Journal of Nanobiotechnology

Extracellular vesicles (EVs) are promising nanocarriers for therapeutic delivery; however, the factors governing EV uptake by recipient cells remain incompletely understood. In this study, we investigated whether EV internalization is primarily influenced by donor-cell origin or recipient-cell phenotype. Fluorescently labeled EVs derived from HEK293T, or SKBR-3 cells were incubated with a range of human epithelial, immune, and murine cancer cell lines at different doses and time points. HEK293T-derived EVs showed highly variable uptake across recipient cells, with hepatocellular carcinoma cell lines Huh7 and HepG2 exhibiting the highest internalization, while parental HEK293T cells showed the lowest. THP-1 immune cells also demonstrated strong uptake, whereas Jurkat cells showed moderate uptake. In murine melanoma models, Yummer cells internalized more EVs than B16F10 cells. Importantly, similar uptake trends were observed using SKBR-3-derived EVs, where Huh7 and HepG2 again displayed the highest uptake despite originating from a different donor cell source. EV internalization increased with dose and incubation time until saturation at higher concentrations. Together, these results demonstrate that EV uptake is predominantly determined by recipient-cell characteristics rather than EV source. These findings provide important mechanistic insight for the development of EV-based therapeutics and suggest that optimizing recipient-cell targeting is essential for efficient vesicle-mediated delivery. Graphical abstractEV uptake is determined by cell membrane properties rather than by the source of the EVs. The image was created by Biorender. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/726167v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@f5c1cborg.highwire.dtl.DTLVardef@860962org.highwire.dtl.DTLVardef@1d20239org.highwire.dtl.DTLVardef@9003af_HPS_FORMAT_FIGEXP M_FIG C_FIG

Detection of micro- and nanoplastics (MNPs) in human tissues has raised growing concern about their biological effects on tissue and cell function. While previous studies have examined MNP-cell interaction, most focused on limited cell and plastic types. Here, we present a comprehensive, quantitative investigation into how different types of nanoplastics (NPs) associate with and affect diverse cell types under physiologically relevant conditions. Using microfluidic-calibrated fluorescence microscopy, we quantify NP accumulation in cells in vitro and match cellular NP concentrations to levels reported in human tissues. While cell-associated NPs could be gradually released in vitro, they persist in vivo for over one month without detectable reduction in a mouse model. We discover that NP exposure at these levels broadly impairs cell proliferation across epithelial, endothelial, fibroblast, and immune cells, with cell type-dependent sensitivity. NP exposure also reduces motility in T cells and fibroblasts, with more complex effects observed in macrophages. Mechanistically, NP-cell association and trans-epithelial transport involved not only classical endocytic regulators but also pathways related to ion and water transport. Notably, NP association and release were highly sensitive to the extracellular fluid environment within the physiological range. By testing inhibitors of these pathways, we identified molecules that reduce NP-cell association and promote release. We further compared common NPs found in human samples and widely used in research: polystyrene (PS), polyethylene (PE), and polypropylene (PP). Although these NPs similarly impaired proliferation and motility, they showed markedly different cellular association and release dynamics. These findings reveal the impact of NPs on tissue cell functions and uncover novel regulatory pathways, establishing a quantitative framework for studying NP-cell interactions in biologically relevant conditions.

Gold is widely distributed in the biosphere, and higher plants growing on geochemically anomalous substrates can accumulate significant amounts of gold. This study reports, for the first time from Goa, the detection, spectroscopic characterisation, and X-ray diffraction analysis of phytoformic gold -- biologically sequestered crystalline gold -- in the above-ground dry litter ash of six tree species (Acacia auriculiformis, Alstonia scholaris, Anacardium occidentale, Artocarpus heterophyllus, Ficus benghalensis, Syzygium cumini) growing on mining dumps within the North Goa Banded Iron Formation (BIF) Belt of the Western Dharwad Craton. Microgravimetric analysis of aqua regia-extracted heavy ash fractions revealed gold concentrations of 275-1100 ppm, two to five orders of magnitude above the crustal background ([~]0.004 ppm). Fourier Transform Infrared (FTIR) spectroscopy of 0.22{square}m membrane-filtered crude extracts confirmed the tetrachloroaurate(III) complex [AuCl{square}]{square} as the dominant dissolved gold species, with the diagnostic 1400-1700{square}cm{square}1 absorption envelope present in all six species. UV-Visible spectrophotometry confirmed chloroauric acid formation with a universal {lambda}max at 372.5{square}nm across all species. Powder X-ray diffraction (XRD) of heavy ash fractions yielded the characteristic FCC metallic gold reflections Au(111), Au(200), and Au(220) in all five species analysed. Application of the Debye-Scherrer equation to the Au(111) reflection (2{theta} = 38.2{degrees}, Cu K) established crystallite sizes of 17.7-31.8{square}nm, confirming that phytoformic gold exists as nanoscale crystalline particles in all species. Ficus benghalensis produced the largest and most crystalline gold nanoparticles (31.8{square}nm) and uniquely exhibited strawberry-shaped isomorphic auriferous siliceous biominerals designated phytoauroliths. The described low-cost protocol -- ashing, aqua regia extraction, membrane filtration, and multi-technique spectroscopic and diffraction confirmation -- constitutes a validated method for rapid biogeochemical gold anomaly detection. Applications in gold phytoextraction and mining waste phytoremediation are discussed.

Extracellular vesicles (EVs) hold significant promise as biomarkers, but their clinical translation is constrained by variability in pre-analytical handling and isolation. EV isolation methods directly shape which EV populations are captured and characterized, yet systematic method comparisons across multiple analytical dimensions are limited. We comprehensively evaluated eleven EV isolation methods to define their performance and applications. EVs were quantified by NanoFCM, profiled for tetraspanins (CD9, CD63, CD81) via MSD assays, and further characterized by LC-MS/MS proteomics. We show that different EV isolation methods recover different EV populations. Our data provide guidance on method selection based on downstream application needs and serve as a look-up tool if a protein of interest is detected. EV isolation methods broadened proteome coverage but showed divergent performance and recover different EV populations. While all methods captured EVs in the 50-150nm range, centrifugation and ultracentrifugation identified the broadest proteomes (up to 1093 proteins) driven by higher plasma protein carryover. Conversely, ExoEasy and qEV 70 isolated larger EVs and achieved stronger depletion of abundant plasma proteins but showed lower proteome coverage. A total of 117 proteins were detected across all isolation methods. Pre-clearing samples removed contaminants but at the cost of protein identifications. We demonstrate that method selection must align with the specific analytical goal: centrifugation for comprehensive proteome profiling, affinity/size-exclusion methods for contaminant-sensitive assays, and precipitation for high-throughput applications. This systematic characterization provides an evidence-based framework and look-up resource for matching isolation strategies to downstream applications and research questions. Graphical Abstract for Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/710675v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@12ad967org.highwire.dtl.DTLVardef@270e4eorg.highwire.dtl.DTLVardef@1c41bcorg.highwire.dtl.DTLVardef@11fb236_HPS_FORMAT_FIGEXP M_FIG C_FIG This study evaluated 11 extracellular vesicle (EV) isolation methods which enriched distinct EV subpopulations with varying degrees of contaminants. No single approach optimized purity or proteome coverage; in this paper we present an Evidence-Based Framework to select plasma EV isolation methods based on downstream application needs.

The isolation of cellulose nanofibrils (CNFs), a promising precursor for sustainable and high-performance materials, has relied on chemically intensive, energy-demanding processes. As these processes were originally designed for the isolation of CNFs from wood, we herein show that the intrinsic ultrastructure of non-structural plant cells provides unique opportunities, namely direct access to loosely organized cellulose nanonetworks. We demonstrate that this loose nanofibrillar tissue can be transformed into CNFs with sizes down to elementary nanofibrils ([~]4 nm) at high yields (reaching [~]32%) under exceptionally mild hydrothermal conditions. Three distinct plants were evaluated and the physicochemical properties of the obtained nanonetworks and corresponding CNFs were thoroughly studied, including the hydrodynamics of the resulting gels. Films prepared from the obtained CNFs showed similar performance to those obtained from conventionally isolated wood-based CNFs. Overall, this study demonstrates that CNFs can be obtained through low-intensity, hazard-free, processes from widely available biomass. Thus, this approach offers a unique shift in the range of opportunities to produce CNFs facilitating the integration of their use into the food supply chain, biomedical applications, and other regulatory-constrained applications.

A programmable 4-input cascade DNA logic gate utilizing toehold mediated strand displacement (TMSD) was implemented on a 3D printed hybrid paper-polymer vertical flow device (3D HPVF) for on/off sensitive and specific fluorescence detection of platelet derived growth factor BB (PDGF BB). Polypropylene was 3D printed directly on paper and thermally cured to create micro paper analytical devices ({micro}PADs). The 3D HPVF comprised of three layers of {micro}PADs enclosed in a casing that clamped each {micro}PAD securely to ensure seamless and efficient wicking between layers. In the presence of PDGF BB, a partially complementary strand to a PDGF B aptamer (PDGF B Apt), cApt, was liberated from a PDGF B Apt/cApt duplex in solution. The solution was then deposited on the 3D HPVF with a dimeric g-quadruplex hairpin. The 4-nucleotide toehold region on the cApt started the hybridization reaction with the dimeric g-quadruplex hairpin (dGH) opening it up allowing formation of a dimeric g-quadruplex structure that binds with thioflavin T (ThT) with enhanced fluorescence intensity at room temperature. The 3D HPVF exhibits a pico molar range of detection from 10pM to 100pM with a 10pM limit of detection (LOD) for PDGF BB concentrations relevant for pregnant women predisposed to early-onset preeclampsia with clear differentiation when compared to similarly competing analytes PDGF AA and AB.

Many agricultural soils are deficient in key macronutrients needed for healthy plant development. Relying on highly water-soluble commercial fertilizers for long durations can be costly and environmentally harmful. This study investigates a phosphorus-loaded Mg/Fe layered double hydroxide (LDH) dispersed on Douglas fir biochar (Mg/Fe-LDH biochar) as a controlled-release fertilizer and evaluates its impact on bush bean (Phaseolus vulgaris L.) growth. Emphasizing sustainability, the work integrates controlled-release fertilizers, biochar, and LDH modification to enhance nutrient use efficiency and mitigate environmental runoff. Mg/Fe-LDH was directly synthesized on biochar via a co-precipitation approach, loaded the composite with phosphate by anion exchange, and characterized the material using elemental analysis, N2 Brunauer-Emmett-Teller (BET) determinations surface area analysis, and x-ray photoelectron spectroscopy to confirm successful LDH modification on Douglas fir biochar, and high surface area with accessible active sites. The synthesis yielded a stable P-Mg/Fe-LDH biochar with enhanced dispersibility and phosphate-buffering capacity, enabling controlled-release fertilization. In greenhouse experiments, bush beans grown with the P-Mg/Fe-LDH biochar exhibited improved growth metrics, including increased yield (beans fresh weight of 31.7 g), biomass (plant dry weight of 6.3 g), plant height (32.8 cm), and improved nutrient uptakes (1.88 mg (P) g-1) at 100.88 kg (P2O5) ha-1 compared with unfertilized controls and conventional P fertilizers, indicating efficient, controlled-release phosphate delivery and sustained nutrient availability. The results demonstrate that integrating LDH-modified biochar can enhance P uptake and plant growth while reducing leaching losses. Overall, this study highlights the strategic significance of combining biochar, layered double hydroxides, and controlled-release formulations to advance sustainable nutrient management and improve crop performance in agroecosystems. The findings offer a promising pathway for environmentally conscious fertilizer design and soil amendment strategies that align with global goals for resource efficiency and food security. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/727001v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@316444org.highwire.dtl.DTLVardef@adcd48org.highwire.dtl.DTLVardef@8068aforg.highwire.dtl.DTLVardef@58d623_HPS_FORMAT_FIGEXP M_FIG C_FIG

Adenoid cystic carcinoma (ACC) of salivary gland is a "immune-cold" tumour. Annexin A3 (ANXA3) is an apoptotic protein found to be participating in immune cell infiltration in tumour microenvironment (TME) of various cancer cases. Significant low expressions of ANXA3 protein found in adenoid cystic carcinoma. We hypothesized overexpressing ANXA3 transforms ACC "cold" TME to "hot". We cultured UM-HACC-2A and UFH2 spheroids on extracellular matrix and co cultured them with peripheral blood mononuclear cells. We functionalized FDA (The Food and Drug Administration) approved Poly(lactic-co-glycolic acid) PLGA nanoparticles with anti-cMyb antibody and ANXA3 recombinant protein using streptavidin-biotin conjugation. Upon overexpressing ANXA3 in ACC spheroids in immune coculture model using functionalized nanoparticles, significant increase of tumour infiltrating lymphocytes and decrease in the size of the ACC spheroids observed. Apoptotic profiler assay further confirmed significant upregulation of apoptotic proteins, some of them participate in immune infiltration. Overall, this project exhibits promising results showing potential approach to convert ACC into an immune "hot" tumour.

In this paper, we aim to present a new intravital cells visualization method, which is based on use of a dye called ABDS ("A Beautiful dye for staining"), which can be prepared using a marker pen and is useful for eukaryotic cell research. Using a wide range of instruments, including optical measurements, microscopy studies and wet biology techniques, we have shown that ABDS is close by properties to Rhodamine 6G dye (R6G), which is well known as endoplasmic reticulum stainer. However, by the careful examination of the ABDS and R6G images (ABDS/R6G), we have proved for the first time that these dyes also stain the cytoplasmic membranes. The significant contrast between ABDS/R6G signal from cell membrane and endoplasmic reticulum allows them to be distinguished in the fluorescence photographs. Other important properties of ABDS are its availability, simplicity in manufacturing, safety for living cells in vitro, and bright stable fluorescence, which in contrast to commercial dye like DiBAC allows us to study cells in space and time with high detalization. The paper includes a method for preparing ABDS, a data set with its characteristics, comparison with other commercial dyes, as well as examples of ABDS usage in cells research. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/717455v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@f1ceacorg.highwire.dtl.DTLVardef@137abd2org.highwire.dtl.DTLVardef@1f19efcorg.highwire.dtl.DTLVardef@1fcbc9e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA protocol for high-resolution vital staining of the cells using an inexpensive dye based on permanent marker ink is proposed. C_LIO_LIThe absorption, emission and Raman spectra of the proposed dye are presented, and a direct comparison with commercial dyes Rhodamine 6G, DiBAC and Deep Red Cell Mask dye is made. C_LIO_LIThe main characteristics of the proposed dye are low toxicity, long-term fluorescence, and the ability to separately stain the endoplasmic reticulum and cytoplasmic membrane. C_LIO_LIThe ability of the Rhodamine 6G dye to stain cell membranes also has been proved. C_LI

Extracellular vesicles (EVs) are cell-secreted biological nanoparticles that play a crucial role in intercellular communication and are gaining increasing attention as diagnostic biomarkers, therapeutic agents, and drug delivery vehicles. Consequently, the development of robust and sensitive methods for their characterization is essential. Herein we present the use of a microscope-mounted nanofluidic device for direct size determination and multi-parametric (3-color) fluorescence-based phenotyping of single biological nanoparticles that are in the size range of 20-200 nm in a method we denote Nano-SMF (SMF; size and multiplexed fluorescence). We demonstrate that it is possible to accurately determine the size of nanoparticles by analyzing their one-dimensional Brownian motion during directional flow through nanochannels, achieving size distributions for monodisperse nanoparticle solutions that are on par with TEM analysis, and size discrimination of nanoparticle mixtures that is significantly improved compared to conventional nanoparticle tracking analysis (NTA). Furter, we demonstrate that the method can be applied to analyze EVs directly in minute volumes of cell supernatant, avoiding pre-isolation or concentration steps. The method was applied to phenotype CD63- and CD81-positive EVs from a human embryonic kidney cell model, demonstrating that vesicle sub-populations defined by these two tetraspanin biomarkers differ significantly in size.

Glioblastoma (GBM) presents significant therapeutic challenges due to its aggressive nature, complex microenvironment and the limitations of conventional drug delivery systems. In this study, hybrid nanoparticles were developed by combining synthetic liposomes with macrophage-derived extracellular vesicles (EVs) to harness the strengths of both platforms. Two distinct liposomal formulations, DPPC:Chol:DSPE-mPEG2000 (F1) and DPPC:DPPS:Chol:DSPE-mPEG2000 (F2), were used as the basis for the synthesis. EVs derived from J774 macrophages were integrated with F1 and F2 to create hybrid nanoparticles (H-F1 and H-F2). Doxorubicin (DOX) was encapsulated using a pH gradient and a remote loading procedure. The mean particle size of H-F1-DOX and H-F2-DOX was 158.2 {+/-} 1 nm and 162.8 {+/-} 9 nm, respectively. The polydispersity index (PDI) was 0.130 {+/-} 0.012 and 0.084 {+/-} 0.033, while the zeta potential values were -14.9 {+/-} 0.7 mV and -26.7 {+/-} 3.1 mV, respectively. H-F2-DOX exhibited the highest encapsulation efficiency (EE%), reaching 76.5{+/-}3.4%. The encapsulated hybrids remained stable up to one week, at +5{degrees}C. The release of DOX from H-F2-DOX in DMEM supplemented with 10% serum showed pH sensitivity, with total DOX release of 64.9 {+/-} 5.3% at pH 7.4 and 90.7 {+/-} 6.5% at pH 5.5. The cell viability assay demonstrated that all formulations exhibited strong cytotoxic effects against GBM cells under normoxic conditions, with H-F2-DOX showing the most potent effect under hypoxia-mimetic conditions.

Polymeric 3D printing of microfluidic devices for biosensing is an appealing fabrication alternative for rapid manufacturing of biosensing devices with complex geometry in a streamlined, repeatable and cost-effective manner without the need for expensive instrumentation such as those employed in photochemical etching and soft lithography. Hybrid 3D printed paper-based microfluidics is an emerging area which harnesses the unique properties of both, merging the construction of microfluidic structures and the inherent capillary-driven flow within paper substrates. In this work, we have fabricated hydrophobic barriers by 3D printing a single layer of machinable wax, thermoplastic polyurethane, polylactic acid and polypropylene directly on chromatography paper to create open microchannels and determine the most suitable material. Characterization of each open microchannel using the four materials revealed polypropylene as the most reliable material with high hydrophobic barrier integrity and resolution. Polypropylene achieved functional microchannels with a resolution of 621 {+/-} 33{micro}m, hydrophobic barrier integrity of (93.75 {+/-} 9.16%), wicking speed of 0.38mm/s and optimal hydrophilicity of channels (51.4 {+/-} 8.36 {degrees}) with minimal embedding during thermal curing. To demonstrate proof of principle, a fluorescence assay demonstrating the formation of a dimeric g-quadruplex structure from a g-rich sequence which significantly enhances fluorescence of thioflavin T was implemented.

With the introduction of dedicated nanoscale flow cytometers, the need for suitable compensation beads has emerged. Here, we present a rapid and cost-effective method to generate [~]100 nm antibody-binding compensation beads compatible with a wide range of antibody species for use in nanoscale flow cytometry. This approach may provide a practical interim solution until commercial alternatives become available.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies due to its dense stroma, which limits drug delivery and therapeutic efficacy. Ultrasound (US) mediated strategies using nanobubbles (NBs) offer a promising approach to enhance treatment, yet the biological effects of NB exposure and the timing of US application remain unclear. Here, we investigated how NB exposure with immediate (0h) or delayed (1h) US affects viability, proliferation, metabolism, and stress signaling in PANC-1 and BxPC-3 cells. Immediate US exposure in the presence of extracellular nanobubbles resulted in a greater reduction in cell viability at 24 h compared to delayed US application. Proliferation analysis showed that Ki67 positivity decreased following USNB treatments in both cell lines. Metabolically, NB treatment alone increased cellular activity, whereas combined USNB treatment reduced metabolic activity over time. Seahorse analysis revealed higher basal respiration in PANC-1 cells compared to BxPC-3 cells, consistent with a more glycolytic phenotype, while USNB treatment enhanced glycolytic responses, particularly in PANC-1. Moreover, stress responses were also more pronounced in PANC-1 cells, with HSP70 expression increasing up to 2-fold in NB incubated group and decreasing in USNB groups compared to untreated, whereas BxPC-3 cells exhibited only modest and opposite changes to PANC-1 in HSP70 expression decreasing with NB incubation. Treatment timing critically influenced outcomes, with immediate US producing stronger antiproliferative and cytotoxic effects, highlighting the importance of sequencing in USNB therapeutic strategies. Moreover, NBs alone stimulated metabolic and stress responses that may promote proliferation, whereas NBs combined with US induced stronger stress responses associated with metabolic reprogramming and reduced proliferation.

Environmental degradation and accumulation of plastics results in micro- and nanoplastics (MNPLs) that are small enough to cross biological barriers, including the blood-brain barrier. Microglia, resident immune cells of brain, are critical regulators of neuroimmune homeostasis and represent a cellular target of nanoplastic exposure. In this study, we assessed the neurotoxic effects of two sizes of polystyrene nanoplastics (PS-NPs; 100 nm and 500 nm) using integrated in vivo and in vitro exposure and washout paradigms. In vivo exposure in mice (60 days; 0.15 or 1.5 mg/day) showed the accumulation of both PS-NP sizes in the cerebral cortex without histopathological damage. However, cortical microglia showed pronounced morphological remodeling, observed as increased expression of Iba1 and GFAP. Transcriptomic profiling of cortical tissue revealed a strong size-dependent response. The 100 nm PS-NP group revealed 18 DEGs (|log2FC| [&ge;] 2, padj < 0.05), whereas the 500 nm PS-NPs showed more than 4,000 DEGs, including upregulation of immune- and microglia-associated genes (CCL5, CXCL10, LCN2, LYZ2) and downregulation of synaptic and neuronal signaling genes (GRIN2B, SYN1, STX1B, MAP1B, ITPR1/2). In vitro assessment, using BV2 microglia cells, showed internalization of PS-NPs via the endolysosomal pathway, with strong co-localization to Rab7- and LAMP2-positive compartments and prolonged intracellular retention following exposure washout. Also, microglial activation markers (Iba1, CD68) exhibited a transient, size- and concentration-dependent increase, correlated with intracellular particle burden rather than cumulative exposure. Overall, these findings demonstrate that PS-NPs accumulate in brain, driving size-dependent microglia activation and transcriptomic reprogramming, even after cessation of exposure to PS-NPs. HighlightsO_LIPS-NPs (100 nm and 500 nm) reach mouse cerebral cortex following 60-day oral exposure. C_LIO_LIPS-NPs were internalized by microglia; accumulated in endolysosomal compartments. C_LIO_LIPS-NP exposure induced transient microglial activation without sustained cytotoxicity. C_LIO_LIMicroglial activation was correlated with intracellular PS-NPs burden. C_LIO_LITranscriptomics revealed disruption of neuroimmune and microglial regulatory pathways. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/712727v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1aba3eaorg.highwire.dtl.DTLVardef@1967641org.highwire.dtl.DTLVardef@12da637org.highwire.dtl.DTLVardef@1fb8441_HPS_FORMAT_FIGEXP M_FIG C_FIG

The tumor microenvironment (TME) is a complex ecosystem composed of tumor cells, cancer-associated fibroblasts (CAFs), immune suppressive cells, and the extracellular matrix (ECM), playing a crucial role in tumor development and CAR-T cell therapy efficacy. CAR-T therapy has shown promise in hematological malignancies but faces challenges in solid tumors due to the TMEs ability to suppress CAR-T cell infiltration, proliferation, and cytotoxicity. Traditional drug evaluation models, such as 2D cell cultures and animal models, have significant limitations due to oversimplification of the in vivo environment or physiological differences between species. Organoid models offer a more biomimetic approach but often fail to fully recapitulate the TMEs complexity and heterogeneity. Our research developed a tumor organoid and CAF co-culture model using the IBAC co-culture chip, demonstrating that CAFs significantly impact CAR-T cell therapy efficacy by forming physical (e.g., fibronectin) and chemical (e.g., IL-10) barriers that prevent CAR-T cell infiltration and cytotoxicity. This model provides a high-biomimetic platform for investigating the TMEs effects on CAR-T therapy and highlights the importance of incorporating a comprehensive stromal component into in vitro models to enhance their predictive power for cancer treatment.

Surface functionalization of biomaterials enables the immobilization of proteins and other molecules and can be utilized to direct the biological response to devices and implants. Fetuin-A is a blood plasma protein involved in numerous physiological processes, including the regulation of mineralization. Notably, many investigations of fetuin-A have explored its cellular interaction when in solution, but limited studies report the role of fetuin-A when used as a surface modifier. The present investigation explores the response elicited by fetuin-A on Saos-2 cells when it is immobilized on a model gold surface through the covalent reaction with dithiobis(succinimdyl propionate) (DSP). Comparative surface characterization using x-ray photoelectron spectroscopy (XPS), atomic force microscopy - infrared spectroscopy (AFM-IR) and surface plasmon resonance (SPR) confirmed the surface modifications but indicate partial inhomogeneity in the functionalizer surface coverage. The interaction of albumin and fetuin-A with the surface was quantified by radiolabeling, quartz crystal microbalance with dissipation (QCM-D) and SPR, demonstrating a higher mass of fetuin-A bound to the surface in comparison to serum albumin. Over 7 days, cells bound to the surfaces with immobilized fetuin-A showed significantly hindered proliferation of osteoblast-like cells compared to the positive control (fibronectin), presumably due to a decrease in cell metabolism. This study provides new insights into the role of fetuin-A in regulating Saos2 cell response and elucidates its potential use in combination with chemical functionalizers for biomedical applications requiring surface modification.

In root nodule symbiosis, symbiosome compartments accommodate nitrogen-fixing rhizobia inside the plant cell. Differentiated into bacteroids, the rhizobia are surrounded by a peribacteroid space and a plant-derived peribacteroid membrane, which separates them from the plant cytoplasm but allows signal and nutrient exchange between host and microbe. The morphological features of symbiosomes are primarily determined by ultrastructural single focal plane imaging, with limited information about spatial details. This study combines 2D and 3D imaging, using transmission electron microscopy and focused ion beam scanning electron microscopy as complementary techniques to analyse the symbiosome ultrastructure and organisation in Lotus japonicus wild-type plants. The 3D model of a mature colonised root nodule cell region demonstrates a dense, puzzle-like arrangement of symbiosomes relative to one another and adjacent plant organelles. The symbiosome shape and size depends on the orientation and number of bacteroids within the compartment and features connective tubular structures. Furthermore, vesicular structures, some likely of bacterial origin, were present at the interface. The study presents a multi-angled analysis of symbiosome-related structures, highlighting their volumes, spatial distribution, and pronounced compactness. Interface associated vesicles, protrusions and connective structures hint towards a dynamic and flexible system that contributes to the plant-microbe crosstalk.

Ion channels are pore-forming transmembrane proteins that allow ions to move down an electrochemical gradient and across the channel pore and regulate many cell functions. Among them, are the G-protein-gated inwardly-rectifying K+ channels 1 (GIRK1) that are ubiquitously expressed with major functions in the brain and heart. Interestingly, significantly higher GIRK1 expression has been found in estrogen receptor positive (ER+) breast cancer patients compared to patients with HER2+ tumors or normal patients, and that was statistically correlated with shorter survival times and metastatic potential. Herein, we report the preparation of [~]4 nm GAT1508-coated poly(ethylene glycol) gold nanoparticle (PEGylated AuNP) biomarker for ER+ breast cancer cell screening through an optical microscope. A urea-based small molecule, GAT1508, with an N-methylpyrazole benzyl group on one side and a bromo-thiophene tail on the other side, has been shown to predominantly bind GIRK1 subunits and specifically activate GIRK1/2 channels. Two derivatives of GAT1508were synthesized and characterized: an ethylamine derivative (GAT1508-EA) with a chain extension from the benzyl ring, and a propylamine derivative (GAT1508-PA) with a chain extension from the pyrazole ring. Electrophysiology (TEVC and whole-cell patch-clump) experiments as well as fluorescence studies (Thallium assay) showed that only GAT1508-PA inhibited GIRK1/2-mediated K+ currents in transfected HEK293GIRK1 cells. Docking studies showed strong binding for the propylamine GAT1508 derivative, both in the amine form (GAT1508-PA) as well as in the amide form (GAT1508-PA-EG2; coupled with PEG as in the AuNPs). GAT1508-PEG-AuNPs (GAT1508-NPs) were synthesized subsequently with [~]65 wt% metal loading. UV-Vis studies revealed the presence of the conjugated ligand at 260 nm. Flow cytometry studies showed binding of Alexa 594-labeled GAT1508-NPs in ER+ MCF-7 breast cancer cells with a strong interaction, while incubation of fixed MCF-7 cells with a GAT1508-NP solution led to optical detection of ER+ breast cancer cells, without the need of fluorescent dyes and additional amplification steps. Detection was not feasible in MDA-MB-231 cells, a triple (-) breast cell line that does not express GIRK1. This is the first study, to our knowledge, that couples nanotechnology with small molecule drug design and electrophysiology to develop ion channel-tracing molecular probes for the detection/screening of ER+ breast cancer.

Rapid and highly selective sensing of ultra-low concentration protein biomarkers remains a critical challenge important for early disease diagnosis and monitoring. Here, we use conical SiO2 nanopore-based biosensing for the rapid detection of heart-type fatty acid binding protein (H-FABP). Antibodies were covalently immobilized on the nanopore surface through siloxane chemistry. The functionalized asymmetric nanopores generate a characteristic rectifying current-voltage response, which shows a distinct shift upon binding to the target protein due to partial neutralization of the negatively charged pore surface. The sensor exhibits excellent sensitivity in the attomolar to nanomolar concentration range with a detection limit (LOD) of [~]0.4 aM. Furthermore, the platform exhibits high selectivity, distinguishing H-FABP from non-target proteins (HSA and Hb) at concentrations six orders of magnitude higher. We also demonstrate that nanopores can be regenerated using sodium hypochloride and O2 plasma treatment, enabling repeated functionalization and reuse.