ACS Nano

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.

Self-assembled peptide-based nanostructures have diverse applications in the pharmaceutical and materials fields, but accurately predicting their self-assembly behavior without time-intensive organic synthesis and characterization remains a significant challenge. Here, we assess the effectiveness of AlphaFold3 (AF3), a deep learning model for protein structure prediction, in modeling peptide-based nanostructures and the interactions driving supramolecular self-assembly. We designed amphiphilic peptides composed of alternating hydrophobic residues (valine, leucine, isoleucine, phenylalanine) and hydrophilic residues (glutamic acid), varying both sequence length and residue order. Using AF3s multimer mode, we modeled assemblies with copy numbers ranging from 10 to 1000, generating diverse morphologies such as micelles and nanotubes. We qualitatively analyzed hydrophobic regions, secondary structures, and intermolecular interactions, while also calculating radii of gyration, packing scores, and aspect ratios using PyRosetta. Our results indicate that AF3 predicts morphologies consistent with hydrophobic driving forces and steric constraints. Increased hydrophobicity correlates with smaller radii of gyration, while higher copy numbers correspond to smaller aspect ratios (more compact structures). Longer hydrophobic segments lead to disordered structures, whereas longer hydrophilic segments promote organization. While AF3 captures systemic trends consistent with biophysical principles, comparisons to literature reveal discrepancies driven by charge effects and secondary structure bias, including an overemphasis on helical propensity (e.g., alanine-rich sequences) and sensitivity to terminal charge repulsion. Additionally, since AF3 is predisposed to predict a single assembled entity rather than higher-order assemblies such as multiple micelles or fibers, finding the optimal copy number for the best prediction requires system-specific iteration. These limitations highlight the need for complementary approaches with controlled chemical potential and environmental conditions, though qualitative agreement with experimental trends in morphology and compactness supports AF3s utility for initial structure generation. Our findings highlight AF3s potential as a user-friendly design tool for structure generation in peptide design, aiding the efficient development of functional self-assembled peptide nanomaterials.

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

While engineered nanomaterials offer unprecedented precision in targeting tumor cells, their efficacy is often limited by rapid clearance from the bloodstream via the mononuclear phagocyte system (MPS). To overcome this limitation, a promising strategy known as MPS-cytoblockade has been developed. This approach involves administering antibodies against host erythrocytes. The resulting saturation of the MPS with erythrocyte clearance creates a critical window, allowing subsequently administered nanoparticles to evade immune surveillance and circulate for a significantly extended period. However, MPS-cytoblockade induces a transient reduction in hematocrit, which can lead to adverse effects. Here, we demonstrate that approaches to restore hematocrit, specifically through the administration of donor erythrocyte suspension or the hormone erythropoietin, effectively prevent this drop while maintaining the efficacy of the MPS-cytoblockade. Notably, these interventions do not compromise the prolonged circulation time of the nanoparticles or alter their biodistribution, preserving high accumulation in tumors. Our findings establish a viable strategy to mitigate a key side effect of MPS-cytoblockade, thereby enhancing its therapeutic potential and safety profile.

The protein corona (PC) that forms on the surface of nanomaterials upon contact with biological fluids provides a molecular snapshot of the hosts physiological and pathological state. Here, we investigate two-dimensional (2D) titanium carbide (Ti3C2Tx) MXene nanosheets as nanobiointerfaces for capturing Alzheimers disease (AD)-associated plasma protein signatures. Ti3C2Tx MXene flakes were incubated with plasma from clinically diagnosed AD patients and age-matched healthy controls (HC), leading to the formation of Ti3C2Tx MXene-PC complexes. Physicochemical characterization using dynamic light scattering, zeta potential analysis, and transmission electron microscopy revealed disease-dependent changes in hydrodynamic size, surface charge, and PC profile. Proteomic analysis of the isolated PC layers quantified 1,611 proteins without prior fractionation, demonstrating effective enrichment of low-abundance plasma components. Principal component analysis (PCA) revealed consistent separation between AD- and HC-derived Ti3C2Tx MXene-PC proteomes despite inter-individual heterogeneity. Differential abundance analysis identified selective enrichment of heterogeneous nuclear ribonucleoproteins (hnRNPs), annexins, and inflammatory mediators in AD-derived PC, implicating dysregulated RNA metabolism, membrane stress responses, and immune activation, hallmark processes in AD pathology. Our findings demonstrate that Ti3C2Tx MXene-PC interfaces act as selective molecular filters that reshape the detectable plasma proteome, enabling disease-associated molecular phenotyping and establishing a versatile nanointerface-driven framework for uncovering AD-related plasma signatures, providing a foundation for future translational diagnostic development.

Clot resistance to pharmacological thrombolysis remains a critical challenge in ischemic stroke (IS) management. Thrombus heterogeneity, particularly the presence of thrombolysis-resistant domains composed of dense fibrin and non-fibrin components, including neutrophil extracellular traps (NETs), significantly limits the efficacy of recombinant tissue-type plasminogen activator (r-tPA) and its variant, Tenecteplase (TNK). Consequently, novel therapeutic strategies are urgently required. Emerging evidence suggests that co-administration of deoxyribonuclease I (DNase I) with r-tPA can degrade DNA fibers and enhance clot lysis. In this study, we optimized a previously developed theranostic agent--iron oxide microparticles coated with polydopamine--by dual-grafting both r-tPA and DNase to target resistant thrombi. Using functional ultrasound imaging (fUS) during the acute phase of IS, we demonstrated accelerated reperfusion with this dual-functionalized platform in a r-tPA resistant IS model. Furthermore, MRI analysis confirmed a significant reduction in lesion volume at 24 hours, correlating with improved functional recovery five days post-ischemia.

A major limitation across nanoparticle delivery platforms is sequestration within endosomal compartments, which restricts access to intracellular targets despite efficient cellular uptake. Here, we show that peptide architecture can be used to control intracellular trafficking and reduce endosomal accumulation in lipid-protein nanocarriers. Specifically, we fuse R6W3 (RRWWRRWRR), an amphipathic cell penetrating peptide, to the N- or C- terminus of the nanodisc scaffold proteins and systematically evaluate its impact on membrane interactions and cellular behavior. Structural and biophysical characterization confirms that R6W3 incorporation preserves nanodisc assembly and protein-lipid interactions, enabling direct attribution of functional differences to peptide-driven interfacial effects. R6W3-functionalized nanodiscs exhibit enhanced binding and cellular uptake, with N-terminal fusion producing the strongest interfacial interactions. In live cells, R6W3-functionalization increases endocytic activity, evidenced by increased formation of clathrin-coated pits and intracellular colocalization with clathrin-coated vesicles. Notably, R6W3-funtionalized nanodiscs display reduced accumulation in early endosomes relative to unmodified nanodiscs, indicating decreased endosomal sequestration following endosomal uptake. These trafficking differences translate to functional outcomes, as doxorubicin-loaded, R6W3-functionalized nanodiscs achieve greater cytotoxicity than unmodified controls at equivalent concentrations. Together, these results establish peptide architecture as a design parameter for controlling intracellular trafficking and overcoming endosomal bottlenecks, providing a broadly applicable strategy for improving nanocarrier- based delivery systems.

Nanopores enable single-molecule analysis by measuring current signals through nanoscale pores in either biological or solid-state membranes. Accurate detection of analyte fingerprints within the pore environment is essential for reading-out the analyte type. We develop a framework for robust and label-free detection of the molecular nanopore events using a graph representation of the measured signals. To this end, we build a graph-based two-stage workflow based on a convolutional and graph neural networks that first perform a fast screening of the nanopore events, followed by a deep validation of these. The learned model can thus efficiently and in an unsupervised manner select possible molecular signatures (the current blockades) in the full signal, denoise, validate, reconstruct these, and predict the morphology of unseen molecular events. We could show that the learned model can efficiently predict the correct event morphology for the same analyte within a 2.4-fold range of transmembrane voltage values not included in the training. The developed graph-based workflow is modular, generalizable, and provided that it is trained on a huge amount of different nanopore experiments has the potential to become a blueprint model for nanopore read-out. Such a read-out model would be able to identify subtle differences in molecules like proteins, as well as their conformational or folding states. The proposed framework is developed using experimental signals from DNA translocation through an aerolysin pore and demonstrates a unified approach linking unsupervised feature learning to raw-signal inference for single-molecule sensing.

Herpes simplex virus 2 (HSV-2) is associated with genital ulcers, neonatal encephalitis, increased risk of HIV infection, and dementia. There is no licensed HSV-2 vaccine. We developed nanoparticles displaying the HSV-2 attachment protein gD and fusion mediation protein complex gH/gL. Immunization of mice and non-human primates elicited high levels of neutralizing antibodies. Vaccination conferred robust protection in mice, preventing disease and nearly eliminating infection and shedding following HSV-2 challenge. While gD induced high neutralizing antibody titers, gH/gL contributed substantially to protection despite lower neutralization titers. Instead, gH/gL immunization generated strong fusion-blocking responses which were an important correlate of protection, showing that standard neutralization assays incompletely capture the importance of fusion-blocking activity. These findings demonstrate that targeting both HSV-2 attachment and fusion elicit complementary mechanisms for protection from infection and that neutralizing antibody alone may be insufficient for protection. Overall, these results present an innovative strategy for an HSV-2 vaccine.

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.

Endosomal escape of mRNA remains a critical bottleneck in oligonucleotide therapeutics, with typically less than 5% of delivered mRNA reaching the cytosol. Precise quantification of this escape is hindered by stochastic variability in uptake, release, and expression, while existing methods lack scalability and accuracy. Here we introduce RNASCAPE, a deep learning framework trained on biologically realistic simulations that estimates unlabelled mRNA cytosolic escape efficiency using only three timepoints of EGFP reporter expression and four lipid nanoparticle (LNP) meta-parameters. Benchmarking on synthetic data shows RNASCAPE achieves a mean absolute percentage accuracy of 78%. RNASCAPE accurately predicts escape efficiencies around 5-9% across diverse cell types and revealed that exchanging cholesterol to {beta}-sitosterol drastically reduces mRNA loading and enhances twofold its functional release. By enabling robust, microscopy-agnostic quantification without requiring specialized cell lines or labeled cargo, RNASCAPE provides a scalable framework for benchmarking and rational design of LNP formulations, advancing nucleic acid therapeutic delivery.

Transport in complex biological tissues is governed by local rheological heterogeneity at the nanoscale, yet probing such environments deep inside living systems remains challenging. Here, we introduce an orientation-sensitive single-particle tracking (SPoT) approach that simultaneously resolves translational and rotational dynamics of individual carbon nanotubes deep within biological tissue. By exploiting the intrinsic dipole-like emission and shortwave infrared luminescence of carbon nanotubes enhanced through the incorporation of quantum color-centers our method enables long-duration tracking with high signal-to-noise ratio in optically dense environments. Crucially, the length of these nanotubes can be precisely shortened down to a few tens of nanometers to adapt to diffusion environmental dimensions, further optimizing the tracking applicability. SPoT of single carbon nanotubes provides access to relative changes in local viscosity, steric constraints, and environmental anisotropy. When applied to the brain extracellular space, SPoT demonstrates that local variations in the translational and rotational diffusion of tracers are heterogeneous and not systematically correlated. This allows to disentangle the local effects of viscosity and spatial tortuosity within the brain extracellular space, which are distinct features that would otherwise remain undetected through translational diffusion analysis alone. By enabling combined translational and rotational tracking of nano-emitters over unprecedented depths and timescales, this work establishes a new framework for probing nanoscale transport and rheological heterogeneity in intact biological tissues and more generally in complex diffusive environments.

Proteins in solution adsorb to the corona of nanoparticles such as single-walled carbon nanotubes (SWCNTs), but these interactions are difficult to predict and analyze due to ambiguities in the structure of the latter. In this work, we employ ss(GT)15-DNA wrapped SWCNTs, a commonly used fluorescent sensor construct, to examine protein adsorption by quantifying binding dissociation constants and characterizing the corresponding photophysical effects. A library of 20 proteins are used to evaluate adsorption-induced changes in photoluminescence (PL) intensity ({Delta}I/I0) and emission wavelength upon solution phase binding. We find that 15 proteins produce monotonic dose-response behavior well described using a single-site Langmuir model. Alternatively, five proteins exhibited more complex, non-monotonic behavior consistent with a two-step binding model representing protein-protein interactions coupled to adsorption. The study reveals that metalloproteins, which comprised 12 of the 20 proteins in the library, induced greater PL quenching compared with metal-free proteins for this system, with maximum binding-associated quenching ({Delta}I/I0) of 94% for metalloproteins versus 20% for metal-free proteins. For metalloproteins, we introduce a proximity-based quenching framework in which protein size provides a coarse proxy for cofactor-SWCNT separation, offering a mechanistic interpretation of the observed quenching variation across proteins. Together, these results establish the use of metal coordination sites, such as those in metalloproteins, to assist the transduction of certain nanoparticle fluorescent sensors, helping with sensor probe design and interpretation in biological environments.

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.

Colorectal cancer (CRC) remains a major cause of cancer death, and advanced disease is still limited by resistance and systemic toxicity. We studied intrinsically active, biomimetic cerium oxide nanoparticles (CeNPs) functionalized with D- or L-cysteine (D-Cys@CeNPs and L-Cys@CeNPs) in three CRC cell lines (COLO-201, DLD-1, and LoVo) and healthy colon fibroblasts (CCD-18Co). We propose these materials act as enantioselective functional keys: cysteine stereochemistry shapes recognition at the nano-bio interface, while productive interactions allow the Ce3-rich surface to drive localized redox exchange. We measured viability, ROS as a downstream phenotypic readout, Annexin V/PI-defined cell fate, and expression of the NF-{kappa}B regulatory genes TNFAIP3 (A20), IKBKG (NEMO), and NFKBIA (I{kappa}B). Across the CRC panel, D-Cys@CeNPs caused earlier and stronger loss of viability, with the clearest effect in COLO-201, and shifted cells toward late apoptosis and necrosis. In contrast, L-Cys@CeNPs produced slower and more heterogeneous fate changes. Gene expression showed enantiomer-dependent differences in NF-{kappa}B feedback, consistent with differential pathway engagement. CCD-18Co fibroblasts were comparatively resistant to both enantiomers. Together, these findings link chiral CeNP surface design to redox-linked pathway regulation and support a materials-based route to selective anticancer activity. INTRODUCTION

DNA double-crossover (DX) molecules, comprising two Holliday junctions connected by two duplex arms, are fundamental building blocks of DNA nanostructures, but their mechanical properties remain poorly understood. Here we investigate the elasticity of isolated antiparallel DX motifs with 18 to 22 base pairs between the crossovers. Using mechanical models parameterized by extensive all-atom molecular dynamics simulations, we demonstrate that the bending rigidity of the duplexes within a DX motif is highly anisotropic, and that this anisotropy results from long-range elastic couplings involving all the duplex base pairs between the crossovers. The duplex stretch modulus decreases due to localized defects, while the twist stiffness is close to that of an isolated duplex. The DX core as a whole follows an analytical beam theory in bending but not in torsion. Our results extend beyond local elastic models of DNA nanostructures and pave the way for probing peculiar mechanical properties of other key motifs for DNA and RNA nanotechnology.

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.

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.