNP) granules as stable, programmable, genetically encoded nanoparticles. Here, we adapt this platform into high-avidity sialogranules that inhibit influenza virus entry. Natural or synthetic 2,6-sialylated proteins and peptides were fused to RNA-binding domains and assembled with synthetic long non-coding RNA to form phase-separated glyco-nanoparticles. Using the sialic-acid-binding lectin Sambucus nigra agglutinin (SNA), we identified a selective transition from sialogranules to tri-component multiphasic biocondensates that occurs only for sialylated constructs. The structural features of this transition quantitatively correlate with predicted sialylation density, enabling extraction of an effective avidity constant. Enzymatic desialylation with neuraminidase or Endo H abolished SNA binding and restored native granule morphology. Guided by this assay, LAMP1-based sialogranules were selected and inhibited influenza entry by [~]50% in cell culture. These results establish sRNP sialogranules as programmable glyco-nanoparticles integrating glycan sensing with antiviral decoy activity.

Periodontitis-associated systemic inflammation makes it a great challenge to explore therapeutic options applicable to periodontitis and atherosclerotic comorbidities. Here, we identify the crucial role of cell-free DNA (cfDNA) that underlies these comorbidities. Hypothesizing cfDNA as a therapeutic target, we engineer polyamidoamine dendrimer-functionalized nanomaterials to modulate such local-systemic inflammatory crosstalk. Periodontium-originated DNA can be systemically captured by cationic nanomaterials, and capturing cfDNA, whether locally or systemically, alleviates both periodontitis and atherosclerosis prior to severe atherosclerotic development in vivo. The transcriptomic and single-cell RNA sequencing analyses together reveal that cfDNA-capturing nanomaterials regulate inflammatory foam cell transformation in macrophages by modulating the expression of lipid-related foamy markers Spp1 and Fabp4. This study provides a proof of concept for cfDNA-driven periodontitis-atherosclerosis crosstalk, and offers a cfDNA-capturing nanoplatform for therapeutic intervention targeting periodontitis and atherosclerotic comorbidities in a holistic fashion.

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.

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

Plant-derived extracellular vesicles (PEVs) are promising candidates for oral drug delivery, yet their clinical translation is hindered by limited targeting precision and inconsistent systemic absorption. While surface engineering can enhance tissue accumulation, strategies that preserve biocompatibility and enable scalable production remain limited. Here, we introduce a simple thermal processing approach, boiling, to structurally reconfigure ginger EVs into functionally enhanced, thermally reassembled nanoparticles (B-GEVs). The surface architecture of B-GEVs is enriched with key vesicle trafficking regulators, including V-type proton ATPase subunit G, ARF1, and {beta}-adaptin-like protein. This specific composition drives their tissue-specific accumulation in the intestine and liver and potentiates clathrin-dependent cellular uptake in intestinal cells by 8.57-fold. Beyond superior intrinsic anti-inflammatory activity through NLRP3 inflammasome suppression, B-GEVs function as an efficient oral delivery platform. When loaded with TNF- siRNA, they enable a synergistic therapy that simultaneously modulates upstream inflammation and silences key downstream mediators, showing potent efficacy in colitis. Our findings position boiling as a natural strategy for enhancing the bioactivity and targeted oral delivery potential of ginger-derived EVs.

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.

Efficient siRNA delivery by lipid nanoparticles (LNPs) is widely attributed to carrier composition, yet how intraparticle packing governs function remains unclear. Here, we developed a single-particle fluorescence microscopy assay that simultaneously quantifies size and siRNA loading of individual, chromophore-labeled LNPs. Imaging [~]0.5M particles per hour uncovered two major packing modes: a high and a low order corroborated by cryo-EM. Quantitative live cell imaging on destabilized eGFP reporter cell line combined with systematic variation of LNPs lipid composition and N/P ratio allowed deconvolution of the interplay between siRNA packing, cell internalization and silencing and its dependance on lipid composition and electrostatics. Our findings surprisingly revealed that low-order particles while encapsulating modest RNA, they mediate more efficient knockdown of the destabilized eGFP reporter than their high-order counterparts. Guided by these findings we predicted and experimentally validated that tuning composition and N/P ratio to favor less compact siRNA packing enhances silencing potency. This framework offers actionable guiding for the rational optimization of LNP formulations for RNA therapeutics.

Post-translational modifications (PTMs) underpin much of protein regulation, yet their single-molecule readout remains a challenge in nanopore proteomics. While biological nanopores have shown exquisite PTM sensitivity, the microscopic mechanisms by which PTMs perturb signals in solid-state nanopores are largely unexplored. Here, we use all-atom molecular dynamics to investigate how three common PTMs, acetylation, phosphorylation, and methylation, modulate the translocation of a cancer-relevant p53 peptide fragment through a bilayer graphene nanopore. We find that PTMs remodel the translocation landscape far more strongly at the level of dwell-time statistics than at the level of mean current blockade. Acetylation enhances peptide-graphene adhesion and substantially slows transport, with adjacent acetylations producing the longest residence times due to cooperative interfacial interactions, while remotely spaced acetylations yield broader, heterogeneous dynamics. Phosphorylation introduces a negative charge that increases dwell time through an electrostatic tug-of-war, while also generating the largest current blockade among the PTMs studied. In contrast, methylation minimally perturbs translocation due to weak pore interactions and preserved charge. Combining dwell time with relative blockade features enables a simple linear SVM classifier to reliably distinguish unmodified, acetylated, and phosphorylated states. These results establish mechanistic design principles for PTM detection using solid-state nanopores and delineate which classes of PTMs are the most amenable to single-molecule detection with these devices.

Size exclusion within biological hydrogels imposes a fundamental constraint on the design of nanocarriers, limiting the transport of cargo-loaded and structurally complex materials through mucus barriers. While surface passivation strategies are commonly used to improve compatibility, they do not address steric limitations imposed by the polymer network. Here, we introduce mechanical flexibility as an independent materials design parameter to expand the functional transport window of nanocarriers in mucus. Using programmable DNA origami to decouple flexibility from size and surface chemistry, we show that increased structural compliance enhances transport under steric confinement by facilitating passage through confined network pores. When surface-driven aggregation dominates, passivation is required to restore transport, after which flexibility provides additional gains. Together, these results establish mechanical flexibility as a general materials design strategy for improving transport under size-constrained conditions, with implications for nanocarrier engineering across biological barriers.

Premature clearance and limited organ targeting remain major barriers for nanoparticle (NP) drug delivery. Hitchhiking NPs on red blood cells (RBCs) can enhance circulation and organ-selective accumulation, but most approaches require ex vivo RBC extraction and reinfusion, limiting clinical translation. Here, we report an in situ RBC-hitchhiking strategy, named i-Bind, which employs polyphenol surface functionalization to enable spontaneous NP attachment to RBCs directly in the bloodstream. Driven by strong interactions of phenolic motifs with RBC membranes, i-Bind NPs exhibited markedly enhanced and more stable hitchhiking onto RBCs under flowing whole blood conditions. In both healthy and diseased mice, i-Bind NPs selectively target the lungs, resulting in an over 20-fold increase in lung-to-liver deposition ratio compared to unmodified NPs. Additionally, i-Bind NPs show preferential targeting to distinct lung immune cell subsets in a pathology-dependent manner, including cDC2s in healthy lungs, neutrophils in acute lung injury, and cDC1s in lung metastases. In a melanoma lung metastasis model, delivery of the STING agonist diABZI via i-Bind NPs significantly inhibited lung metastasis progression by reprogramming the lung immune microenvironment. Collectively, i-Bind provides a simple and versatile platform for organ-selective drug delivery and immune reprogramming. TeaserSurface functionalization of nanoparticles enables in situ red blood cell hitchhiking, unlocking new paths for organ-selective immune reprogramming

DNA-encoded library (DEL) technology enables high-throughput small-molecule discovery but is typically performed using purified proteins under in vitro conditions that do not reflect native intracellular environments. Here, we present a microfluidic agarose -droplet platform for cellular-context DEL screening. The porous hydrogel droplets provide mechanically stable yet permeable microenvironments that protect weak protein-ligand interactions while enabling efficient buffer exchange and ligand diffusion. Importantly, mild cell permeabilization within droplets selectively retained chromatin-associated proteins, allowing screening directly in a cellular context. Using BRD4 as a model target, we validated intracellular ligand engagement by fluorescence imaging and super-resolution microscopy. Small-scale DEL screening selectively enriched JQ1 in both bead-based and cell-based formats, and large-scale DEL screening across millions of encoded compounds successfully identified hit molecules by sequencing. This agarose -droplet-based strategy expands DEL technology toward biologically relevant and chromatin-associated targets under near-native conditions.

Lipid nanoparticles (LNPs) are a versatile platform for in vivo delivery of biomolecules, yet systemically administered LNPs predominantly accumulate in the liver, limiting extrahepatic applications. This tropism arises from LNP adsorption of serum proteins, particularly apolipoprotein E (ApoE), which binds to LDL receptors (LDLR) on hepatocytes. Here, we overcome this tropism with two compatible strategies. First, we engineer dead ApoE mutants (dApoE) with five receptor-binding domain substitutions that selectively disrupt the ApoE-LDLR interaction but retain lipid binding. In cultured cells, pre-coating with these dApoE markedly inhibited LDLR-mediated uptake. Second, we pretreat cells with hyperactive PCSK9 (haPCSK9) to internalize surface LDLR, similarly reducing the LDLR-mediate uptake of LNPs. In vivo, both strategies substantially reduced liver LNP transduction without inducing redistribution to other major organs. To retarget LNP to new cell types we combined antibody conjugation with dApoE or haPCSK9, effectively engineering tropism to T cells, brain and lung tissues in vivo with substantially reduced hepatic background. In pilot studies, this strategy enabled specific delivery of reporter mRNAs to additional tissues, including megakaryocytes, hematopoietic progenitor cells, and cardiac tissue, and in aged T cells, to deliver miRNA cargos that produced a sustained reduction in DNA damage markers following a single systemic dose. dApoE coated CD5-targeted LNPs generated CAR+ T cells that retained cytotoxicity against CD19+ targets, while simultaneously reducing hepatocyte transduction by 90%. These findings establish a modular framework that integrates dApoE and haPCSK9-mediated detargeting with antibody-based retargeting, allowing for improvements in LNP specificity and broadening the therapeutic scope of LNPs.

BODIPY dyes are widely used in bioimaging, yet their aggregation behavior within the plasma membrane (PM) remains poorly exploited for single-molecule localization microscopy (SMLM). Here, we design a series of green-emitting PM-targeted BODIPY dimers engineered to undergo spontaneous and transient H- and J-aggregation. By tuning the linker length between the fluorophores, we identify dimers that form intramolecular H-aggregates in polar media and emissive J-aggregates ({lambda}em {approx} 535 nm) through membrane-driven intermolecular interactions. In lipid bilayers, all dimers aggregate above a probe/lipid ratio of 1/100, exclusively generating J-aggregates. In live-cell SMLM, the monomeric MB-488 provides high event numbers via diffusion-driven emission, whereas dimers exhibit stable blinking and yield brighter red-shifted J-aggregate events with improved localization precision. Red-channel events localize to specific PM regions, suggesting preferential J-aggregation within distinct membrane microdomains. HaloTag constructs targeted to cell-surface PDGFR further confirm that intramolecular J-aggregation is possible but strongly amplified by the membrane through intermolecular collisions. These results demonstrate that green BODIPYs and their J-aggregates enable robust live SMLM in green and red channels, and reveal the PM as a privileged environment for emissive J-aggregation.