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

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.

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.

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

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.

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.

Agriphotovoltaics (APV) combines crop production with solar energy generation to address increasing demands for food and energy while reducing land-use competition. Unlike conventional opaque photovoltaic systems, semitransparent organic photovoltaics (OPVs) selectively absorb light, potentially improving efficiency but also altering both light quantity and spectral quality, key factors affecting plant growth. Here, we developed a rapid bioassay based on hypocotyl elongation to evaluate plant responses to OPV-filtered light using Arabidopsis thaliana and Cardamine hirsuta, two species with contrasting shade strategies. Screening a diverse set of OPV materials revealed that plant growth responses depend more on spectral composition than on total light intensity alone. Certain materials, such as PTB7-Th and D18, produced growth patterns similar to neutral shading, while others promoted elongation. Our analyses identified blue light wavelengths, linked to cryptochrome activity, as more critical than red light wavelengths, linked to phytochrome activity, for maintaining normal development. These findings provide a scalable framework to assess OPV-plant compatibility and demonstrate that optimizing spectral quality alongside light intensity is essential for designing efficient APV systems that sustain crop performance while generating renewable energy.

Biomarkers in sweat and saliva offer a promising avenue for non-invasive health monitoring. Electrochemical sensors have the potential to measure such biomarkers simultaneously. However, they are limited in discriminating individual biomarkers in mixtures, as redox potentials often overlap, resulting in current signatures that cannot be deconvoluted. This study focuses on differentiating biomarkers using orthogonal sensing materials combined with machine learning. We introduce a flexible electrochemical sensor array comprising carbon flower electrodes modified with poly(vinylidene fluoride) (PVDF) or poly(4-vinylpyridine) (P4VP) for the detection of estradiol (E2), ascorbic acid (AA), serotonin (5-HT), and melatonin (Mel). The two polymers act by altering the redox potential and current response of each biomarker, thereby enhancing signal diversity and enabling peak separation. Using multi-output regression models on 450 single and mixture measurements, the array accurately predicts concentrations (R2 = 0.95) over a wide dynamic range spanning nanomolar to micromolar levels. Polymer-resolved analysis reveals that PVDF-modifications enhance E2 and Mel detection, while P4VP-modifications improve AA and 5-HT quantification, highlighting the benefit of complementary orthogonal sensing electrodes. This finding is further supported by feature attribution analysis, which shows that the machine learning model relies on polymer-specific electrochemical signatures, directly linking improved performance to distinct polymer-analyte interactions. Overall, these results demonstrate that combining polymer-modified orthogonal electrodes with machine learning enables accurate, multiplexed sensing in complex mixtures, advancing selective detection strategies for future sensor platforms.

Cancer is a significant contributor to global mortality and places a substantial burden on healthcare systems, underscoring the need for improved strategies for developing and evaluating new therapies. Electrochemical impedance monitoring of in vitro cancer models is a promising technique for evaluating treatment effectiveness, particularly for evaluating how well a drug may kill cancer cells. This approach is advantageous over conventional end-point assays because it is non-destructive, label-free, and can provide temporal information on cell behavior and drug kinetics. However, traditional impedance devices are limited in that they do not support three-dimensional cell culture that has become standard in cancer studies. Typical devices are planar substrates that support monolayer culture, which has been shown to overestimate drug effectiveness. In this work, we propose 3D printed bioelectronic scaffold devices that provide 3D cancer cell culture while functioning as an on-chip readout for monitoring changes in cell characteristics via impedance. We describe device development and demonstrate reproducible fabrication, stable electrochemical properties, cell detection by impedance, and proof-of-concept monitoring of cytotoxicity in response to a chemotherapeutic drug. Overall, this technology offers a promising platform that could be further developed for compound screening as part of drug development or precision medicine.

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

Polydimethylsiloxane (PDMS) is an excellent material for the construction of biomimetic leaf replicas which reproduce leaf surfaces with high fidelity. This allows for the study of leaf surface-colonizing bacteria and the impact of the leaf topology on bacterial distributions and behavior. However, their application is limited to short-term experiments, as long term survival of microorganisms on their surface is not possible due to a lack of nutrient replenishment. On living leaves, nutrients diffuse across the cuticle via leaching, a process not yet replicated in biomimetic systems. Here, we explore whether water and fructose can be supplied to microbial colonizers on PDMS membranes by mimicking leaching. We created hybrid membranes by incorporating polymers (Carbopol, Pemulen, cellulose microfibers, cellulose nanocrystals, and polyvinylpyrrolidone) to enhance nutrient transport. We determined that bulk diffusion of water correlated negatively with membrane thickness and positively with polymer concentration. Further, fructose diffusion across hybrid membranes reached similar rates compared to isolated Populus x canescens leaf cuticles. Under high relative humidity, these membranes supported long-term bacterial survival. Our findings represent important steps towards the development of topomimetic leaf surfaces that sustain microbial life, enabling further investigation into the microbe-microbe interactions that take place on leaves.

Lipid nanoparticles (LNPs) have demonstrated strong potential in COVID-19 mRNA vaccines nevertheless they still face the challenges in low mRNA delivery efficacy. Virus-like porous silica (VLPSi) nanoparticles (NPs) represent a promising biomimetic delivery platform because their spiked morphology may enhance cellular internalization and promote endosomal membrane disruption. However, the application of VLPSi for mRNA has been rarely explored. In this study, hybrid lipid-VLPSi NPs were developed by combining VLPSi with either lipoplexes (LPs) or LNPs. The effects of lipid types, mass ratio of different compositions, and amine modifications of VLPSi on mRNA delivery were studied. The results demonstrated that both LP and LNP could be successfully integrated with VLPSi to form hybrid delivery systems for mRNA transfection. VLPSi could significantly enhance mRNA delivery of both LPs and LNPs due to improved cellular uptake, structural stabilization of the mRNA complex, and enhanced endosomal escape mediated by the rigid virus-like surface architecture. Among the tested lipid formulations, the ionizable lipid ALC-0315 and helper lipid DOPE with mass ratio of 5:3 was the most effective lipid composition to be integrated with VLPSi, showing the highest mRNA delivery performance. In addition, amino modification of VLPSi was found to be a critical factor for efficient mRNA delivery. Hybrid LNPs containing amino-modified VLPSi showed significantly higher transfection efficiency than those containing unmodified VLPSi. Notably, amino-modified LNP-VLPSi achieved up to fivefold higher gene expression than conventional LNPs. Overall, this study establishes VLPSi as an efficient platform for amplifying lipid-mediated mRNA delivery. Owing to its straightforward integration into widely used LNP systems, VLPSi offers an adaptable and effective strategy for advancing next-generation mRNA therapeutics.

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

Infections caused by multi-drug-resistant organisms (MDROs) pose a significant public health threat, responsible for over 2 million hospitalizations and 23,000 deaths annually in the United States. Microbiome dysbiosis (imbalance) is considered one of the main causes for MDRO colonization and the resulting infections. Rapid detection and intervention of MDRO outbreaks are crucial to alleviating strain on patients and healthcare facilities. Current diagnostic methods for MDRO detection are too slow and costly to provide the rapid MDRO detection necessary for patient care facilities. Here we present a rapid, accurate and cost-effective electrochemical sensor capable of MDRO detection down to [~]104 colony forming units (CFU)/g in mice and human stool samples. Our novel sensor utilizes probe-modified Screen-Printed Electrodes (SPEs) capable of hybridizing target gene sequences associated with MDROs. The resulting probe/target complex generates a unique and highly sensitive signal detectable down to 10 atto molar or 10 CFU/mL of target TEM-1 gene. The use of these pre-functionalized SPEs reduces individual sample analysis time to less than an hour. Several target sequences from two chromosomal target genes (AmpC and AcrB found in E. coli) have been identified and successfully detected in clinical stool samples with results comparable to the standard quantitative PCR method. Additional target genes associated with antibiotic resistance (TEM-1, VanA, KPC and SHV) have also been successfully detected in vitro and are ready for clinical evaluation. Future development includes multiplexing the sensor to simultaneously detect up to three MDROs target genes, including {beta}-lactamases that hydrolyze {beta}-lactams, the most commonly used antibiotics in clinical settings. This novel sensor platform will be a rapid, economical, point-of-care device with little requirement of reagent handling or technical training.

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 {micro}m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions. HighlightsO_LIDevelopment of size-controlled core-shell microparticles to protect phytase C_LIO_LIPhytase-chitosan microparticles are surrounded by an alginate-{kappa}-carrageenan shell C_LIO_LIOptimization achieved 32.7% loading capacity with a uniform size of 3.2 {micro}m C_LIO_LICore-shell microparticles retained 62.7% enzyme activity after heat and digestion C_LIO_LIPhytase powder (2 mg) is required for a single maize meal C_LI

This work aimed to establish a translationally viable, xeno-free, serum-free platform and protocol for the isolation and expansion of human salivary stem/progenitor cells (hS/PCs) suitable for regulatory qualification and future FDA-approved first-in-human autologous regenerative therapy trials for the treatment of hyposalivation disorders. Parotid gland specimens from non-cancerous regions/tissues were collected from consented surgical patients. Primary hS/PCs were isolated from tissue specimens, cultured in animal-component-free conditions, expanded to produce millions of cells, then enriched for CD44+ stem/progenitor cells by magnetic cell sorting. Normal epithelial purity was assessed using cytokeratins 5/14. Anti-CD133/PROM1 (cancer marker) and anti- fibroblast (clone TE-7) antibodies were used to demonstrate a lack of contaminating cells. Phenotype validation was performed by flow cytometry and immunocytochemistry on both CD44+ sorted and unsorted populations. Senescence-associated beta-galactosidase (SA-{beta}-gal) assays were performed across serial passages (P1-P6). Pluripotency was demonstrated by culture under conditions supporting lineage-specific differentiation. Primary hS/PCs demonstrated consistent expansion and epithelial morphology under serum-free conditions. CD44 expression remained high (>95%) throughout expansion, with negligible detection of CD133 or fibroblast markers, confirming epithelial purity and absence of tumorigenic or stromal contamination. Immunocytochemistry corroborated these expression profiles. SA-{beta}-gal staining revealed only a minor, passage-dependent increase (5-16%) in senescent cells from multiple donors, indicating retention of proliferative potential. Our defined, animal-free culture system supports stable expansion of pure low passage hS/PCs under conditions compatible with good manufacturing practice (GMP).

Conventional fluorescent imaging probes, including organic dyes and semiconductor quantum dots, suffer from inherent limitations such as photobleaching, cytotoxicity, poor aqueous dispersibility, and complex synthetic routes, necessitating the development of next-generation nanoscale fluorophores suitable for biological imaging. Carbon dots (CDs) have emerged as a compelling alternative owing to their nanoscale dimensions, tunable photoluminescence, excellent biocompatibility, and amenability to green synthesis from biomass-derived precursors. Herein, we report a comparative synthesis and systematic physicochemical evaluation of nitrogen-doped and undoped carbon dots derived from chamomile (Matricaria chamomilla L.) extract, prepared via solvothermal and microwave-assisted routes. Among the four synthesized variants--CM ST-U, CM ST-N, CM MW-U, and CM MW-N--the solvothermally synthesized nitrogen-doped carbon dots (CM ST-N) exhibited markedly superior optical performance, characterized by a high fluorescence quantum yield of 57.2%, which is among the highest reported for biomass-derived nitrogen-doped carbon dots. Comprehensive characterization using UV-visible spectroscopy, photoluminescence (PL) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), zeta potential analysis, and atomic force microscopy (AFM) confirmed the nanoscale dimensions (~8.3 nm), surface-rich functional groups, successful nitrogen incorporation (10.86 %), and moderate colloidal stability (zeta potential: -17.3 mV). Photoluminescence stability studies across seven solvent systems including biologically relevant media--phosphate-buffered saline (PBS), Dulbeccos modified Eagles medium (DMEM), and serum-free medium (SFM) demonstrated sustained fluorescence emission over 72 hours. In vitro cytotoxicity assessment using the MTT assay on RPE-1 retinal pigment epithelial cells confirmed high cell viability (>70%) across a broad concentration range (10-500 {micro}g mL-1) over multiple exposure durations. Collectively, these results establish CM ST-N as a highly fluorescent, biocompatible, and colloidally stable nanoprobe with strong potential for fluorescence-based bioimaging applications.

Red-emitting carbon quantum dots (HP-CQDs) were synthesised for the first time from aqueous leaf extracts of Hamelia patens through single-step, reagent-free microwave-assisted carbonisation (750 W). The resulting nanoparticles displayed a narrow hydrodynamic size distribution centred at 3.9 nm, consistent with atomic force microscopy measurements showing a maximum height of 2.81 nm. Under 400 nm excitation, the CQDs exhibited a characteristic red emission maximum at 675 nm, representing a rare example of long-wavelength-emitting green CQDs derived from plant biomass. UV-Vis absorption bands at 224 and 256 nm were assigned to {pi}-{pi}* transitions of aromatic carbon domains and n-{pi}* transitions associated with carbonyl-containing surface groups, respectively. X-ray photoelectron spectroscopy (XPS) indicated a carbon-rich composition (C: 67.24%, O: 31.25%, N: 1.52%) with prominent C-O (42.67%) and C-C/C=C (42.64%) contributions. ATR-FTIR further confirmed the retention of hydroxyl, ether, and aliphatic functionalities following carbonisation. The excitation-wavelength-independent emission peak position implicates discrete surface molecular states rather than a heterogeneous distribution of emitters. HP-CQDs exhibit potent DPPH radical scavenging activity (IC50 = 141.8 {micro}g mL-1), comparable to ascorbic acid (IC50 = 114.8 {micro}g mL-1), and maintain >95% cell viability in both HeLa and RPE-1 cells up to 250 {micro}g mL-1. Confocal microscopy demonstrates concentration-dependent cytoplasmic accumulation and selective perinuclear localization at 300 {micro}g mL-1. In vivo biodistribution in zebrafish larvae confirms systemic uptake with statistically significant fluorescence enhancement at 500 {micro}g mL-1 (p < 0.01), establishing HP-CQDs as biocompatible red-fluorescent probes with dual imaging-antioxidant functionality. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/724069v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1dbe864org.highwire.dtl.DTLVardef@763ed0org.highwire.dtl.DTLVardef@115e9b9org.highwire.dtl.DTLVardef@1a3941e_HPS_FORMAT_FIGEXP M_FIG C_FIG