ACS Nano

The co-occurrence of per- and polyfluoroalkyl substances (PFAS) and volatile organic compounds (VOCs) in industrial environments poses complex toxicological risks that standard additive models fail to capture. This study elucidates a novel "metabolic blockade" mechanism wherein PFAS competitively inhibits the renal excretion of VOC metabolites, thereby amplifying neurotoxic burdens. Utilizing a Double Machine Learning (DML) framework on data from National Health and Nutrition Examination Survey (2005-2020), we analyzed a final intersectional cohort of 1,975 participants. We identified a robust inhibition of VOC metabolite clearance by serum PFAS. Specifically, PFNA significantly suppressed the excretion of the benzene metabolite URXPMA (Causal {beta}TMLE = -0.219, p < 0.001), with efficacy dependent on perfluorinated chain length. Molecular docking simulations revealed the biophysical basis of this antagonism: long-chain PFNA exhibited superior binding affinity to the Organic Anion Transporter 1 (OAT1) ({Delta}G = -6.333 kcal/mol) compared to native VOC metabolites ({Delta}G = -4.957 kcal/mol), confirming high-affinity competitive inhibition at the renal interface. In a neurocognitive sub-cohort (N = 1,200), this interference translated into functional synergism; high-PFNA exposure magnified VOC-associated cognitive impairment by 1.5-fold and significantly exacerbated the negative association between VOC burden and processing speed ({beta}int = -0.263, p = 0.004). These findings define PFAS as a "metabolic amplifier" of co-contaminant toxicity, necessitating a paradigm shift toward mixture-based hazardous material regulations that account for transporter-level interactions.

Accurate characterization of the nanoparticle (NP) protein corona is essential for predicting biological fate, safety, and therapeutic efficacy, and for enabling robust biomarker discovery. Standard isolation techniques, most commonly centrifugation and magnetic separation, are widely used, yet they rarely account for co-isolating endogenous biological NPs such as extracellular vesicles (EVs). This oversight can distort the apparent "biological identity" of the NP. Here, we quantitatively demonstrate the magnitude and impact of EVs on the perceived protein corona composition. We incubated highly monodisperse polystyrene NPs (50-1000 nm) and superparamagnetic beads in either standard human plasma or plasma depleted of EVs by immunoaffinity capture targeting 37 EV surface epitopes. Mass spectrometry revealed that EV depletion reduced the number of proteins identified on polystyrene NPs by 60-75% and on magnetic beads by 45-50%. Importantly, EV depletion also altered the apparent abundance hierarchy; it restored the expected relative abundance and rank of major plasma proteins such as albumin and shifted the top-ranked proteins from intracellular cytoskeletal component, consistent with EV carryover, to genuine soluble plasma adsorbates (e.g., apolipoproteins, complement factors). These results highlight that standard corona workflows can inadvertently co-isolate a vast array of EV-associated proteins, yielding inaccurate proteomic profiles. Discriminating genuine corona proteins and EV-associated contaminants is critical for advancing nanomedicine, ensuring predictive safety and efficacy profiles, and enhancing the precision of NP-based biomarker discovery.

Efficient integration of proteins into amphiphilic polymer membranes offers new opportunities in synthetic biology and nanotechnology. Long-term protein reconstitution into artificial membranes remains challenging due to a lack of stabilising protein-membrane interactions found in native lipid bilayers. Here, we redesigned the transmembrane region of a CytK-4D {beta}-barrel nanopore for stable insertion into 3.5-6.6 nm thick PBD-PEO (poly(1,2-butadiene)-b-poly(ethylene oxide)) bilayers. PBD-PEO membranes offer high mechanical and chemical stability and low electrical noise, but the thick membrane hinders anchoring of biological nanopores. By systematically investigating the elongation of the {beta}-barrel, we engineered nanopore constructs suitable for PBD11PEO8 and PBD22PEO14 membranes. Efficient insertions were observed by adding amino acids that stabilised the transmembrane {beta}-barrel structure and enhanced anchoring of the nanopore into the membrane. Molecular dynamics simulations and single-molecule assays revealed that nanopores folded naturally into PBD-PEO bilayers, enabling successful detection of cyclodextrins and translocation of polypeptides and full-length proteins. Our study offers important lessons for the reconstitution of membrane proteins into artificial membranes. Moreover, these highly robust nanopore-membrane interfaces can be readily integrated into biosensing devices, enabling peptide and protein analysis directly from complex solutions.

mRNA vaccines have transformed prophylactic immunization against infectious diseases as well as therapeutic interventions for cancer. However, their effectiveness against emerging viral variants and a range of malignancies continues to be hindered by suboptimal induction of T cell-mediated immunity. To overcome this limitation, here we developed a polymer-lipid hybrid nanoparticle (PLNP) platform engineered to improve mRNA delivery to antigen-presenting cells (APCs) and to potentiate T cell responses. Relative to conventional mRNA lipid nanoparticle (LNP) vaccines, mRNA PLNP vaccines demonstrated markedly improved lymph node targeting, APC activation, Th1-biased pro-inflammatory cytokine response, and antigen-specific T cell expansion while retaining robust humoral immunity. Remarkably, mRNA-PLNP vaccines generated approximately 50% more antigen-specific CD8+ T cells than mRNA-LNP vaccines across multiple antigens, including SARS-CoV-2 spike, influenza hemagglutinin, and ovalbumin. In prophylactic applications, mRNA PLNP vaccine provided complete protection against SARS-CoV-2 variants. As a therapeutic approach in a melanoma model, mRNA PLNP vaccination resulted in enhanced tumor control and significantly prolonged survival compared to LNP-based formulations. Collectively, these results establish PLNP as a versatile and broadly applicable platform for augmenting mRNA vaccine efficacy through improved mRNA delivery and T cell priming, offering promising implications for infection prevention and cancer immunotherapy.

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

Plasmonic nanosensors - spanning nanopores, nanoantennas, and metasurfaces - achieve extreme electromagnetic (EM) field confinement that amplifies molecular interaction signals by orders of magnitude. Yet the full diagnostic potential of these platforms remains unrealised because the non-linear coupling between geometry, near-field physics, stochastic binding kinetics, and signal transduction is poorly characterised in biologically relevant systems. Here we propose the Plasmonic-AI-Organoid (PAO) framework: a modular, systems-level architecture linking (i) geometry-controlled plasmonic structures parameterised by Gaussian-process (GP) surrogate electromagnetic models; (ii) Bayesian inference of molecular kinetic parameters - association and dissociation rate constants, analyte concentrations - from noisy time-series sensor data using Metropolis-Hastings Markov chain Monte Carlo (MCMC); and (iii) human induced pluripotent stem cell (iPSC)-derived and patient-derived organoids as meso-scale biological validators. We formalise a forward model mapping geometry to EM field maps to reaction propensities to observable localized surface plasmon resonance (LSPR) signals, and an inverse model recovering posterior distributions over kinetic and geometric latent variables. A closed design loop employing active learning with expected-improvement acquisition functions iteratively proposes optimal geometries and assay conditions. Multi-objective Pareto optimisation balances analytical sensitivity, specificity, and manufacturability. Computational benchmarks demonstrate that active learning reduces the number of FDTD simulations required to identify near-optimal geometries by 3.2-fold compared with random search, while MCMC inference recovers kinetic parameters with sub-log-unit accuracy from synthetic time-series. The PAO framework provides a conceptual and fully reproducible computational roadmap for next-generation, AI-augmented plasmonic biosensing.

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.

Proteins exist in diverse biochemical states, including oligomers, complexes, heterogeneous proteoforms and shed fragments that encode functional and regulatory information. Yet, these molecular states remain difficult to resolve with existing analytical techniques, which typically require labelling, immobilisation or amplification and collapse this information into a single readout. Here, we introduce a modular platform that integrates programmable DNA nanostructures with single-molecule mass photometry for rapid, label-free and multiplexed protein analysis in native and complex media, without washing, enrichment or immobilisation, making native biochemical states observable directly in serum and plasma. DNA nanostructures act as nanosensors whose mass and mobility on supported lipid bilayers provide orthogonal identifiers for target identity and biochemical state, thereby decoupling recognition from readout. We define the analytical specificity and response window, demonstrate quantitative affinity determination, and resolve oligomeric and proteoform differences under native conditions. The nanosensors are rapidly reprogrammable to new targets, support multiplexed detection with internal controls for non-specific interactions, and enable selective resetting via strand displacement. Together, these capabilities establish nanoscale programmability as a route to state-resolved single-molecule protein profiling adaptable to both diagnostic and mechanistic applications.

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.

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.

We present a comprehensive analysis of an efficient lipid nanoparticle (LNP) formulation that exhibits strong nucleic-acid delivery and potent inhibition of targeted RNAs. Using a combination of coarse-grained and atomistic molecular dynamics simulations, we characterized the pH-dependent structure of both unloaded and RNA-loaded LNPs, elucidated the mechanism of RNA encapsulation, and used these models to propose a plausible mechanism of endosomal escape. Consistent with prior experimental and computational studies, our simulations reproduce an inverted-hexagonal-type morphology in RNA-loaded LNPs, in which a hydrated core provides a polar environment suitable for accommodating RNA, whose charge is neutralized mainly by ionizable lipids that remain protonated near the RNA even at high pH, thereby bridging the RNA with the surrounding lipid environment. This structural picture is consistently observed across our multiscale simulations, with smaller self-assembled LNP mimetics reproducing the same local organization at both coarse-grained and atomistic resolution. A potential mechanism of endosomal escape emerges spontaneously from the simulations, involving stalk formation between the LNP and the endosomal membrane, followed by the opening of a water-filled pore that permits slow RNA diffusion, in line with the low efficiency and slow kinetics reported for endosomal escape. The rate-limiting step of endosomal escape arises from persistent electrostatic coupling between RNA and protonated ionizable lipids maintained by the immediate RNA-lipid environment, hindering cargo disengagement even after pore formation. This delayed release is consistent with the experimentally observed time-dependent inhibitory activity of the loaded LNP. Together, these results highlight the importance of local pK and protonation effects near the RNA, which are not captured by global apparent pK measurements.

Mechanical forces at the cell-substrate interface govern processes from migration to differentiation, yet mapping these forces at high spatial resolution remains challenging. Traction force microscopy (TFM) addresses this by quantifying substrate deformations using fiducial markers, which are conventionally fluorescent beads. Here, we introduce fluorescently labeled DNA nanostructures (FluoroCubes) as alternative fiducials grafted onto polydimethylsiloxane (PDMS) substrates. Co-anchored with RGD peptides, FluoroCubes remain stably tethered, resist internalization, and enable dense, minimally perturbing labeling. This surface-functionalized platform is compatible with TIRF microscopy and leverages tunable biotin-NeutrAvidin chemistry for precise control of fiducial density. Using a modified multi-channel optical flow algorithm, we achieve improved displacement sensitivity and force reconstruction resolution compared to conventional algorithms. FluoroCube-functionalized substrates provide a reproducible, high-resolution method for traction force mapping and offer a versatile foundation for future integration with DNA-based molecular sensors to probe interfacial forces at biointerfaces.

Nanoplastics (NPs) are increasingly recognized as pervasive environmental toxicants, however, their interactions with gut and renal barriers, and the resulting systemic consequences remain poorly understood. Here, we studied the uptake of 50 nm polystyrene (PS) nanoparticles using a multi-scale approach integrating zebrafish models, isolated perfused mouse kidneys, and in vitro assays to delineate uptake and barrier-dependent organ distribution. In zebrafish larvae, PS-NPs were efficiently absorbed via the intestinal tract, as visualized by confocal and label-free stimulated Raman scattering (SRS) microscopy, leading to gut microbiota dysbiosis and systemic inflammatory responses. Despite widespread systemic dissemination, renal accumulation was minimal under physiological conditions, whereas both zebrafish and isolated perfused mouse kidneys exhibited substantial PS-NPs retention only when the glomerular filtration barrier was disrupted. In vitro glomerular endothelial cells and podocytes readily internalized PS-NPs without altering key glomerular identity markers, highlighting their intrinsic uptake capacity that is normally restricted in vivo by barrier integrity. Our findings establish the glomerular filtration barrier as a crucial gatekeeper that prevents renal nanoplastic deposition. Furthermore, we revealed a microbiota-mediated axis that may prime the kidney for the environmentally induced stressing in long term.

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.

Understanding the intracellular fate of nanoparticles is essential for designing safer and more effective nanomedicines, yet most studies rely on static observations and lack high-resolution, near-native volumetric information. Here, we establish a synchrotron-based correlative X-ray microscopy framework to investigate how fluorescent silica nanoparticles (SiNPs) redistribute within macrophages as a function of concentration and successive cell-division cycles. SiNPs were internalized by RAW 264.7 macrophages at different concentrations and analyzed using a synchrotron-based correlative X-ray microscopy workflow integrating cryogenic soft X-ray tomography (cryo-SXT), cryogenic structured illumination microscopy (cryo-SIM), and coherent X-ray ptychography, with confocal fluorescence microscopy used to establish population-level uptake tendencies. Cryo-SXT reveals a concentration-dependent redistribution of nanoparticle-containing vesicles from peripheral endosomes toward the perinuclear region, while correlative cryo-SIM confirms strict vesicular confinement, with no evidence of free nanoparticle diffusion into the nucleoplasm. At higher doses, nanoparticles approach the nuclear region via vesicles extending into nuclear-envelope invaginations, rather than by true nuclear entry. Successive cell divisions redistribute the intracellular nanoparticle load and promote stable perinuclear clustering, identifying a long-term sequestration route in macrophages. Coherent X-ray ptychography further reveals nanoscale deformations of the nuclear envelope associated with dense perinuclear vesicles. Together, these results establish synchrotron-based correlative X-ray microscopy as a mechanistic, multiscale platform for unveiling the dynamic intracellular fate of nanoparticles and providing mechanistic insight into their apparent nuclear localization.

Conventional subunit vaccines suffer from premature clearance and poor synchronization of antigen and adjuvant, limiting coordinated immune activation. Here, we develop an amphipathic polymer (YAXA) with acid-labile imine bonds for acid-sensitive degradation and terminal NHS-activated esters for antigen conjugation. YAXA is co-assembled with hydrophobic TLR7 agonist 3M-052 and conjugated to protein antigen, forming nanoparticle vaccine YM3.7. Following aerosolized intratracheal inoculation into the lung, YM3.7 is efficiently internalized by antigen-presenting cell and trafficked into lysosome, where acidic conditions trigger its dissociation and co-release of antigen and adjuvant. This spatiotemporally synchronized delivery promotes robust immune activation, including cell maturation, germinal center formation, systemic and lung-resident B/T cell response, and IgG/sIgA production. In lethal pneumonia models induced by either Pseudomonas aeruginosa or Staphylococcus aureus, YM3.7 markedly improves survival over conventional antigen and adjuvant mixture. This study presents an inhalable nanoparticle platform that coordinately activates innate, humoral, mucosal, and cellular immunity for enhanced protection.

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.

Inflammatory bowel diseases (IBDs) are frequently accompanied by anxiety and depression, largely driven by perturbed gut-brain axis signaling. However, current oral therapies remain constrained by the spatial and functional separation between intestinal inflammation and central nervous system dysfunction. Here, we present a comprehensive gut-brain dual region integrated therapeutic strategy based on functionalized Bifidobacterium longum hydrogel (INPs@BL@Gel), in which baicalin and tyrosine are coordinated with Fe(III) to form infinite coordination polymers (ICPs), coated with inulin, assembled onto Bifidobacterium longum (BL), and subsequently encapsulated within a pH- and matrix metalloproteinase-responsive silk fibroin-gelatin hydrogel. INPs@BL@Gel exhibits high drug-loading, effective gastric protection, inflammation-triggered release, and long-term intestinal colonization. Within the inflamed intestine, BL and components synergistically suppress inflammatory responses, restore gut microbiota homeostasis, and promote intestinal barrier repair through multi-target integrated therapy. Importantly, BL combined with components markedly enhances the production of beneficial neuroactive metabolites such as homovanillic acid and short-chain fatty acids, which integrated regulate neuroinflammation, preserve synaptic function, and facilitate blood-brain barrier repair via the gut-brain axis. In vivo studies demonstrate that INPs@BL@Gel not only exert potent therapeutic efficacy against colitis and effectively alleviate associated depression, but also reshape the gut microbiota and restore barrier integrity, achieving an remarkable comprehensive therapeutic effect. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/710940v1_fig1a.gif" ALT="Figure 11"> View larger version (59K): org.highwire.dtl.DTLVardef@1ceb3ceorg.highwire.dtl.DTLVardef@17ed1b4org.highwire.dtl.DTLVardef@f98f8corg.highwire.dtl.DTLVardef@3f6a46_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO (a) Schematic diagram of the design and preparation of functionalized Bifidobacterium longum hydrogel. (b) Exploration of the mechanism of INPs@BL@Gel in treating colitis-associated anxiety and depression through a dual-site multi-target synergistic strategy. C_FIG