Journal of Nanobiotechnology

Previously, we demonstrated the biosafety of polyethylene glycol-coated iron oxide nanoparticles (MAG@PEG) in a cellular model of viral oncogenesis, supporting their further preclinical evaluation. Herein, we evaluated the safety and biodistribution of MAG@PEG in tumor-bearing mice and assessed the influence of an external magnetic field on nanoparticle distribution. Animals were randomly assigned to three groups: G1, control (PBS); G2, MAG@PEG (10 mg/kg); and G3, MAG@PEG (10 mg/kg) with magnetic exposure for 1 h. Doses were administered intraperitoneally weekly for 1 month. Results showed that animal behavior and body weight were unaffected by MAG@PEG, while tumor growth was significantly reduced only in G2. Biodistribution studies based on tissue magnetization revealed higher magnetic iron levels in the stomach followed by the spleen, lungs, and tumors, particularly in G2. Histological analysis detected Prussian blue-positive staining mainly in the spleen and in lymphoid-associated structures in the lung, stomach and intestine, while a fraction of MAG@PEG remained within the peritoneal cavity. Serum biochemistry showed no evidence of nephrotoxicity. Furthermore, hemocompatibility studies using human blood samples demonstrated that MAG@PEG was non-hemolytic according to the ASTM F756 standard. Taken together, the biodistribution, histological, biochemical, and hemocompatibility findings provide a comprehensive preclinical assessment of the biosafety of MAG@PEG and identify the route of administration and magnetic targeting as key determinants of nanoparticle biodistribution.

Nanoplastics (NPs) are widespread environmental contaminants, yet most toxicity studies rely on short-term, single-dose exposures that poorly reflect daily human exposure. The kidney, particularly the proximal tubule, is a likely site of repeated NP exposure given its role in filtration and reabsorption, but chronic exposure effects are largely unexplored. Here, a long-term in vitro model was developed in which human proximal tubule (HK-2) cells were continuously exposed to 20 nm or 100 nm carboxylated polystyrene NPs (100 {micro}g/mL) over 12 days. Continuous 20 nm NP exposure caused progressive loss of viability whereas 100 nm NPs left viable cell numbers largely unaffected. RNA-sequencing showed 20 nm NPs drove extensive, time-dependent transcriptional dysregulation (1,548 differentially expressed genes at day 6, rising to 4,431 at day 9), far exceeding the minimal alterations in gene expression (35 genes) caused by 100 nm NPs. Cell-cycle analysis and pathway enrichment revealed KRAS pathway activation and S-phase arrest in 20 nm-treated cells, with ANGPTL4 the most upregulated gene. Validation with qRT-PCR confirmed increased ANGPTL4, HMOX1 and FN1 expression, genes implicated in renal fibrosis and chronic kidney disease. These findings show that continuous, size-dependent NP exposure elicits progressive cytotoxic and transcriptional responses in kidney proximal tubule cells and indicate ANGPTL4 as a candidate early biomarker of NP-induced renal stress.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. This clinical burden is predominantly attributable to atherothrombotic events resulting from platelet hyperactivation, a process integral to the pathophysiology of atherosclerosis. Cilostazol (CLZ) is a clinically established phosphodiesterase-IIIA inhibitor with antiplatelet and vasodilatory activity. However, its extremely low aqueous solubility results in dissolution-limited and variable oral absorption, while dose-dependent adverse effects and cardiovascular safety restrictions may constrain its clinical use. Lipid-polymer hybrid nanoparticles (LPHNPs) represent a highly promising drug delivery system to address these biopharmaceutical limitations. However, their interactions with blood components remain poorly understood, with surface charge among the most controversial physicochemical parameters implicated in platelet toxicity. Here, we applied an integrated ex vivo and in silico screening strategy to design LPHNPs based on poly(lactic acid) and poly(ethylene glycol)-modified lipids displaying positive (amine), nominally neutral (methoxy), and negative (carboxyl) surface charges to systematically identify safe boundaries at the nano-human platelet interface and evaluate their suitability as CLZ carriers. Positively charged nanoparticles precipitated immediately during self-assembly due to uncontrolled ionic pairing and were therefore structurally unviable. Neutral nanoparticles maintained a stable size (~113 nm hydrodynamic diameter) but unexpectedly induced spontaneous platelet aggregation, limiting their applicability. In contrast, negatively charged nanoparticles exhibited exceptional colloidal stability (hydrodynamic size of ~120 nm and zeta potential of -48 mV), achieved significant inhibition of ADP-induced platelet aggregation, and adhered to the platelet surface without inducing activation or membrane disruption. Steered molecular dynamics simulations, performed under identical non-equilibrium pulling conditions, showed the highest interfacial resistance to CLZ extraction for the negatively charged model, qualitatively consistent with the sustained CLZ release profile observed in vitro. These findings highlight the importance of precise surface charge tuning in the development of safe cardiovascular nanomedicines based on LPHNPs, emphasizing the need for comprehensive platelet compatibility testing before further biological evaluation.

Extracellular vesicles (EVs) are nanosized, lipid bilayer-enclosed particles that mediate intercellular communication and hold considerable promise as biomarkers and therapeutic agents. However, their functional heterogeneity remains incompletely understood, largely due to the absence of reliable methods for isolating specific EV subpopulations. Here, we established a Nanoscale Flow sorting (NanoFACS) workflow and evaluated its potential for EV detection, sorting, and molecular characterization. We assessed critical parameters, including fluorescent staining and labeling approaches, background signal discrimination, dilution and flow-rate effects, and scatter triggering. We achieved high-purity sorting of CFSE-stained and antibody-labeled EVs, preserving vesicle integrity and sufficient RNA yields for small RNA sequencing of sorted populations. We identified EV subtype-specific small RNAs despite partial cargo overlap in single-marker sorted populations. Our results establish a reproducible antibody-based NanoFACS workflow that defines key operational parameters for reliable EV detection and sorting, with potential adaptability across conventional and spectral flow cytometry platforms. By reducing background and non-EV-associated events, the workflow enables the isolation of phenotypically defined EV subpopulations suitable for downstream molecular analysis, providing a practical approach to investigate EV heterogeneity.

Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three- port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to- protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum- free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.

Non-small-cell lung cancer (NSCLC) diagnosis is challenged owing to the need for reliable, non-invasive biomarkers. Circulating cell-free microRNAs (ccf-miRNAs) are promising candidates, but their low levels and short sequences in blood complicate detection. In this study, a novel nano-hybrid fluorescent array was developed to enable rapid and sensitive identification of NSCLC-related ccf-miRNAs. The array consists of poly(T)-modified fluorescent nano-polystyrene beads for capturing target miRNAs, and fluorophore-labeled, sequence-specific locked nucleic acid (LNA) probes for their detection. This design forms a sandwich structure with the target miRNA. Nano cytometry and fluorescence microscopy assessed both the capture and specificity of detection. The assay was tested on plasma samples for miR-16-5p and U6, evaluating selectivity, sensitivity, and reproducibility. The results showed that the poly(T)-modified beads efficiently captured the target miRNAs, and the LNA probes accurately distinguished the sequences. The assay enabled direct detection of miR-16-5p and U6 from plasma without amplification, demonstrating high selectivity, sensitivity, and reproducibility. Combining enrichment with nano-polystyrene beads and sequence-specific LNA probes addressed the main challenges of ccf-miRNA detection, namely their low abundance and short sequence length. As ccf-miRNAs are linked to NSCLC progression, this method could assist in early diagnosis, disease monitoring, and assessment of treatment response. The target-independent nature of the poly(T) capture layer allows for easy calibration to new miRNAs by changing the LNA probes, rendering it suitable for multiplex detection in clinical settings. However, further preclinical and clinical studies are needed before adoption in routine practice. This nano-hybrid fluorescent array presents a rapid and reliable approach for detecting low-abundance ccf-miRNAs in plasma. Its encouraging performance and flexible design show potential for future application in NSCLC diagnosis and monitoring, subject to additional confirmation.

Cationic lipid nanoparticles have demonstrated unique potential for extrahepatic mRNA delivery, particularly enabling selective targeting of the pulmonary endothelium. However, their translational development has been hampered by reports of infusion-related immune reactions and innate immune system activation, most notably transient complement activation. Here, we present a case study illustrating the discovery and translational advancement of a selected cationic LNP into non-human primates (NHPs) for initial pharmacokinetic assessment and evaluation of potential immunostimulatory side effects. We show surface charge dependent organ-selective expression of reporter mRNAs from different LNPs in vivo. An mRNA encoding the Tie2 agonist COMP-Angl, was formulated with LNP002, and respective pharmacokinetic and pharmacodynamic readouts were analyzed in two independent non-human primate studies. Notably, dose-dependent transient complement activation could be abrogated by extending the infusion time. Finally, we identified the blood-borne pharmacodynamic biomarker PDGFB for LNP002/mRNA-76 treatment reflecting activated Tie2-signalling in healthy pulmonary endothelium in vivo supported by single cell sequencing and cluster-alignment of downstream effector genes with the same spatial profile as the delivered mRNA.

Perovskite quantum dots (PQDs) are exceptionally promising next-generation optical materials for bioimaging applications. This paper presents an optical PQDs nanocomposite method to identify bacterial species associated with breast cancer, employing a LSTM deep learning model. The results indicate that the developed PQD-based nano-sensor offers impressive sensitivity, selectivity, and practical application. The four bacterial species identified in the study, Pseudomonas aeruginosa, Vibrio parvula, Acinetobacter baumannii, and Streptococcus vestibularis, were considered the most relevant indicators of breast cancer. With rapid detection, an intuitive design, and improved reliability, the PQD sensor could be an excellent breast cancer diagnostic tool for a point-of-care application. Furthermore, it has the potential to be integrated into healthcare systems, specifically in resource-limited settings, thereby improving the accessibility and efficiency of early breast cancer detection.

Animal-derived surface coatings present the gold standard in stem cell culture. However, they come with major drawbacks such as lot-to-lot inconsistency and an ethically problematic production. Approaches to develop synthetic coating alternatives have largely failed to recapitulate biological functions essential to PSC-self renewal and differentiation. In this study, we have investigated the suitability of a fully synthetic, chemically defined extracellular matrix-mimicking coating named BIOINSTRUCT for hiPSC culture. BIOINSTRUCT was employed during maintenance culture of IMR90-4 cells, as well as directed differentiation of HIHCNi008-A, ACTN2-GFP, and UKKI-011-A cells. BIOINSTRUCT preserved hiPSC characteristics over the experimental period of 10 passages similar to Matrigel. Spontaneous differentiation resulted in the formation of ectodermal, mesodermal and endodermal derivatives, confirming the retention of pluripotency. Directed differentiation on BIOINSTRUCT enabled the generation of neuronal, cardiac and pancreatic endocrine cells. Altogether, our data underscore BIOINSTRUCT as a promising alternative to animal-derived extracellular matrix coatings.

Our prior works have demonstrated the therapeutic potential extracellular vesicles (EVs) derived from batch cultures of brain endothelial cells (BECs) owing to their ability to transfer mitochondria to injured recipient cells. However, conventional batch/flask cultures limit scalable and reproducible production of EVs. Here, we evaluated a CELLine AD1000 bioreactor as a platform for sustained production of EVs from mouse b.End3 BECs and compared bioreactor-derived (BR) EVs with EVs obtained conventional batch cultures (BT). Large EVs (lEVs) and small (sEVs) were isolated using a sequential centrifugation protocol and were characterized using dynamic light scattering, nanoparticle tracking analysis, microBCA assay, western blotting, proteomics analysis, and their ability to rescue bioenergetics in oxygen-glucose deprived (OGD) BECs. Bioreactor cultures maintained high cell viabilities (~90%) for up to eight weeks and produced approximately three- to four-fold greater EV protein yields than batch cultures. BR EVs showed reproducible particle characteristics consistent with EV subtypes derived from BT cultures. CD63, CD9 and Arf6 were detected in EV fractions, whereas the mitochondrial outer-membrane protein TOMM20 was enriched in lEVs. Proteomic analyses revealed that bioreactor-derived lEVs retained the mitochondrial cargo signature observed in batch-derived lEVs. Both bioreactor and batch EVs restored relative ATP levels in ODG-exposed BECs. These findings suggest that bioreactor BEC cultures are a scalable approach for generating functionally active BEC-EVs for downstream characterization and therapeutic development.

Microplastics are pervasive environmental pollutants that pose a growing concern for human health. Oral ingestion is a common route of human microplastic exposure, yet the proteomic response of the gut epithelium to microplastics remains unclear. This study aimed to investigate the cellular effects of pristine and artificially digested microplastic exposure in primary rat duodenal epithelial cells using untargeted proteomics. Cells were exposed to pristine or digested 0.5 um polystyrene microplastics at 10 or 100 ug/mL for 72 hours and were then analyzed by tandem liquid chromatography and mass spectrometry (LC-MS). Proteins that were both significantly different in intensity compared to controls, with a threshold change of 1.3 or greater, were considered to be differentially expressed. This criterion identified 41 differentially expressed proteins after 100 ug/mL pristine MP exposure, with 19 downregulated and 21 upregulated. Following exposure to 100 ug/mL digested MP, only 3 differentially expressed proteins were upregulated and 7 were down regulated, demonstrating the impact of microplastic physicochemistry. FGSEA pathway analysis revealed that 270 Reactome pathways were significantly altered following microplastic exposure in either condition at both concentrations. These pathways contributed to functional domains including protein synthesis, DNA replication, cell cycle control and aerobic respiration. Overall, microplastic exposure was associated with upregulated mitochondrial respiration, and downregulation of nuclear-related processes including DNA synthesis, transcription and cell proliferation. This study provides targets for future investigation (mitochondria and nucleus) and emphasizes the need to consider biological and environmental conditions for in vitro models of microplastic exposure.

Glial cells play essential roles in maintaining neural tissue homeostasis and limiting the spread of injury-induced neuroinflammation, yet their dysfunction can also exacerbate neurodegenerative, neuroimmune, and neurodevelopmental disorders. Therapeutic targeting of specific glial cell populations would open up new treatment possibilities, but currently available viral and non-viral delivery vehicles are limited by carrier toxicity as well as poor delivery efficiency and specificity. To work towards new glia-targeted, non-viral delivery vehicles, we performed an in silico analysis on neural cell specific RNA-Seq datasets in mice to identify surface molecules differentially enriched on neurons, astrocytes, and microglia. We identified Slc2a5 (Glut5) as a microglia-enriched transporter and validated its microglia specific expression in healthy murine neural tissue using RNA in situ hybridization. By employing a sequential thiol-ene and enzymatic catalyzed reaction scheme, we synthesized polyelectrolytic dendrimers with multivalent presentation of fructose, the natural ligand for Glut5, and formulated nano-sized polyionic complexes (PICs) loaded with protein or nucleic acid cargo by mixing oppositely charged dendrimers. Fructose functionalized PICs lacking cationic surface charge enabled fructose-dependent intracellular protein delivery to Glut5-expressing human breast cancer cells (MCF-7) and mouse fibroblasts, while restricting uptake by Glut5-negative mouse astrocytes, confirming transporter-dependent selectivity in vitro. Fructose functionalized PICs resulted in minimal neuroinflammation when injected into mouse striatum at 1 mg/ml but elicited concentration-dependent neural tissue toxicity at higher doses. Fructose-PICs failed to achieve microglia specific intracellular delivery of functional proteins when injected into healthy neural tissue but aided in directing protein delivery to microglia and astrocytes at ischemic stroke lesions. Together, our findings demonstrate proof-of-principle for using in silico RNA-seq screening workflows to identify cell specific surfaceome targets and incorporating that insight into a modular biomaterial platform for targeted drug delivery.

Extracellular vesicles (EVs) are membrane-bound particles secreted by all cells and serve as critical mediators of intercellular communication in both physiological and pathological contexts. However, elucidating the precise functions of endogenous EVs remains challenged by a fundamental technical bottleneck: current isolation methodologies depend on shared physical properties or generic surface markers, preventing the selective enrichment of cell-type-specific EVs within complex tissues. Consequently, EVs recovered from biofluids or tissue extracts represent heterogeneous populations, confounding the direct attribution of biochemical signatures and biological activities to distinct cellular origins. Here, we describe a versatile genetic-labeling and affinity-based purification strategy that enables the selective isolation of cell-specific primary EV. Using cardiomyocyte-derived EV (CM-EVs) as a proof-of-concept, we crossed Myh6-Cre mice with the ROSA26-mTmG reporter line to restrict membrane-targeted GFP expression to cardiomyocytes. Following myocardial infarction, cardiac EV were recovered and GFP-positive CM-EVs were selectively purified by anti-GFP nanobody-based magnetic pulldown. This approach yields highly enriched, functional cardiomyocyte-derived extracellular vesicles (CM-EVs). To accommodate diverse analytical needs, we established two distinct elution workflows: (i) a denaturing protocol optimized for downstream proteomics and immunoblotting, and (ii) a gentle, non-denaturing protocol that preserves vesicle integrity and biological activity for downstream in vitro and in vivo functional assays. Using this method, we characterized CM-EVs size distribution and proteomic signature, and demonstrated their capacity to transfer functional cargo to target cells in vitro. Because the purification approach relies solely on targeting a genetically restricted membrane tag rather than assumed surface markers, it is immediately adaptable to virtually any cell type for which a validated Cre driver exists. This method removes a long-standing bottleneck in EV research and provides a broadly applicable platform for deciphering the biochemical composition and in-vivo functions of cell-type-specific primary EV in physiological and pathological settings.

Therapeutic siRNAs are a major clinical modality for hepatic gene silencing, but efficient extrahepatic delivery and multi-gene modulation remain significant barriers. Nucleic acid nanotechnology offers programmable assembly, but conventional structures suffer from enzymatic instability in vivo . Here we report fully chemically modified RNA nanostructures (FMRNs): a vehicle-free platform entirely composed of stabilized therapeutic nucleic acids that integrate structural precision with metabolic durability. Following systemic administration, FMRNs exhibit enhanced pharmacokinetics and extrahepatic accumulation, with in vivo behavior influenced greatly by hydrodynamic size rather than molecular weight. Using triangular and square geometries, we demonstrate programmable control over siRNA stoichiometry to achieve sustained, simultaneous silencing of up to four genes from a single entity. Nanostructure assembly alone drives productive delivery, especially to heart and muscle, matching lipophilic conjugation. Furthermore, cerebrospinal fluid administration reveals that larger FMRNs improve central nervous system distribution and retention revealing physicochemical parameters that impact brain exposure. FMRNs establish a modular and effective platform combining improved pharmacokinetics, extrahepatic delivery, and multi-gene silencing for complex diseases.

Background Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, and emerging T cell engager (TCE) immunotherapies require predictive preclinical models that capture patient-specific tumour biology and tumour-immune interactions. Although patient-derived organoid (PDO)-immune co-culture systems show promise for evaluating immunotherapy responses, many rely on matrix-embedded cultures that limit scalability and reproducibility. We therefore developed and validated a low-matrix organoid-T cell co-culture platform for functional assessment of TCE activity in patient-derived CRC models. Methods Patient-derived CRC organoids representing diverse molecular and genetic backgrounds were co-cultured with activated allogeneic CD3+ T cells in a suspension low-matrix format. Matrix concentration, medium composition, T cell activation status, and assay duration were optimized. Tumour killing, T cells activation, cytokine secretion, apoptosis, and motility were assessed using flow cytometry, live-cell imaging, cytokine profiling, and immunofluorescence. The platform was evaluated using EGFR- and HER2-targeting bispecific T cell engagers across 21 CRC organoid models and multiple healthy donor-derived T cell populations. Results Optimization identified 1% matrix and a 1:1 organoid: T cell medium that preserved organoid integrity while maintaining T cell viability, activation, and motility. The final workflow enabled reproducible co-culture of 5-day mature organoids with 7-day activated T cells for 72 hours. EGFR-targeting TCEs induced tumour killing, T cell activation, and IFN-{gamma} secretion across donor-organoid combinations. Screening of 21 CRC organoid models revealed substantial inter-patient heterogeneity, with 12 models maintaining [≥]50% baseline viability and supporting functional TCE evaluation. EGFR- and HER2-targeting TCEs produced potent dose-dependent cytotoxicity, with IC50 values ranging from 0.0188 to 54.77 nM across responsive models and up to 75%-85% tumour killing in the most sensitive organoids. These responses were accompanied by increased effector cytokine secretion. Real-time imaging confirmed dynamic T cell engagement and apoptosis-driven organoid destruction following TCE treatment. Conclusions We establish a robust, scalable, human-relevant low-matrix organoid-T cell co-culture platform for functional screening of T cell engagers in patient-derived CRC models. By enabling integrated assessment of tumour killing, immune activation, cytokine responses, and tumour-immune interactions while preserving inter-patient heterogeneity, this system provides a translational framework for immunotherapy development. The platform may support candidate prioritisation, biomarker discovery, patient stratification, and preclinical evaluation of immune-engaging therapeutics.

Nanoplastic contamination is increasingly recognized as a biochemical stressor, yet the molecular determinants governing how polymer fragment size dictates protein-level interactions remain poorly understood. Here, we combine molecular docking and all-atom molecular dynamics simulations to characterize the interaction of polyethylene (PE) oligomers of increasing length (n = 3, 5, 7, and 14 repeat units) with human catalase, a tetrameric heme enzyme central to cellular antioxidant defense. Single-chain simulations reveal that short PE fragments (3-, 5-, and 7-mer) migrate from an initial domain-connector pose toward a heme-proximal, previously characterized inhibitor-binding site, whereas the 14-mer remains kinetically confined to a peripheral surface site, implicating short-chain PE as a potential catalase inhibitor. In multichain corona systems, backbone dynamics show a non-monotonic, chain-length-dependent destabilization, with the 3- and 5-mer inducing the greatest structural perturbation and the 14-mer best preserving native-like dynamics despite forming an extensive, percolating surface network across all four subunits. MM-PBSA free energy decomposition identifies van der Waals interactions as the dominant thermodynamic driver of binding, while electrostatic surface mapping indicates that cationic surface patches instead govern long-range spatial targeting of PE chains. These findings establish a framework linking nanoplastic fragment size to protein-level toxicity, with direct implications for assessing the biochemical risk of environmentally weathered polyethylene.

Biological systems such as extracellular vesicles (EVs) are highly sophisticated and functionally diverse. Despite growing recognition of EVs' roles in health and disease through mediating intercellular communication, their inherent heterogeneity and challenges in purity and yield hinder their mechanistic research and clinical translation. To address this gap, we sought to translate essential EV properties into a controllable and simplified platform by engineering biomimetic synthetic vesicles (SVs) incorporating defined proteins and lipids. Using microfluidics, we generated SVs loaded with bulk membrane and cytosolic proteins from cancer cells, as well as individual organotropism-associated integrins. Bulk membrane protein incorporation into SVs reduced surface charge, modified lipid profiles, and enhanced cellular uptake, while integrin incorporation altered biodistribution. To investigate protein loading, we profiled input materials and SVs by mass spectrometry, identifying features linked to high incorporation, such as low molecular weight, positive charge, high isoelectric point, and predicted stability, globularity, lipidation, and amphipathic helices. This work presents a synthetic platform for dissecting the roles of EV-associated features and cargo, while providing insights into protein properties relevant to the optimization of delivery systems.

Expansion microscopy (ExM) physically magnifies preserved biological specimens by using a specimen-permeating hydrogel to pull biomolecules, such as proteins, apart from each other. ExM protocols exhibit a tension between the need to initially fix proteins to each other during sample preservation, to retain proteins and their nanoscale organization, and the need to separate proteins from each other isotropically, to achieve physical magnification. We here propose to address this tension by fixing proteins directly to the hydrogel network itself, which then serves as a common substrate for both initial fixation and final separation. We equip proteins with chemical handles, then form a specimen-permeating hydrogel that binds the handles, in a ~15-minute chemical process conducted entirely at -20oC. This network fixation ExM (nfExM) procedure enables excellent epitope retention and nanoscale organization preservation, in cultured cells. nfExM may help support ultrastructural investigations of protein complexes and interactions.

The delivery of small-molecule therapeutics across cellular membranes remains a fundamental challenge in drug development, particularly for compounds whose physicochemical properties limit intracellular bioavailability. Inspired by the low toxicity, biodegradability, and synthetic accessibility of triethanolamine-derived lipids, we investigated a series of C18 aminotriester lipids as excipients for the codelivery of anticancer therapeutics. Four structurally related triester lipids bearing saturated, cis-monounsaturated, trans-monounsaturated, and di-unsaturated C18 tails were synthesized through straightforward esterification reactions with triethanolamine. These lipids were evaluated for their ability to enhance the antiproliferative activity of several anticancer agents, including paclitaxel, doxorubicin, amonafide, homoharringtonine, camptothecin, and SN-38. Across multiple drug classes, the aminotriester lipids exhibited minimal intrinsic cytotoxicity and generally produced limited enhancement of drug potency. However, the fully saturated tris-stearate aminotriester lipid selectively enhanced the antiproliferative activity of amonafide in HCT-116 colorectal cancer cells, particularly at lower concentrations. In contrast, unsaturated lipid analogues displayed negligible enhancement effects, and the activity of the tris-stearate lipid was substantially attenuated in A549 lung adenocarcinoma cells, weakly observed in HT-29 colorectal cancer cells, and absent in CT26 murine colorectal cancer cells. Lactate dehydrogenase release assays indicated that these effects were not associated with membrane lysis or nonspecific cytotoxicity. Further, fluorescence microscopy time course experiments suggest improved membrane trafficking of amonafide when codelivered stoichiometrically with fully saturated C18 aminotriester lipids. Collectively, these findings demonstrate that saturated triethanolamine-based aminotriester lipids may function as selective and biocompatible codelivery excipients for certain small-molecule therapeutics. The observed dependence on both lipid saturation state and cellular context suggests that membrane organization and lipid packing may play important roles in mediating these effects. These results establish a foundation for further mechanistic investigations into aminotriester lipid-assisted small-molecule delivery systems.

Finite-bandgap charge-based FET biosensors are known to be insensitive to electronic tunneling noise in the semiconductor and interference from weakly-charged proteins in physiological samples. However, their signal is still corrupted by pH fluctuations and condensing counterions in the bulk electrolyte. Here, we demonstrate that electrolyte noise is screened by the Stern layer beyond the linear Debye-Huckel limit, when the interfacial potential drop is larger than the thermal voltage. Using a fully nonlinear Gouy-Chapman-Stern (GCS) theory, a universal calibration curve is derived and shown to collapse the low-noise FET signals for miRNA, extracellular vesicles (EV), and protein, captured either directly or through silica and magnetic nanoparticle charge reporters, over eleven decades of analyte concentration. The theory involves the dissociation constant KD and a gain parameter {chi}, which represents the competing effects of the reporter charge and buffer Debye screening that must be large to produce the low-noise GCS condition. The limit of detection (LOD) is shown to be KD/{chi}, which can be optimized by varying the probe density, charge reporter, and buffer solution. An optimized hybridization buffer then yields {chi} ~ 22 (LOD ~1 pM) for miRNA detection, and multivalent capture reduces KD by four orders of magnitude for EV detection (relative to protein detection) with {chi} ~ 34 (LOD ~10 fM).