Nanoscale Advances

Extrahepatic delivery of lipid nanoparticles (LNPs) to non-phagocytic cells is a major challenge, with the leading strategy involving surface functionalization with target-specific monoclonal antibody (mAb) ligands. We investigate the stability of mAb-conjugated LNPs using two anchoring systems: the commonly used DSPE-PEG2kDa-maleimide and a block copolymer, PCL5kDa-b-PEG2kDa -maleimide, with the hypothesis that conjugation to a 150,000 Da antibody could overwhelm the relatively small ~600 Da aliphatic anchor on the PEG-lipid in vivo. Shedding of the mAB would compromise targeting. Conjugation integrity following IV injection was assessed by tagging LNPs and mAbs with metal ion tracers that could be quantified by ICP-MS. Results show that DSPE-PEG-mAb rapidly (within 1h) dissociates from LNPs in blood, leading to accelerated LNP clearance. In contrast, mAbs conjugated using PCL-b-PEG remained stably associated with the LNP over the 24h circulation and clearance of the construct. Results are connected to a thermodynamic model that reproduces experimental findings for PEG-anchor(-mAb) shedding in vitro and in vivo. This study identifies anchoring strength as a critical, unconsidered parameter for in vivo performance when conjugating mAbs to LNPs for extrahepatic delivery.

The protein corona influences the in vivo biodistribution of ionizable lipid nanoparticles (LNPs) in nucleic acid delivery, yet its structural architecture remains poorly defined. Using cryo-transmission electron microscopy, we visualized LNP-protein interactions in their native state. We show that, unlike the discrete "fuzzy" shells observed on hard nanoparticles, LNPs displayed no peripheral protein shell. Instead, controlled incubation and competitive "dual-particle" assays, supported by molecular dynamics simulations, indicate that LNP membranes undergo localized thickening and electron-dense remodeling consistent with lipoprotein integration rather than surface adsorption. Similar features were observed in extracellular vesicles, suggesting this behavior is shared among lipid-based carriers, and proteomic analysis identified apolipoproteins as the dominant associated proteins. Together, these findings support a model in which the biological identity of LNPs arises through membrane remodeling rather than shell-like adsorption, and provide a framework for the rational design of targeted nanomedicines. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/695162v2_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@5275d3org.highwire.dtl.DTLVardef@1b59ae4org.highwire.dtl.DTLVardef@1cc290eorg.highwire.dtl.DTLVardef@9b7bfb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Single-particle tracking (SPT) over time enables direct observation of molecular transport and interactions in living cells. Fluorescence-based SPT has provided insights into intracellular processes such as endocytosis, receptor signaling, and drug delivery. Extending the observation window to several hours and beyond is critical for capturing slow intracellular dynamics, including the full course of endosomal trafficking, the long-term accumulation of particles within subcellular compartments, and transitions between transport modes that occur on hour-scale timescales. However, long-term intracellular SPT under visible-wavelength excitation remains challenging because fluorescence probes generally suffer from photobleaching and phototoxicity. While near-infrared (NIR) excitation can simultaneously mitigate these issues, generally weak emission of NIR-emitting dyes has hindered its wide application in long-term SPT. Here, we demonstrate long-term NIR SPT using atomically precise gold quantum needles, Au42(PET)32 (PET = 2-phenylethanethiolate). Continuous tracking of intracellular particles in living HEK293 cells was achieved for up to 12 h. Trajectory analysis revealed temporal transitions between directional and diffusive transport, as well as the accumulation of multiple particles within localized intracellular domains over several-hour timescales. The high photostability of Au42, combined with low phototoxicity of NIR excitation, enables visualization of intracellular transport dynamics over timescales difficult to access using conventional visible fluorescent probes. These results establish Au42-based NIR imaging as a platform for long-term, low-phototoxicity intracellular SPT and provide a framework for investigating slow intracellular dynamics in living systems.

Light scattering in scintillators is a pervasive problem and a key factor limiting X-ray imaging resolution. Here, we shift scintillator radioluminescence from the traditional visible range into the short-wave infrared (SWIR) or near-infrared II (NIR-II, 1000-3000 nm) window to mitigate light scattering and thereby enhance light penetration and X-ray imaging resolution. We present an NIR II MgGa2O4:Ni2+ scintillator with peak emission at 1340 nm, achieving a threefold improvement in X-ray imaging resolution compared with visible scintillators owing to reduced light scattering. This heavy-metal-free NIR-II scintillator exhibits intense radioluminescence comparable to that of conventional visible-emitting CsI:Tl, achieving a detection limit of 56 nanograys per second, ~100-fold lower than typical doses used in medical imaging. We show that this NIR-II scintillator enables high-resolution X-ray radiography of electronic circuit boards and biological tissues.

Modulation of immune cells as therapeutic tools has gained significant clinical relevance in the treatment of cancer. Among them, macrophages represent a promising immunotherapeutic platform not only because they can internalize tumor material, but also because they profoundly shape the tumor microenvironment through cytokine production, antigen presentation, metabolic regulation, and modulation of other immune and stromal populations. Lipid Nanoparticles (LNPs) have enabled RNA therapies to the bedside and are thus considered the gold standard for gene delivery. However, optimizing LNPs for RNA delivery to macrophages remains an active area of investigation. Here, we propose the surface modification of unPEGylated LNPs using the Layer-by-Layer (LbL) approach for enhanced RNA delivery to macrophages. Specifically, we show that fucoidan, a sulfated polysaccharide, when at the outermost layer in the LbL process provides two physicochemical advantages to unPEGylated LNPs: (1) stability in PBS and (2) resistance to lyophilization in the presence of cryoprotectant. Additionally, fucoidan improves macrophage targeting and RNA transfection efficiency compared to previously synthesized hyaluronan-decorated LbL LNPs. Fucoidan LbL LNPs (Fuc-LNPs) preferentially accumulated in CD11b+ macrophages when co-cultured with U87 glioblastoma cells, which was not observed for control PEGylated LNPs. Furthermore, Fuc-LNPs induced a higher transfection of mRNA in primary human macrophages when compared to PEGylated control LNPs. Using the model mRNA encoding CAR@CD19, Fuc-LNPs generated CAR macrophages which mediated CD19 cell ablation in vitro. Altogether, these findings highlight the potential of the LbL strategy to modulate the targeting properties of LNPs, improving RNA delivery to human macrophages and encouraging further studies using LbL LNPs for the generation of CAR-Macrophages in the context of solid tumors.

Although microplastics and nanoplastics (MP/NP) are pervasive environmental contaminants, our understanding of cellular toxicity remains incomplete, as adverse effects are often attributed to long-term intracellular accumulation, while the spatiotemporal onset of cellular damage remains poorly defined. Here, we employ chemical-bond-selective stimulated Raman scattering (SRS) microscopy and cell models that decouple continuous exposure from intracellular retention to directly visualize clinically derived MP/NP-cell interactions. Cellular stress occurs primarily during MP/NP exposure, accompanied by alterations in lipid droplet (LD) composition. In contrast, following extracellular removal, intracellularly retained MP/NP become largely inert, with recovery of lipid metabolism and cellular functions. Lipidomics identifies arachidonic acid (AA) as a key dysregulated metabolite, and SRS imaging further reveals transient, spatially confined AA enrichment in MP/NP-proximal LDs during uptake. Importantly, phospholipid coating of MP/NP attenuates LD alterations and cytotoxicity while preserving particle internalization, establishing uptake-driven metabolic stress, rather than long-term intracellular retention, as primary source of MP/NP-induced damage.

Underwater adhesion research increasingly draws on bioinspired systems to uncover the molecular mechanisms that enable strong interfacial binding in aqueous environments. The biofilm adhesin Bap1 from Vibrio cholerae contains a short peptide motif, SYWFFGWHTK (CP), which exhibits exceptional adhesive performance, surpassing mussel foot protein mfp5 under comparable conditions. Despite its promise, the roles of ionic environments and aggregation behavior in governing CP adhesion remain unclear. In this study, we investigate how ion identity influences CP aggregation, film formation, and interfacial properties. Using dynamic light scattering, we identify the formation of micron-scale assemblies of aggregated molecular clusters (AAMCs), with size distributions modulated by salt type. Quartz crystal microbalance with dissipation and liquid atomic force microscopy reveal that CP film formation is both surface- and ion-dependent. On gold substrates, AAMCs preferentially adsorb and collapse into rigid, smooth nanofilms, consistent with hydrophobic-driven compaction. In contrast, silicate surfaces inhibit such collapse, yielding distinct morphologies and interfacial energetics. These findings demonstrate that surface chemistry and ionic conditions jointly regulate peptide aggregation and adhesion. This work provides mechanistic insight into hydrophobic-rich peptide systems and informs the rational design of next-generation wet adhesives, with broader implications for biomaterials and peptide-based formulations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/733527v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1bd012aorg.highwire.dtl.DTLVardef@1977892org.highwire.dtl.DTLVardef@16cf79borg.highwire.dtl.DTLVardef@f405bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Innate immune agonists are promising therapeutic agents to induce immune responses against cancer. However, these agents have been limited by toxicity associated with systemic accumulation and activity in off-target cells. In this work, a targeted nanoparticle (NP) platform to encapsulate and protect the Toll-like receptor 3 (TLR3) agonist polyinosinic-polycytidylic acid (poly(I:C)) and promote its specific delivery to antigen presenting cells (APCs), macrophages and dendritic cells, for activation of this cell population was designed. To determine NP physiochemical properties that promote APC delivery, we developed a library of NP surface chemistries formed by electrostatic adsorption of polyanion coatings onto liposomes using layer-by-layer (LbL) assembly and screened the particles on APCs and off-target cells. Dextran sulfate was identified as a promising coating to enhance specific APC delivery. We applied these design parameters to develop a poly(I:C)-loaded NP for an APC-targeted immunotherapy. In a model of metastatic ovarian cancer, the LbL NP prolonged poly(I:C) retention in the peritoneal space--with 2-fold remaining 24-48hr after administration compared to free poly(I:C)--ultimately reducing systemic accumulation and associated toxicities. Compared to free drug, the NP reduced the increase in serum levels of TNF, IL-6, and CXCL10 by 9-, 4-, and 31-fold respectively. NP-treated mice experienced lower weight loss and recovered more quickly at a higher poly(I:C) dose, indicating a widening of the therapeutic window. The NP formulation enhanced accumulation of poly(I:C) in the tumor 2-fold and activation of the target APC population compared to free drug, and ultimately slowed tumor growth and extended survival in combination with doxorubicin chemotherapy. Overall, this work demonstrates a modular NP delivery strategy to improve the delivery, safety, and therapeutic window of a TLR3 agonist.

The first and critical reaction in magnetic hyperthermia to induce the death of cancer cells is the production of ROS (reactive oxygen species). We previously showed that it is possible to specifically deliver iron oxide magnetic nanoparticles (IONPs) in the lysosomes of cancer cells and eradicate them by targeted magnetic intra-lysosomal hyperthermia (MILH) via the application of a high frequency alternating magnetic field (AMF) without macroscopic temperature elevation. The mechanism involves a local temperature elevation at the IONPs surface which enhances the ROS production through the Fenton reaction; ROS then peroxide the proteins and lipids of the lysosomal membrane, inducing its permeabilization and leading to lysosomal enzymes release and cell death. Fe ions, critical to produce ROS in MILH, were assumed to be released by IONPs. We thus developed PEGylated multi-cores IONPs called NanoFlowers (NF@PEG) presenting or not a SiO2 shell (NF@SiO2@PEG), the later preventing the Fe3+ release from IONPs. NF@PEG released Fe ions and produced ROS production in vitro, in acidic medium mimicking lysosome upon AMF exposure, whereas NF@SiO2@PEG did not. Surprisingly, both nanoparticles increased the ROS production in cells, induced lysosome permeabilization and cell death, and slowed down the proliferation of cancer cells with the same efficacy, upon AMF application, indicating that MILH was efficient in absence of Fe3+ release from IONPs. In contrast, Ferristatin-II, an iron uptake inhibitor, prevented the ROS production and cell death in MILH induced by both IONPs, elucidating the role of endogenous iron cations responsible for the ROS production ROS in MILH to kill cancer cells.

Low response rates to immune checkpoint inhibitors (ICIs) in solid tumors are often driven by insufficient tumor-infiltrating CD8 T cells and immunosuppressive tumor microenvironment (TME). Although interleukin-2 (IL-2) potently expands and activates CD8 T cells, its clinical use is limited by rapid clearance, dose-limiting toxicity, and regulatory T cell (Treg) stimulation. Engineered IL-2 variants have not yet achieved meaningful clinical efficacy. Here, polymer-modified mesoporous silica nanoparticles displaying dense, unmodified wild-type IL-2 on their surface (IL2-NP) are developed, conferring proteolytic stability and tumor retention. IL2-NP enables avidity-mediated CD8 T cell binding and enhances proliferation and effector function without increased Treg binding or proliferation. Intratumoral IL2-NP expands CD8 T cells, increases CD8/Treg ratios, and reprograms TME through dendritic cell activation and M1-like macrophage polarization. IL2-NP induces regression of both treated and untreated distant colorectal tumors in a CD8 T cell-dependent manner. IL2-NP synergizes with ICIs and leads to complete tumor regression and immunological memory that protect against rechallenge. Treatment is well tolerated, with strong efficacy also observed in triple-negative breast and metastatic ovarian cancer models. Overall, intratumoral IL2-NP elicits robust systemic antitumor immunity, offering a promising strategy to enhance ICIs, cancer vaccines, and adoptive T-cell therapies. Graphical abstractThis work introduces a nanoparticle platform that overcomes major shortcomings of IL-2 immunotherapy by presenting wild-type IL-2 at high density on the nanoparticle surface, thereby increasing binding avidity to effector T cells. The resulting IL-2 nanoparticles enhance cytotoxic T cell expansion, reprogram the tumor microenvironment, and augment responses to immune checkpoint blockade to achieve robust ant-tumor immune response in mouse tumor models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/738558v1_ufig1.gif" ALT="Figure 1"> View larger version (82K): org.highwire.dtl.DTLVardef@12f8c8corg.highwire.dtl.DTLVardef@b46b1forg.highwire.dtl.DTLVardef@e4efc5org.highwire.dtl.DTLVardef@3993e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

The widespread use of synthetic food dyes, such as Acid Yellow 23 (AY 23), in the food, cosmetics, and pharmaceutical industries raises questions about their potential effects on biological systems and public health. The concentration-dependent interaction between AY 23 and bovine serum albumin (BSA), a crucial model protein for understanding pharmacokinetics and protein-ligand behaviour, was examined in this study. We demonstrate that, under physiological conditions, increasing dye concentrations from 50 M to 200 M results in notable conformational changes, increased surface hydrophobicity, and protein aggregation using a multimodal biophysical approach that includes fluorescence spectroscopy. Direct visualisation verified these structural changes and aggregate formation, whereas hemolytic assay confirmed the high hemolytic nature of AY 23-induced fibrils. Additionally, this study provides a mechanistic basis for the toxicological effects of AY 23, underscoring the implications of food dyes for public health.

Mycobacterium tuberculosis (Mtb) infection kills more people worldwide than any other pathogen. While the Bacille Calmette-Guerin (BCG) vaccine for Mtb has been widely used for over a century, it provides insufficient protection to eradicate this disease. One of our labs has recently established that a protein antigen (H1) can be combined with a STING pathway agonist to achieve strong protection against Mtb in mice, with performance that exceeds that of the BCG vaccine. However, its reliance on a synthetic cyclic dinucleotide (CDN) with relatively poor cell uptake requires higher dosing levels, thus increasing costs. To increase the efficiency of this vaccine and provide a delivery strategy that could also be used in humans, the H1 Mtb antigen and CDN adjuvant were conjugated to genome-free MS2 viral capsids that included cationic mutations to increase cell uptake. Specifically, the H1 antigen was conjugated to the external surface of MS2 using a tyrosinase-mediated oxidative coupling reaction, and the native STING agonist cGAMP was coupled to internal cysteine residues through a reductively cleavable disulfide linker. The resulting MS2-H1 and MS2-cGAMP conjugates were then co-delivered for three doses of vaccination in mice before exposure to Mtb. The MS2-based vaccine platform was observed to have comparable efficacy to the original H1/CDN formulation, but its enhanced uptake properties enabled 57-fold less CDN and 3-fold less H1 antigen. Additionally, this vaccine elicited immune responses that have been previously demonstrated to correlate with protection. The ability of the capsid shells to protect the CDN cargo during transport allowed enzymatically produced, and thus readily accessible, cGAMP to be used instead of more costly CDNs that require many synthetic steps. This, combined with the reduced overall amount of CDN and H1 that was required, could lower the production costs of future vaccines substantially. Finally, the ability of the capsid-based carriers to bypass the membrane transporters for CDNs suggests that this enhanced vaccination platform is likely to exhibit improved human efficacy in future studies.

The early detection of breast cancer currently relies on expensive mammography, followed by pathology that uses biopsied, fixed, and immunohistochemically stained tissues. A live-cell detection approach could be highly beneficial as a supportive diagnostic and research tool to better understand and resolve the dynamic nature of breast cancer cells and their response to treatment in real time. Here, we present a single-walled carbon nanotube (SWCNT) near-infrared fluorescence spectral fingerprinting approach combined with machine learning to precisely detect the heterogeneity of breast cancer cells in live culture. We introduced DNA-functionalized SWCNTs to MCF-10A (a non-tumorigenic healthy control) and cancer cell lines spanning known extrinsic disease subtypes: MCF-7 (luminal A), HCC1954 (HER2+), MDA-MB-231, and MDA-MB-468 (both triple-negative). The NIR fluorescence spectra of DNA-SWCNTs across 600 individual cells within each type showed significant differences in emission peak intensities, center wavelengths, and peak intensity ratios, attributable to variations in cellular uptake and biomolecular interactions. These spectral changes likely arise from complex SWCNT cellular interaction fingerprint that includes redox-mediated modulation of the local nanotube environment, rather than from a single biomarker response. The extracted spectral features were used to train an ensemble machine learning model. The model achieved 98% classification accuracy for breast cancer detection and 95% classification accuracy for breast cancer cell subtyping. Moreover, Raman microscopy further showed that MDA-MB-468 cells exhibited the highest SWCNT uptake, whereas MCF-10A cells showed greater SWCNT aggregation, consistent with their lower broadband NIR fluorescence intensity. These results demonstrate that SWCNT NIR fluorescence fingerprints can capture cell line-specific optical signatures. This platform provides a foundation for nanomaterial-enabled biosensing strategies aimed at real-time monitoring of cancer-associated cellular states.

Potent antineoplastics, such as afatinib and freebase doxorubicin, are associated with systemic toxicity. To address this issue, we developed a carrier that releases drugs, including afatinib and doxorubicin, specifically at the focus of low-intensity ultrasound. This remotely triggered and focal approach enables the release of drugs specifically at the ultrasound focus, thus mitigating undesirable off-target effects, and at concentrations governed by the duration of the applied ultrasound. We produced ultrasound-sensitive microdroplets with high encapsulation efficiencies (39.6% for afatinib and 46.6% for doxorubicin). The microdroplets consist of an ultrasound-sensitive drug delivery system based on a methoxy poly(ethylene glycol)-poly(D, L-lactide) diblock copolymer (mPEG-PDLLA) and perfluorooctyl bromide (PFOB). Antineoplastic agents were encapsulated within these microdroplets via co-evaporation during particle synthesis. The microdroplets released doxorubicin and afatinib in an ultrasound-pressure-dependent manner, with fitted half-maximal release pressures (P50) of 0.61 MPa and 0.72 MPa, respectively. Together, the effective encapsulation of hydrophobic antineoplastic agents and the dose-dependent ultrasound-triggered release provide a new method for targeted drug delivery and a foundation for future targeted chemotherapies.

Interleukin-6 (IL-6) is a pleiotropic cytokine whose aberrant elevation drives life-threatening conditions, including sepsis, cytokine storm, and autoimmune disorders, yet existing clinical detection methods demand centralized laboratory infrastructure and multi-hour assay times incompatible with rapid point-of-care decision-making. Here, we report an impedimetric aptasensor built on a programmable tetrahedral DNA nanostructure (TDN) interface anchored to a disposable gold screen-printed electrode (Au-SPE) for the ultrasensitive, label-free detection of IL-6. By systematically varying the number of thiolated base vertices from zero to three, we establish a clear and previously unreported structure-function relationship between multipodal anchoring geometry and charge-transfer resistance modulation: tripodal thiolation yields the most rigid, upright, and electrochemically responsive interface, producing the steepest analytical signal gain upon IL-6 binding at the apex-localised aptamer. Under optimised conditions (pH 7.0, 0.05 uM TDN, MCH passivation), the aptasensor exhibits a linear dynamic range of 0.0001-0.001 pg/mL, a limit of detection of 55 ag/mL, and a sensitivity of 1.55x107 ohm (pg mL-1)-1. Selectivity evaluation against seven physiologically relevant interferents such as TNF-, BSA, glucose, urea, ascorbic acid, glycine, and cysteine confirms negligible cross-reactivity, with relative responses ranging from 0.57% to 14.35% of the IL-6 signal. Spike-recovery experiments in human serum yield recoveries of 74.0-87.6% (%RSD < 4.5%), and the sensor retains functional activity for at least 21 days under refrigerated storage. This work demonstrates that thiolated vertex number is a critical and tunable design parameter for TDN-based biosensors, offering a modular, disposable platform for sub-femtogram cytokine detection with direct applicability to early sepsis diagnosis and inflammatory disease monitoring.

Nanoplastics (NPs) are emerging environmental contaminants able to cross biological barriers, disrupt cellular and organelle homeostasis, and alter the brain microenvironment. This study investigated whether NPs affect microglia to neuron communication, a key mechanism underlying neuronal resilience, via the nuclear factor erythroid 2 like 2 (NFE2L2) pathway. Using an in vitro model, we evaluated the effects of polystyrene nanoplastics on microglial metabolic fitness and microglia-mediated neuronal stress responses. Increasing NP concentrations induced a dose dependent biphasic effect. Low to intermediate concentrations increased intracellular adenosine triphosphate (ATP) levels in microglia and enhanced microglia-mediated activation of neuronal NFE2L2. In contrast, high NP concentration impaired microglial metabolism, reduced ATP availability, and decreased microglia to neuron communication. These findings indicate that NPs alter microglial energetic status and modulate neuroprotective signalling, potentially contributing to impaired neuron to microglia interactions and increased susceptibility to neurotoxicity.

Efficient delivery of molecular therapies to the central nervous system (CNS) remains a major barrier to treating neurogenetic disorders such as Niemann Pick type C (NPC) disease. Focused ultrasound-mediated blood-brain barrier opening (FUS-BBBO) has emerged as a non-invasive strategy to enhance delivery of systemically administered therapeutics. In this study, we evaluated whether FUS-BBBO could enable delivery of lipid nanoparticle (LNP)-packaged modified mRNA (modRNA) to the cerebellum in an NPC mouse model. A pilot study in wild-type mice demonstrated successful FUS-mediated BBB opening, delivery of LNP-packaged GFP mRNA, and subsequent protein expression in the cerebellum. We then performed a controlled study in NPC mice comparing delivery of LNP-GFP and LNP-NPC modRNA using intravenous administration with and without FUS-BBBO. BBB opening was confirmed by contrast-enhanced MRI in FUS-treated animals. Quantitative PCR revealed the presence of GFP mRNA in the cerebellum following FUS-BBBO, whereas NPC mRNA was minimal or undetectable across groups. However, no GFP or NPC1 protein expression was detected in the cerebellum by western blot in any experimental group. Consistent with this, no therapeutic effect on Purkinje cell survival was observed. These results demonstrate that while FUS-BBBO reliably induces BBB opening and can facilitate limited delivery of LNP-packaged mRNA to the brain, this did not translate into detectable protein expression or therapeutic benefit in the NPC model under the conditions tested. This discrepancy between successful delivery in wild-type mice and lack of efficacy in diseased animals points to potential important biological and/or formulation-dependent barriers that must be addressed to enable effective CNS delivery of LNP-based mRNA therapies.

Lipophilic dyes are widely used to track extracellular vesicles (EVs), yet their labeling efficiency toward bona fide small EVs (sEVs) remains poorly defined. Here, we critically reassess this efficiency using a serum-free HEK293F system that generates endogenously fluorescent protein-tagged sEVs (sEVs-FPT) as an unambiguous positive reference, thereby minimizing interference from co-isolated, dye-labelable non-vesicular extracellular particles (NVEPs). Two orthogonal methods, nanoflow cytometry and fluorescence microscopy, were employed for cross-validation. We found that PKH26, PKH67, and DiD labeled <0.5% of sEVs-FPT, regardless of vesicle heterogeneity. In vivo tracking confirmed that dye-derived signals were far weaker than FPT signals and strikingly failed to colocalize with them. Preliminary mechanistic evidence indicates that this failure is due to an inability of sEVs to actively internalize dye aggregates. Our findings raise serious concerns about the validity of lipophilic dye-based EV tracking and call for a critical reevaluation of the relevant literature.

Liquid crystalline mesophases exhibit structurally programmable internal architectures that enable co-loading of chemically orthogonal molecules within a single composite material. Realizing the potential of these materials for drug delivery requires a quantitative understanding of how tuning the composition affects internal mesophase architecture and consequently performance metrics such as payload release. Here, Flash NanoPrecipitation with hydrophobic ion pairing is used to prepare nanocarriers containing liquid crystalline mesophases co-encapsulating two compounds from widely different chemical classes: hydrophilic polymyxin B (logP -6) with one of four hydrophobic co-core materials (logP 7-11), achieving >75% encapsulation efficiency and up to 32% and 50% mass loadings for polymyxin and co-core. Synchrotron SAXS is used to quantify characteristic mesophase repeat spacing, which is found to be tunable as a function of composition. A strong correlation between d-spacing and polymyxin release rate is presented. Co-core chemistry and weight fraction jointly govern mesophase architecture, and repeat distance emerges as a structural metric linking these to the hydrophilic payload release kinetics. Mucus diffusivity and antibacterial efficacy are assessed as independent performance metrics, and results corroborate the release behavior. These findings establish a quantitative framework connecting material composition, mesophase architecture, and functional performance that can be applied toward rational co-formulation design. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/734853v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1d722eborg.highwire.dtl.DTLVardef@135adb7org.highwire.dtl.DTLVardef@11fe29eorg.highwire.dtl.DTLVardef@571165_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOToC Graphic TextC_FLOATNO Flash NanoPrecipitation yields liquid crystalline nanocarriers co-encapsulating with high efficiency payloads with widely distinct physicochemical properties. Synchrotron SAXS establishes characteristic repeat spacing as a quantitative structural metric directly governing hydrophilic release kinetics, providing a rational design framework linking mesophase architecture to functional performance across a range of payload structures. C_FIG

Background: Gadolinium-based contrast agents (GBCAs) are routinely used in magnetic resonance imaging (MRI). Although macrocyclic GBCAs were initially considered biologically inert, it is now known that a fraction of patients retains gadolinium (Gd) for prolonged periods in tissues such as blood, bone, and brain. Because the first cellular interactions of GBCAs occur in the bloodstream, this study aimed to elucidate the uptake mechanism but also the intracellular persistence and release dynamics of gadoterate meglumine, one of the most widely used macrocyclic agents, in white blood cells (WBCs). Methodology and principal findings: WBCs and K562 cells were incubated with gadoterate meglumine under different conditions to investigate its cellular entry mechanisms. Uptake of the contrast agent was quantified by measuring intracellular Gd using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Time and concentration-dependent incubation of K562 cells revealed saturable uptake kinetics consistent with a Michaelis-Menten model which is independent of the phase of the cell cycle. Gadoterate meglumine uptake in both WBCs and K562 cells was shown to be an active process, as uptake was strongly reduced or abolished at low temperature (16C and 4C) and in the presence of metabolic inhibitors (sodium azide and 2-deoxyglucose). Co-incubation with multiple endocytosis inhibitors (Dyngo 4a, Dynole 2-24 and chlorpromazine) did not significantly decrease intracellular Gd levels in K562 cells and caused only a slight reduction in WBCs, indicating that endocytosis is not the main entry pathway for gadoterate meglumine in these cells. Furthermore, we assessed the retention time of the Gd inside the cells, showing that only after 24 hours post incubation 80% percent of the intracellular Gd was released through an active process. Finally, we demonstrate that one of the mechanisms of Gd release from WBCs involves extracellular vesicles, which may substantially increase its potential for downstream accumulation in different tissues, including immunoprivileged tissues like brain. Significance: The observed time-dependent accumulation, temperature and energy dependence of gadoterate meglumine uptake demonstrate that active cellular mechanisms are primarily responsible for GBCA internalization. Furthermore, our results indicate that macropinocytosis, phagocytosis, and clathrin-mediated endocytosis are not the primary routes of gadoterate meglumine entry. Hereby, we also describe that Gd externalization is an active process involving extracellular vesicles which may influence the Gd distribution in different tissues and its consequent long-term retention. Further studies are required to explore strategies to block this process in order to mitigate potential long-term gadolinium retention.