ACS Nano

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.

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.

T-cell engagers (TCEs) for cancer immunotherapy have traditionally relied on high affinity single chain fragment variable (scFv) domains to target CD3, specifically the {varepsilon} chain, for the activation of T-cells. Despite their clinical success, there have been reports of TCEs driving systemic toxicity, non-specific T-cell activation, on-target off-tumor effects, and severe inflammation due to cytokine release. To address these limitations, we designed multivalent TCEs using Chemically Self-Assembled Nanorings (CSANs) that target the /{beta} constant region of the T-cell receptor (TCR) in the TCR/CD3 complex using a moderate affinity TCR nanobody (TCRVHH). Nanobodies offer superior physical and chemical properties over scFvs- including higher solubility, stability and lower production cost- making them increasingly popular as structural units of TCEs. We compared the efficacy and safety profile of this moderate affinity, nanobody-based TCR binder against high affinity CD3scFv based CSANs across EGFR and PSMA expressing solid tumor models. While the CD3scFv CSANs offered potent cytotoxicity, they also induced antigen independent T-cell activation bypassing the requirement of tumor crosslinking for cytotoxicity. In contrast the TCRVHH CSANs required strict antigen engagement to trigger cytotoxicity, significantly reducing non-specific T-cell activation and thus enhancing the safety profile. Although the initiation of cytotoxicity was kinetically slower than the CD3scFv counterpart, TCRVHH CSANs achieved comparable end point cytotoxicity across multiple antigen densities, as well as in 3D tumor spheroids. Through this study we demonstrate the applicability of nanobodies as T-cell targeting domains, enhanced specificity and safety of moderate affinity T-cells binders and the diversification of T-cell targeting epitopes without compromising the efficacy of TCEs.

Integrating autonomous electronics within single cells has remained beyond the reach of modern bioelectronics. Miniaturization at this scale could transform our ability to study and actuate biological processes at the cellular level, complementing existing molecular and fluorescent approaches. As these devices approach the sub-100-{micro}m length scale, volumetric constraints demand fundamentally new approaches to power delivery and telemetry. Here, we report an optically powered 10-picoliter complementary metal oxide semiconductor (CMOS) mote that operates with a power density of 1 pW/pL, comparable to the metabolic rate of cellular systems. These fully CMOS motes can be manufactured at scale yielding 1000 motes from a 4-mm2 silicon die. Multiple motes can be simultaneously powered and interrogated within a single optical field of view using epifluorescence microscopy. We demonstrate intracellular implantation of these motes within the single-celled mixotrophic dinoflagellate Noctiluca scintillans with negligible cytoplasmic displacement, pushing the boundaries of active CMOS bioelectronics to the intracellular domain and establishing a next-generation of truly cell-scale bioelectronic interfaces TeaserA 10-pL autonomous CMOS mote with fluorescence-based backscatter communication enables cell-scale sensing.

Antibody and protein-drug conjugates (XDCs) have emerged as promising cancer therapeutics, yet their clinical utility remains constrained by dose-limiting toxicities and narrow therapeutic windows. These safety challenges stem primarily from two factors: premature payload release during systemic circulation, and poor physicochemical properties inherent to the hydrophobic cytotoxic drugs they carry. Prior strategies attempted to address these limitations by appending water-soluble tags to reduce overall conjugate hydrophobicity, but achieved only modest improvements. As a result, the hydrophobic nature of cytotoxic payloads has remained a persistent obstacle in XDC development. Here, we report a fundamentally different chemical strategy that reframes this liability as a design opportunity. Rather than masking drug hydrophobicity, we exploit it as the driving force for self-assembly of facially amphiphilic protein-drug conjugates with programmable drug moieties (PDCs). In this architecture, the hydrophobic cytotoxic drug and the hydrophilic protein serve as the core and shell, respectively, spontaneously assembling into monodisperse, well-defined spherical protein nanotherapeutics of controlled size. This design principle transforms a longstanding physicochemical challenge into a functional engineering tool, enabling precise nanostructure formation without sacrificing potency. In vitro studies confirm that the resulting nanotherapeutics effectively kill cancer cells, establishing a strong foundation for further therapeutic development.

Antibody-based therapeutics have revolutionized disease treatment, and recent advances in messenger RNA (mRNA) technologies have opened new opportunities for their intracellular production. In particular, in vitro-transcribed mRNA encapsulated in lipid nanoparticles (LNPs) enables targeted delivery to specific cells, where it can enable the synthesis of therapeutic antibodies with prolonged half-lives in a cost-effective manner. Despite rapidly growing experimental data, a modeling framework that integrates mRNA delivery, intracellular expression kinetics, and whole-body antibody disposition remains unavailable. To address this gap, we extended a Physiologically Based Pharmacokinetic model with a novel multiscale layer describing mRNA trafficking, cellular uptake, translation, and degradation. The integrated model was calibrated and validated using five datasets of mRNA-based cancer therapeutics, demonstrating strong predictive performance for the biodistribution of mRNA-encoded antibodies. The newly introduced mRNA layer, while minimally parameterized, effectively represents complex intracellular and systemic processes, enabling quantitative investigation of antibody biodistribution, optimization of dose scheduling, and providing an initial framework for future exploration of how LNP-mRNA formulation influences delivery and pharmacokinetics.

Tetrahedral DNA nanostructures (TDNs) are promising nanocarriers due to their structural precision, biocompatibility, and efficient cellular uptake. However, their stability under physiological conditions remains a key challenge. In this study, TDNs were synthesized via a one-pot thermal annealing method and characterized using native PAGE, dynamic light scattering (DLS), and zeta potential analysis, confirming uniform size ([~]13 nm) and negative surface charge. Their stability was systematically evaluated across different biological media (DMEM complete, serum-free DMEM, and E3), temperatures (4 {degrees}C, 25 {degrees}C, and 37 {degrees}C), and pH conditions (4.0, 7.0, and 8.5) over 24 h. Results revealed rapid degradation in serum-containing medium, increased instability at higher temperatures, and reduced stability under acidic conditions, while serum-free, lower-temperature, and neutral to mildly basic environments enhanced structural integrity. These findings highlight the strong environmental dependence of TDN stability and provide insights for optimizing their design for biomedical applications.

Spatial organization and temporal regulation of membrane components are essential for achieving complex functions in artificial cells, such as cell division and signalling. DNA-based molecular tools provide a powerful means to control biomolecular interactions with high precision. Here, we investigate the phase behavior of cholesterol-modified, star-shaped DNA nanomotifs anchored to the lipid bilayers of giant unilamellar vesicles (GUVs), by using fluorescence confocal microscopy and cryo-electron microscopy. These motifs spontaneously anchor to the lipid bilayers via hydrophobic interactions and exhibit distinct spatial organization depending on their sticky end sequences. Motifs with complementary sticky end sequences interact and distribute uniformly, while orthogonal motifs with different sticky end sequences segregate into isolated gel-like domains with limited lateral mobility. Notably, the phase separation of motifs does not require lipid phase separation, indicating that DNA-driven organization can take place independently of lipid phase separation. The behavior of this system is governed by the interplay of three key parameters: (i) hydrophobic anchoring via cholesterol, (ii) electrostatic repulsion between negatively charged DNA nanomotifs, and (iii) sticky end interactions. The observed two-dimensional phase separation of orthogonal DNA nanomotifs at the GUV interface presents a novel strategy for controlling lateral membrane organization in GUV systems. This approach would offer flexibility in membrane composition and enables molecular positioning, thereby achieving a high degree of organization on the surface in artificial cell models.

The optimization of mRNA-lipid nanoparticles (mRNA-LNPs) for therapeutic applications is limited in part by the inadequate characterization of mRNA payload heterogeneity. One current challenge is accurately measuring the number of mRNA copies within individual LNPs, where the standard method of intensity-based mRNA number determination is sensitive to fluorescent dye-dye interactions and heterogeneity of mRNA labeling. Here we present a single-particle microscopy method that combines direct counting of the mRNA copies per LNP with LNP size measurements. While confined in microwells, individual mRNA-LNPs are lysed to release their cargo and stained with a dye such that the number of mRNA molecules in each well can be directly counted using fluorescence microscopy. Since the method stains the mRNA cargo in situ, it enables characterization of LNPs formulated with therapeutic grade (e.g., unlabeled) mRNA. We applied this approach to two Onpattro(R)-based LNP formulations prepared using different formulation buffers, where the two formulations had different average mRNA copy number, particle size, and fraction of LNPs lacking mRNA. The ability to directly count the number of mRNA molecules in LNPs establishes a complimentary method to intensity-based mRNA number determination and supports the characterization and screening of clinically relevant LNP formulations.

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

Nanoscale lipid bilayer mimetics are powerful tools for research on lipid bilayer, membrane proteins or for drug delivery. Established nanoscale bilayer systems that are stabilized by short peptides or polymers produce a broad size distribution and are difficult to customize. Here we introduce a DNA nanotechnology-based lipid bilayer mimetic, in which we covalently conjugated established nanodisc-forming amphiphilic peptides to oligonucleotides. These peptide-DNA conjugates were then hybridized with a circular single-stranded scaffold to form stiff, circular PDC minicircles with 14 peptide modifications at the inner rim of the torus. Lipid reconstitution yielded defined nanodisc with a tightly controlled circumference and component stoichiometry. Molecular dynamics simulations further validated the structural stability and reveal an asymmetric migration of the DNA to one rim of the bilayer. To mimic membrane protein insertion, we co-reconstituted a transmembrane peptide coupled to a bulky quantum dot. In future applications, the size and peptide arrangement can easily be modified in these DNA-templated PDC nanodiscs.

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

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

TRPA1 is a polymodal ion channel receptor known for its role in nociception. TRPA1 can be activated by local mechanical perturbations in the surrounding plasma membrane (PM) by molecules that insert in the lipid bilayer. Here, we tested whether TRPA1 function can be modulated by lipid nanoparticles (LNPs) while interacting with the target cell plasma membrane. We found that LNP induce irregular Ca2+ transients in heterologous and native TRPA1-expressing cells, which may reflect stochastic LNP-PM interactions. By using different cell types and applying selective and non-selective TRPA1 inhibitors, we revealed that the cytosolic [Ca2+] is elevated transients arise as a result through multiple mechanisms: TRPA1-dependent Ca2+ influx, TRPA1-independent Ca2+ influx, and Ca2+ mobilization from the endoplasmic reticulum. Our results describe a novel, non-canonical TRPA1 activation mechanism by LNPs, that may be relevant in the context of the development of cancer and nasal vaccines.

Engineered living materials (ELMs) harness the adaptive and self-replicating capabilities of biological systems to create functional materials for sensing, catalysis, and biomineralization. While most ELM strategies rely on static microbial assemblies, the role of bacterial motility in structuring living materials remains unexplored. Here, for the first time, we demonstrate how swarming motility in Escherichia coli MG1655 can be induced to guide spatio-temporally organized calcium phosphate mineralization. The mineralized calcium phosphate is characterized by scanning electron microscopy and elemental analysis. By systematically varying phosphate sources and their concentrations in calcium-rich media, we observe the emergence of regularly spaced concentric mineralized patterns. The previously undocumented observation of the concentric patterns was rationalized through a continuum model that captures the spatiotemporal coupling between swarm expansion and mineral deposition. The model shows that this coupling can generate recurrent front arrest and restart, leading to concentric ring formation. Finally, we show that altering the phosphate species results in distinct mineral morphologies. Together, this work establishes a novel framework for integrating bacterial swarming with biomineralization, enabling dynamic and programmable pattern formation in ELMs.

The gut microbiome can contribute to anti-tumour immunity and cancer therapy responses, but translating live microbe-based interventions remains challenging due to safety, controllability, and delivery constraints. Bacterial extracellular vesicles (BEVs) are an attractive cell-free alternative, as they package bacterial cargo into a nanoscale format capable of host-cell engagement, immunological activation, and systemic distribution. Here, we investigated the anti-tumour potential of BEVs derived from the human gut commensal Bacteroides thetaiotaomicron (Bt). We show that delivery route is a major determinant of efficacy. Intravenous, but not intraperitoneal, administration produced robust anti-tumour activity in a B16F10 melanoma mouse model. Intravenously delivered Bt BEVs suppressed primary tumour growth in a dose-dependent manner and reduced metastatic outgrowth in the lung. Bt BEVs did not directly impair tumour-cell viability in vitro, but they activated NF-{kappa}B and Toll-like receptor signalling in innate immune reporter systems and localised to tumour tissue following systemic administration. Together, these data support a model in which Bt BEVs act via host immune modulation rather than direct tumour cytotoxicity. These findings identify naturally produced commensal-derived Bt BEVs as a potential microbial therapeutic modality and as an alternative to the use of live bacterial administration in cancer therapy.