ACS Nano

Environmental degradation and accumulation of plastics results in micro- and nanoplastics (MNPLs) that are small enough to cross biological barriers, including the blood-brain barrier. Microglia, resident immune cells of brain, are critical regulators of neuroimmune homeostasis and represent a cellular target of nanoplastic exposure. In this study, we assessed the neurotoxic effects of two sizes of polystyrene nanoplastics (PS-NPs; 100 nm and 500 nm) using integrated in vivo and in vitro exposure and washout paradigms. In vivo exposure in mice (60 days; 0.15 or 1.5 mg/day) showed the accumulation of both PS-NP sizes in the cerebral cortex without histopathological damage. However, cortical microglia showed pronounced morphological remodeling, observed as increased expression of Iba1 and GFAP. Transcriptomic profiling of cortical tissue revealed a strong size-dependent response. The 100 nm PS-NP group revealed 18 DEGs (|log2FC| [&ge;] 2, padj < 0.05), whereas the 500 nm PS-NPs showed more than 4,000 DEGs, including upregulation of immune- and microglia-associated genes (CCL5, CXCL10, LCN2, LYZ2) and downregulation of synaptic and neuronal signaling genes (GRIN2B, SYN1, STX1B, MAP1B, ITPR1/2). In vitro assessment, using BV2 microglia cells, showed internalization of PS-NPs via the endolysosomal pathway, with strong co-localization to Rab7- and LAMP2-positive compartments and prolonged intracellular retention following exposure washout. Also, microglial activation markers (Iba1, CD68) exhibited a transient, size- and concentration-dependent increase, correlated with intracellular particle burden rather than cumulative exposure. Overall, these findings demonstrate that PS-NPs accumulate in brain, driving size-dependent microglia activation and transcriptomic reprogramming, even after cessation of exposure to PS-NPs. HighlightsO_LIPS-NPs (100 nm and 500 nm) reach mouse cerebral cortex following 60-day oral exposure. C_LIO_LIPS-NPs were internalized by microglia; accumulated in endolysosomal compartments. C_LIO_LIPS-NP exposure induced transient microglial activation without sustained cytotoxicity. C_LIO_LIMicroglial activation was correlated with intracellular PS-NPs burden. C_LIO_LITranscriptomics revealed disruption of neuroimmune and microglial regulatory pathways. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/712727v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1aba3eaorg.highwire.dtl.DTLVardef@1967641org.highwire.dtl.DTLVardef@12da637org.highwire.dtl.DTLVardef@1fb8441_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bacterial Outer Membrane Vesicles (OMVs), spherical bilayered nanoparticles naturally released by all Gram-negative bacteria, are gaining increasing interest not only in the design of prophylactic vaccines but also in cancer immunotherapy. In particular, thanks to their potent built-in adjuvanticity and to their intrinsic capacity to directly kill tumor cells, OMVs have been successfully tested in intratumoral in situ vaccination (ISV), a strategy in which immunostimulatory formulations are injected directly into tumors to convert the tumor microenvironment (TME) into an immune-reactive state. Previous studies have shown that OMVs induce robust inflammation and a Th1-skewed immune response, resulting in complete tumor remission in a substantial fraction of mice bearing syngeneic tumors. Here, we show that OMVs from our Escherichia coli {Delta}60 strain can be efficiently engineered with multiple cytokines and chemokines. Moreover, CCL3, Flt3L, TNF, and IL-2 not only accumulated on the OMV surface but also retained their in vitro biological activity. Furthermore, OMVs displaying these cytokines exhibited potent antitumor activity, and in particular the intratumoral injection of the combined TNF- and IL-2-engineered OMVs eradicated tumors in over 95% of mice across several syngeneic models. Immunostaining and flow cytometry analyses revealed that injection of engineered OMVs markedly remodeled the TME, promoting the recruitment of inflammatory myeloid cells and {gamma}{delta} T cells, the persistence of local CD8 and CD4 {beta} T cells, and the reduction of regulatory T cells. Overall, these results highlight cytokine-bearing OMVs as a versatile and highly effective platform for intratumoral immunotherapy.

Detection of micro- and nanoplastics (MNPs) in human tissues has raised growing concern about their biological effects on tissue and cell function. While previous studies have examined MNP-cell interaction, most focused on limited cell and plastic types. Here, we present a comprehensive, quantitative investigation into how different types of nanoplastics (NPs) associate with and affect diverse cell types under physiologically relevant conditions. Using microfluidic-calibrated fluorescence microscopy, we quantify NP accumulation in cells in vitro and match cellular NP concentrations to levels reported in human tissues. While cell-associated NPs could be gradually released in vitro, they persist in vivo for over one month without detectable reduction in a mouse model. We discover that NP exposure at these levels broadly impairs cell proliferation across epithelial, endothelial, fibroblast, and immune cells, with cell type-dependent sensitivity. NP exposure also reduces motility in T cells and fibroblasts, with more complex effects observed in macrophages. Mechanistically, NP-cell association and trans-epithelial transport involved not only classical endocytic regulators but also pathways related to ion and water transport. Notably, NP association and release were highly sensitive to the extracellular fluid environment within the physiological range. By testing inhibitors of these pathways, we identified molecules that reduce NP-cell association and promote release. We further compared common NPs found in human samples and widely used in research: polystyrene (PS), polyethylene (PE), and polypropylene (PP). Although these NPs similarly impaired proliferation and motility, they showed markedly different cellular association and release dynamics. These findings reveal the impact of NPs on tissue cell functions and uncover novel regulatory pathways, establishing a quantitative framework for studying NP-cell interactions in biologically relevant conditions.

The superior stealth properties and high information density make DNA a sought-after candidate in the field of molecular steganography. Here, we developed the InfinMark end-to-end DNA steganography framework for anti-counterfeiting applications by combining the characteristics of both the Internet of Things (IoT) and DNA-of-Things (DoT). InfinMark includes five modules: Information Transcoding, Fingerprint Writing, Nano-encapsulation, Invisible Marking, and Multi-level Rapid Authentication. It ensures precise anti-counterfeiting information reading and writing through a dynamic DNA-compatible transcoding algorithm, achieves seamless embedding by developing scalable nanoparticle manufacture methods, and supports cross-scenario on-site verification, ultimately granting it comprehensive anti-counterfeiting capabilities spanning from source labeling to terminal tracing. By addressing the bottlenecks in IoT and DoT integration, lifecycle tracking, as well on-site product authentication, this research constructs a full-chain bimodal anti-counterfeiting system, thereby showcasing the practical application of informational DNA nanoparticles in various aspects of production and daily life.

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.

Cathodoluminescence (CL) microscopy has the potential to achieve a key goal in biological imaging: the simultaneous visualization of proteins and cellular ultrastructure. This goal can be attained by tagging proteins of interest with spectrally distinct cathodoluminescent probes for detection in electron microscopy. To this end, lanthanide nanoparticles (LNPs) are promising probe candidates due to their stability under the electron beam and their distinct ion-dependent emission spectra suitable for multiplexed detection. However, the hydrophobic surface chemistry of LNPs limits their use in biological samples and requires surface functionalization compatible with aqueous environments and EM sample preparation protocols. Here, we use a DNA-based ligand exchange strategy that renders cathodoluminescent LNPs hydrophilic and compatible with further functionalization for specific protein labeling. We characterize the CL emission of DNA-functionalized LNPs following aqueous transfer and common EM preparation steps, including osmium tetroxide staining and drying protocols based on hexamethyldisilazane and critical point drying, and show that LNPs retain their CL emission under all tested conditions. Finally, we demonstrate multicolor CL imaging of spectrally distinct, DNA-functionalized LNPs on the surface of mammalian cells, enabling simultaneous visualization of cellular ultrastructure via secondary electrons and LNPs via multiple CL color channels.

Continuous biochemical sensing provides valuable insights into an individuals physiological state and the mechanisms underlying pathophysiological changes. However, most existing bioanalytical methods are not compatible with continuous biochemical sensing. A major technical challenge lies in achieving rapid measurement readouts while maintaining high specificity and sensitivity in complex biological fluids. Sensitive molecular detection typically requires slow analyte-binder dissociation and long incubation to reach equilibrium, whereas rapid and frequent measurements demand fast association-dissociation kinetics that are difficult to reconcile for low-abundance analytes. To address this challenge, we introduce a sensing mechanism termed photothermal recycling (PTR), which mimics the thermal cycling process in polymerase chain reaction. Using plasmonic photothermal effects, PTR rapidly recycles binders to enable frequent measurements. We demonstrate a digital PTR assay capable of multi-hour biochemical monitoring with subpicomolar(pM) sensitivity in buffer, diluted serum, and saliva. This approach leverages localized thermal energy to dynamically modulate biomolecular recognition, offering a new bioanalytical paradigm for continuous biochemical sensing across diverse application settings.

Ingestible electronics enable the tracking and treatment of gastrointestinal and systemic diseases. However, bulky batteries and circuit boards require large capsules that can result in bowel obstruction, a medical emergency. Here, we engineered a 9 x 26 mm electronic pill capable of triggered severing into tiny pieces with sizes clinically proven to reduce obstruction risk. Our capsule enables multicomponent circuit boards to connect with separately encapsulated powering elements via conductive, interlocking connections. Heat induced softening of polyethylene glycol/polycaprolactone channels activates a spring to separate encapsulated components into inert 9 x 15 mm segments, facilitating intestinal passage. Separation triggers included closed-loop sensors and time-delay circuits. In vivo swine studies demonstrate the ability of our capsules to sense luminal oxygen changes via an optoelectronic sensor, locally trigger upadacitinib delivery, and facilitate safe excretion.

Super-resolution microscopy with DNA-PAINT enables molecular-scale, multiplexed, and quantitative imaging, but its throughput is limited by slow binding kinetics and elevated background at high probe concentrations. Recent speed-optimized and fluorogenic probes improve performance but impose strong constraints on sequence design, revealing a fundamental tradeoff between fast binding and efficient quenching. Here, we introduce a modular probe architecture that spatially decouples binding kinetics from fluorophore-quencher interactions by integrating speed-optimized sequence motifs with PEG spacers. Using DNA origami nanostructures, we demonstrate enhanced localization rates, signal-to-background ratios, and imaging efficiency compared to state-of-the-art probes. We validate our approach in cells, demonstrating its capability to image nuclear targets and enabling three-dimensional imaging of the endoplasmic reticulum using standard widefield illumination. Our work establishes a general framework for fast, multiplexed, and low-background super-resolution imaging.

Over the past decades, the frequency of viral outbreaks has increased substantially worldwide, driven in part by global travel and resulting in millions of deaths each year. This trend underscores the urgent need for rapid, simple, and accessible diagnostic tools for infectious disease detection. Here, we present a nanofluidic digital chip (Nano-dChip) for point-of-care viral RNA detection that delivers results within 30 minutes at a cost of less than $0.50 per chip. The Nano-dChip employs reverse transcription loop-mediated isothermal amplification (RT-LAMP) for highly sensitive and specific target amplification. Reaction reagents are compartmentalized into numerous nanofluidic reservoirs, enabling highly quantitative detection while minimizing contamination risks. Using a single chip, we successfully detect both SARS-CoV-2 and Influenza H3 RNA with a detection limit of 10 fM, demonstrating the Nano-dChips potential as a rapid, low-cost, and scalable diagnostic platform for timely outbreak control.

Early interactions between viruses and live cells are difficult to resolve due to rapid extracellular motion, 3D nature of the cell membrane, and the fast, nanoscale interactions involved. While actin is a central regulator of viral entry, direct observations of actin-aided trafficking have been restricted to membrane protrusions on glass surfaces given the limitations of conventional methods. Here, high-speed 3D Tracking and Imaging microscopy (3D-TrIm) is integrated with highly photostable StayGold-labeled SARS-CoV-2 virus-like particles to capture long-term, high-resolution single-virus trajectories in live cells. This approach revealed distinct regimes of viral dynamics, including extracellular diffusion, protrusion-based surfing, and an unreported linear trafficking mode along the plasma membrane that precedes viral internalization. This work demonstrates that this membrane trafficking is actin-driven and positively correlated with ACE2 expression. These findings reveal new actin exploitation by viruses and demonstrate the utility of 3D-TrIm for dissecting dynamic virus-cell interactions at high spatiotemporal resolution.

Rapid and highly selective sensing of ultra-low concentration protein biomarkers remains a critical challenge important for early disease diagnosis and monitoring. Here, we use conical SiO2 nanopore-based biosensing for the rapid detection of heart-type fatty acid binding protein (H-FABP). Antibodies were covalently immobilized on the nanopore surface through siloxane chemistry. The functionalized asymmetric nanopores generate a characteristic rectifying current-voltage response, which shows a distinct shift upon binding to the target protein due to partial neutralization of the negatively charged pore surface. The sensor exhibits excellent sensitivity in the attomolar to nanomolar concentration range with a detection limit (LOD) of [~]0.4 aM. Furthermore, the platform exhibits high selectivity, distinguishing H-FABP from non-target proteins (HSA and Hb) at concentrations six orders of magnitude higher. We also demonstrate that nanopores can be regenerated using sodium hypochloride and O2 plasma treatment, enabling repeated functionalization and reuse.

Multimode fibers enable minimally invasive, high-resolution imaging through ultrathin probes, thereby enhancing diagnostic precision and facilitating real-time monitoring in delicate anatomical regions. In this work, we introduce HYFEN, a hydrogel-based endomicroscopic imaging platform for flexible, biocompatible, and subcellular-scale fluorescence microscopy. HYFEN leverages the unique properties of hydrogel materials, adaptive optics, and pixel-wise image enhancement to address challenges associated with silica-based fibers, including mode scrambling, limited field of view, and mechanical rigidity. The technique achieves precise mode threading, rapid diffraction-limited focusing at kilohertz speeds, and high-fidelity fluorescence signal acquisition with subcellular resolution. Notably, the approach extends fluorescence imaging under enhanced fiber dimensions and bending conditions that are unachievable with conventional modalities. Together, these advances establish HYFEN as a versatile platform for next-generation biointerfacing and minimally invasive imaging across biomedical and clinical settings.

Targeted lipid nanoparticles (tLNPs) represent the next frontier in nucleic acid therapeutics, enabling cell-specific delivery through covalent attachment of targeting ligands that drive receptor-mediated uptake. tLNPs are particularly promising for pregnancy-associated applications where precise on-target delivery is required to minimize maternal toxicity and protect fetal health. Yet, their rational design is limited by an incomplete understanding of how tLNP physicochemical properties influence biological performance. Conventional LNPs already exhibit pronounced heterogeneity in size, composition, and RNA loading, which is further amplified in tLNPs by variability in ligand attachment and surface density. Because traditional analytical methods report only ensemble-averaged properties, the nanoscale diversity of tLNPs remains unresolved. Here, we find that tLNP functional behavior is governed by previously inaccessible, structurally distinct tLNP subpopulations that are not captured by bulk measurements. We utilize asymmetric flow field-flow fractionation integrated with in-line UV spectral analysis, light scattering, and synchrotron small-angle X-ray scattering (AF4-UV-DLS-MALS-SAXS) to resolve ligand-dependent tLNP subpopulations that differ in size, shape, composition, and relative abundance. We find that protein conjugation preserves the internal lipid-RNA nanostructure of base LNPs but substantially increases particle heterogeneity, particularly for larger and multivalent targeting ligands. Despite increased heterogeneity, tLNPs functionalized with higher-avidity ligands achieve more effective targeted placental RNA delivery in mice, suggesting that binding avidity can offset the functional consequences of polydispersity. Chemometric SAXS analyses reveal that only SAXS-resolved tLNP subpopulations, not ensemble-averaged parameters, correlate with targeted placental transfection in vivo, whereas bulk-derived physicochemical metrics more strongly associate with nonspecific hepatic delivery. Together, this work harnesses a separation-coupled biophysical platform to resolve previously inaccessible tLNP subpopulations and demonstrates that subpopulation nanoscale structure, rather than bulk-averaged properties, dictates targeted RNA delivery. These insights provide a mechanistic foundation for rational engineering of next-generation precision targeted RNA LNP therapeutics.

Immune cell receptor - ligand interactions are key to cancer immunotherapy. However, receptor-ligand affinities often fail to predict T-cell mediated cancer killing, while immune-target cell binding strength measurements are limited by low precision and high non-specific binding. Here we present bilayer acoustic force spectroscopy (BAFS), a method to quantify the binding strength of receptors in immune synapses that virtually eliminates non-specific binding and increases the resolving power by up to 50-fold. By replacing target cells with a supported lipid bilayer functionalized with antigens, BAFS avoids antigen-independent interactions and target cell heterogeneity, while maintaining the spatial self-organization of receptors that typifies active immune synapses. We demonstrate the high sensitivity and control by showing how CAR T-cell synapse strength depends on CD19 antigen density, and by revealing that CD8 synergistically strengthens {beta}TCR-pMHC synapses independently of Lck recruitment to CD8. BAFS is a general method that can be used broadly in immunotherapy screening and to dissect the complex molecular interactions that underpin immune synapse activation.

Understanding how airborne particulates disrupt the alveolar barrier requires in vitro systems that recapitulate both the structure and transport properties of the lung air-blood interface. Here, we report a biodegradable lung alveoli-on-a-chip enabled by porous poly(lactic-co-glycolic acid)/polycaprolactone (PLGA/PCL) membranes with an interconnected porous architecture generated via porogen-assisted phase separation process. The membrane exhibits tunable degradation behavior, allowing progressive increases in surface porosity ([~]40%) and reduction in thickness ([~]3 {micro}m) during culture, while PCL maintains mechanical integrity under dynamic conditions. These degradation-driven structural changes regulate membrane transport properties, leading to enhanced permeability and supporting the formation of a functional epithelial-endothelial barrier under air-liquid interface (ALI) culture with breathing-mimetic cycling strain. Primary human alveolar epithelial and microvascular endothelial cells formed confluent, junctional monolayers on opposing membrane surfaces, exhibiting stable barrier function and high viability throughout the culture period. As a functional application, the platform was used to assess diesel particulate matter (DPM)-induced alveolar injury. Apical exposure to DPM induced dose-dependent cytotoxicity, increased barrier permeability, elevated reactive oxygen species, and DNA damage in both epithelial and endothelial layers, demonstrating trans-barrier propagation of particulate-induced injury. Pharmacological modulation with roflumilast-N-oxide (RNO), a phosphodiesterase-4 (PDE4) inhibitor, selectively attenuated oxidative stress and inflammatory responses, with limited effects on barrier integrity. Together, this work establishes degradable PLGA/PCL membranes as tunable interface materials for lung-on-a-chip systems, where structural evolution during degradation directly governs transport and barrier function. The resulting platform provides a physiologically relevant approach for studying particulate toxicity and therapeutic modulation at the alveolar interface.

Stimulator of interferon genes (STING) is a promising therapeutic target for cancer immunotherapy, but agonists are often rendered ineffective by the loss of STING expression in cancer cells. Here we engineer a multivalent peptide-polymer conjugate material that can easily be delivered to the cytosol, where it mimics key protein interactions from the missing STING protein to directly activate downstream innate immune signaling. While previously developed STING mimicking therapeutics use nearly the full STING protein, this material contains only a 39 amino acid peptide from the STING C-terminal tail that includes interaction motifs for downstream kinase TBK1 and transcription factor IRF3. Conjugation of multiple peptide copies to a negatively charged polymer backbone mimics the multivalent protein-protein interactions of the oligomerized STING signaling complex, activating TBK1 and IRF3 as well as the transcription of downstream genes in both STING-proficient and STING-silenced cancer cell lines. We optimize a lipid nanoparticle formulation to deliver this conjugate material intracellularly, allowing for its application as an immunotherapy for ovarian cancer. Treatment with the STING mimicking conjugate material promoted the production of type I interferons, repolarization of myeloid cells to an anti-tumor phenotype, and recruitment of T cells to tumors in mice. This treatment ultimately led to tumor regression and extended survival in multiple mouse models of metastatic ovarian cancer. Overall, this work highlights the potential of peptide-polymer conjugate mimics of STING to therapeutically activate innate immune signaling.

Novel strategies for treating bacterial infections are needed to combat the growing threat of antibiotic resistance. Here we sought to engineer and produce phage-like particles to either harness the microbiome to secrete therapeutics or to hijack pathogenic bacteria for treatment and prevention of disease. For this, we used the P2/P4 system to design, produce and test P4 phage-mediated single- and dual-action antimicrobial prototypes. Upon successful completion of the in vitro proof of concept experiments, we focused on optimizing early-stage bioprocessing for in vivo studies, leading to 1011 plaque forming units (PFU) per mL and 0.25 endotoxin units (EU) per 109 PFU. We also challenged the P4 viral vector packaging limit by deleting the sid gene to package the payload into P2-sized capsids ([~]25.8 kb cargo capacity). Importantly, repressing the therapeutic payload during the production of particles improved viral titers about 2 logs, maintained viral payload sequence integrity and improved post-transduction functional activity. Altogether, this study demonstrates the potential of novel phage-based antimicrobials to go beyond elimination of bacteria. The in vitro optimized P2/P4 system constitutes a promising platform technology for in vivo evaluations of targeted antimicrobial candidates paving the way for future antimicrobial research in animal models of infection.

Antimicrobial peptides (AMPs) are emerging as promising alternatives to conventional antibiotics, and growing evidence indicates a fundamental link between antimicrobial activity and amyloid-like self-assembly. Many AMPs are known to form amyloid-like fibrils, while several amyloidogenic peptides exhibit intrinsic antimicrobial properties, suggesting shared underlying physicochemical determinants such as amphipathicity, {beta}-sheet propensity, and charge distribution. However, the rational design of peptides that simultaneously encode these dual functionalities remains a significant challenge. Here, we present amyAMP, a generative deep-learning framework based on a Wasserstein generative adversarial network with gradient penalty (WGAN-GP), designed to learn and generate peptides with integrated antimicrobial and amyloidogenic properties. Trained on curated datasets of antimicrobial and amyloid-forming peptides, amyAMP captures the latent sequence-property relationships governing dual functionality. Statistical and latent-space analyses demonstrate that the generated peptides closely overlap with biologically relevant peptide space while remaining distinct from random sequences, indicating successful learning of key biochemical features. To validate functional behavior, we performed extensive coarse-grained molecular dynamics simulations to probe membrane interaction, peptide self-assembly, and membrane disruption. The simulations reveal rapid membrane adsorption, stable amphipathic insertion, and strong peptide-peptide aggregation. Notably, cooperative clustering of peptides on membrane surfaces induces membrane thinning and curvature perturbations, highlighting a mechanistic coupling between aggregation and antimicrobial activity. Collectively, these results establish that amyAMP effectively captures the shared physicochemical principles underlying antimicrobial action and amyloid-like self-assembly. This work provides a generalizable framework for the AI-guided design of multifunctional peptides to advance the development of next-generation therapeutics targeting antimicrobial resistance.

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.