ACS Applied Materials & Interfaces

A major barrier to scaling marine restoration and aquaculture is the lack of reliable tools to induce invertebrate larvae to settle and metamorphose when and where needed. Although microbial cues are known to induce metamorphosis in many invertebrates, existing methods rely on natural biofilms that are variable, difficult to standardize, and unsuitable for large-scale deployment. Here we introduce ReefTiles, a non-living bacterial coating that preserves inductive activity from metamorphosis-stimulating marine bacteria in a stable, reproducible format. Using both tubeworm and coral larvae, we show that dried and inactivated bacterial films retain full settlement-inducing capacity, matching or exceeding live biofilms while eliminating concerns associated with releasing viable microbes into the environment. Viability assays confirm inactivation, and the coating adheres reliably to common substrate materials. Because ReefTiles can be manufactured and stored at scale and tailored to different inductive strains, they provide a practical microbe-based biotechnology for enhancing larval settlement in reef restoration, sustainable aquaculture, and engineered marine infrastructure.

Two-photon polymerization (2PP) enables fabrication of hydrogel constructs with submicron, cell-scale resolution, but hydrogel-based bioinks are markedly more sensitive to process variability than conventional photoresists, and this sensitivity is further amplified when living cells are embedded in the resin. Post-processing evaluation, performed only after development, occurs too late to enable any corrective action. A full-factorial design of experiments across laser power and scan speed shows that fabrication outcome depends on both parameter choice and cell presence, with cells shifting and broadening the range of conditions yielding structurally sound constructs. However, substantial variability persists within each nominal condition and cannot be resolved by parameter refinement alone, indicating that outcome is governed by what occurs during each individual print rather than by the parameters set. To capture this, a layer-wise polymerization score is derived from pairwise comparisons of same-layer coaxial images, grounded in the psychophysics of relative judgment, and assembled into a Layer-wise Image Trajectory (LIT) for each print. Applied to both acellular and cell-laden formulations, LIT curves separate cleanly by post-processing outcome without any outcome label used in training, showing that fabrication quality can be predicted early in the build. Building on this signal, individual LIT curves are compared against statistical control limits derived from confirmed successful prints, enabling early detection of anomalous fabrication behavior at early-to-mid layers, well before development. To the best of the authors knowledge, this is the first application of in situ quality prediction and anomaly detection to cell-laden two-photon polymerization.

Graphene oxide (GO) has primarily been investigated as a carrier for intracellular delivery of therapeutic molecules. In previous work, we identified a cell type-dependent interaction pattern in which GO remained predominantly associated with the plasma membrane of cancer cells but was internalised by non-cancerous epithelial cells. Here, we explored whether plasma membrane-associated GO can be used as a platform to present bioactive ligands and influence cell-surface receptor signalling in cancer cells. To test this hypothesis, we targeted integrin receptors at the plasma membrane in glioblastoma cell models using an RGD-containing peptide non-covalently complexed with GO. We assessed GO-peptide interactions, cellular interactions/uptake, motility, and focal adhesion signalling readouts. Peptide association was quantified using a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay, and GO was characterised by atomic force microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and colloidal measurements. Immediately after complexation, ~70% of RGD was associated with GO. Peptide association increased the nitrogen signal and shifted the principal GO XRD peak while retaining nanosheet morphology. Biological responses were examined in U87 and U251 glioblastoma cells with different integrin-positive fractions, and in non-cancerous BEAS-2B bronchial epithelial cells. Confocal microscopy showed that GO and GO:RGD remained predominantly localised on the plasma membrane in U87 and U251 cells, whereas greater intracellular localisation was observed in BEAS-2B cells. Importantly, GO:RGD significantly reduced key indicators of cell motility: cell velocity in U87 and U251 cells, with trajectory and mean-square-displacement analyses supporting restricted cellular movement. Free RGD had no significant effect, while GO alone produced a smaller reduction in motility only in U251 cells. No treatment significantly altered BEAS-2B motility. Flow cytometry also showed a reduced pFAK-associated signal in GO:RGD-treated U87 cells. These findings establish a proof of concept that the cell-line-dependent plasma membrane localisation of GO can be exploited as a membrane-associated nano-bio interface for cell-surface-active ligands, opening the way for the development of GO-based platforms that modulate receptor-mediated signalling and cell behaviour.

Conducting polymers are widely used in bioelectronic interfaces because of their mixed ionic-electronic conductivity, mechanical compliance, and compatibility with biological systems. However, their electrochemically driven structural dynamics have received little attention as a mechanism for mechanical cell stimulation. Here, we show that electrochemical actuation of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) generates mechanical cues capable of activating endogenous mechanosensitive pathways in HEK293T cells. Transparent PEDOT:PSS films deposited on ITO exhibited a heterogeneous granular morphology and underwent potential-dependent microscopic deformation during electrochemical modulation. Direct optical tracking revealed displacement of the polymer boundary, with structural changes occurring preferentially in polymer-dense regions and propagating toward the film edge. When HEK293T cells were cultured directly on PEDOT:PSS, repeated electrochemical stimulation at -240 mV produced reproducible intracellular Ca2+ responses. Pharmacological inhibition with GsMTx4 attenuated the calcium response, whereas blockade of voltage-gated sodium channels with tetrodotoxin largely preserved it, supporting the involvement of mechanosensitive pathways in the cellular response. These findings identify PEDOT:PSS as an electromechanical biointerface in which electrochemical modulation can introduce a mechanical component alongside the established electrical function of the interface. This mechanical contribution should therefore be considered when interpreting cellular responses to conducting polymer- based electrical stimulation and provides a basis for engineering bioelectronic interfaces that deliberately couple electrical control with mechanotransduction.

Three-dimensional (3D) spheroid culture provides a physiologically relevant alternative to conventional two-dimensional culture, but reliable spheroid formation in microwells depends on limiting cell-substrate adhesion. Pluronic F127 is an amphiphilic triblock copolymer that forms a hydrated surface layer, reducing protein adsorption. Here, we evaluated whether this intrinsic anti-fouling property could restore an anti-adhesive surface in used microwell plates to promote spheroid formation. Using chondrogenic ATDC5 and pancreatic {beta}-cell INS-1 cells, we characterized spheroid assembly kinetics, F127 cytotoxicity, surface hydrophilicity, protein adsorption, and spheroid morphology including size and shape factor. Both cell types formed compact spheroids within 24 hours on commercial anti-adhesive microwells. F127 coating markedly reduced water contact angle and protein adsorption, confirming increased surface hydrophilicity and reduced protein fouling. In microwells stripped of their original surface coating, F127 coating amounts of approximately 0.011-0.045 mg/cm2 consistently promoted spheroid formation across both cell types. Soluble F127 concentrations were confirmed to be non-cytotoxic up to 0.625% (w/v), while even complete dissolution of the highest tested coating amount would correspond to only 0.025% (w/v) F127. This simple, reproducible, and low-cost surface-modification strategy may provide an accessible approach for re-functionalizing microwell platforms for 3D cell culture.

Precise control of growth factor delivery remains a challenge for directing stem cell differentiation in three-dimensional (3D) engineered tissues. In this study, engineered cell-like vesicles are used as programmable microenvironments to enable sustained and localized delivery of growth factors within visible-light-crosslinked GelMA hydrogels. Giant unilamellar vesicles (GUV) loaded with FGF-2 and TGF-{beta}3 were incorporated into bioinks with BM-MSC to drive keratocyte differentiation without repeated soluble growth factor supplementation. ELISA measurements confirmed the removal of non-encapsulated growth factors and the release of the vesicle cargo following induced vesicle rupture. Fluorescence monitoring showed a progressive reduction in detectable FGF-2- and TGF-{beta}3-loaded GUV during culture, while droplet-scale analysis demonstrated the co-deposition of cells and vesicles after printing. After 14 days of differentiation, differentiated cells expressed ALDH1A1, ALDH3A1, lumican, keratocan, and collagen I without induction of -SMA. Interestingly, keratocyte-associated differentiation was retained after drop-on-demand bioprinting, confirmed by qPCR analysis. These findings establish growth factor-loaded vesicles as bioprintable instructive niches capable of supporting localized keratocyte differentiation within 3D corneal constructs.

Antimicrobial resistance motivates the development of approaches capable of probing nanoparticle-bacterium interactions with nanoscale sensitivity. Here, synchrotron infrared nano-spectroscopy (SINS) is applied to investigate interactions between carbohydrate-coated silica nanoparticles and the Gram-negative model bacterium Escherichia coli at the single-cell level. Silica nanoparticles (SiO2) were coated with mannose, maltose, or trehalose to evaluate how surface carbohydrate chemistry influences their interactions with the bacterial envelope. Correlative electron microscopy revealed pronounced association of carbohydrate-SiO2 with the bacterial envelope, with features consistent with localization within the periplasmic region, whereas bare-SiO2 showed no detectable association. SINS measurements acquired directly on bacterial cells and at bacterium-nanoparticle interfaces revealed distinct, carbohydrate-dependent spectral signatures. Quantitative analysis of the amide I band used the I/I{beta} ; ratio, which describes the relative contributions of -helical and {beta}-sheet protein secondary-structure components, together with interface-dependent band-position analysis to characterize local spectral perturbations. Carbohydrate-SiO2 produced systematic changes in the I/I{beta} ; ratio, including at locations where nanoparticles were not directly observed, indicating that their effects extend beyond the sites of nanoparticle association. Comparison of measurements acquired on bacterial surfaces and at bacterium-nanoparticle interfaces further revealed that carbohydrate chemistry modulates both the magnitude and spatial extent of these spectral perturbations. Trehalose-SiO2 produced the largest interface-dependent amide I band shifts and a spectral component consistent with random-coil structures. Overall, these results demonstrate that carbohydrate surface chemistry modulates nanoscale protein conformational perturbations at the nano-bio interface and highlight SINS as a powerful approach for resolving chemically localized molecular responses at single-cell interfaces.

The red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), is a globally invasive quarantine pest threatening palm cultivation across 49 countries and inflicting annual economic losses estimated at over USD 100 million. Weevil larvae burrow into palm trunks, causing progressive internal structural damage that rarely produces visible external symptoms until lethal injury has occurred, rendering early detection exceptionally challenging. In the absence of effective early-warning surveillance technologies, tens of thousands of infested palm trees have been removed across major palm-cultivation regions in the Middle East and Mediterranean basin. Rapid, sensitive detection of volatile organic compounds (VOCs) emitted by weevil colonies and infested palm trees therefore represents a critical unmet need for timely pest surveillance and intervention. Existing artificial gas sensors lack the chemical selectivity required to discriminate among structurally similar VOCs, and no validated field-deployable early-detection platform has been established to date. Here, we report a portable biohybrid sensor array that mimics insect olfaction by exploiting two classes of diagnostic chemical signatures: the male-released aggregation pheromone (4RS,5RS)-4-methylnonan-5-ol (ferrugineol) and ethyl ester volatile blends emitted by weevil-infested palm trees. The R. ferrugineus odorant receptor RferOR1 was stabilised in lipid nanodiscs and co-immobilised with two in vivo-synthesised odorant-binding proteins (RferOBP1768 and RferOBP23) on quartz crystal microbalance (QCM) transducers to construct the biohybrid sensing platform. The sensor array achieved selective detection of airborne ferrugineol at a limit of detection of approximately 60 parts per billion (ppb) under field conditions, distinguishing infested from healthy palms. OBP- and OR-functionalised sensors retained full functional activity for 12 and 7 months, respectively, under ambient storage, confirming operational robustness and shelf life suitable for long-term field deployment. This work translates the molecular architecture of the insect olfactory system into a practical, field-validated chemical sensor platform with direct applicability to early-stage R. ferrugineus infestation monitoring and sustainable integrated pest management. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/744605v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@69f81forg.highwire.dtl.DTLVardef@120e05dorg.highwire.dtl.DTLVardef@16a0cb6org.highwire.dtl.DTLVardef@16889a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

The outer blood-retinal barrier (OBRB) is the primary interface through which systemically circulating drugs reach the retina. A tool that measures delivery across this barrier would support the development of targeted therapies as alternatives to repeated intravitreal injection, and the screening of drugs that reach the retina as an off-target toxicity. Such a tool should deliver drugs fluidically through a vascular compartment, measure transport across the retinal pigment epithelium (RPE), and display disease phenotypes relevant to efficacy. Here we adapt the SiM platform, which places epithelium and endothelium in direct juxtaposition across a permeable, optically transparent silicon nitride nanomembrane. ARPE-19 and human umbilical vein endothelial cells (HUVECs) were used as development cell sources. ARPE-19 monocultures reached a transepithelial electrical resistance of 68 {+/-} 26 {Omega} cm2 by 28 days, and ARPE-19 + HUVEC co-cultures reached a small-molecule permeability of 6.34 {+/-} 1.3 x 10-4 cm min-1 within 14 days, a state reported elsewhere only after longer culture. The barriers developed an intervening basement membrane. Drugs perfused through the basal vascular channel crossed into an open apical well, where sampling and mass spectrometry showed transport correlating with lipophilicity, as reported in vivo. The device also displayed two clinically relevant phenotypes. Digoxin at a clinically toxic concentration reduced viability in the co-barrier by about half and doubled permeability. In a vascularized configuration, VEGF drove endothelial invasion of the RPE layer, as seen in neovascular AMD. The SiM-OBRB therefore satisfies basic design criteria for measurement of drug bioavailability, toxicity, and efficacy.

Understanding the physicochemical factors that govern siRNA nanocarrier assembly is essential for the rational design of effective delivery systems. By optimizing various lipid compositions, cholesterol content and PEG length we created a peptide-functionalized cationic liposomal platform made of DOPE/TAP lipids with cholesterol-anchored nona-arginine (R9-Chol) for siRNA complexation, intracellular transport and effective silencing of the target EGFR gene. Analysis of {zeta}-potential and dynamic light scattering allowed to rationally design formulation of stable, monodisperse nanoscale lipoplexes with a positive surface charge. With fluorescence polarization, circular dichroism and agarose gel electrophoresis we found an optimal siRNA:liposome complexation ratio of 1:77, which protected siRNA from ribonuclease-mediated degradation. Morphological imaging confirmed a shift from discrete vesicular structures to organized multilamellar lipoplexes, consistent with electrostatically driven self-assembly. In cellular studies, the optimized nanocarrier promoted efficient uptake of fluorescent siRNA in MDA-MB-231 cells and achieved functional delivery of anti-EGFR, leading to substantially reduced expression of the target gene at both transcript and protein levels. This work offers mechanistic understanding of peptide-assisted lipid:siRNA assembly and positions R9-functionalized DOPE/TAP liposomes as a promising platform for siRNA delivery.

Hydrogels are widely used in sensing, delivery, and tissue engineering because their transport properties can be tuned through material design. However, while hydrogel permeability is often characterized using small molecules, many practical applications depend on the uptake and retention of much larger species, including protein conjugates and nanoparticles. Here, we systematically investigate how polyethylene glycol (PEG)-acrylate hydrogel microparticle formulation influences accumulation of signal-generating probes spanning a broad size range. We fabricated particles across a 36-condition design space varying nominal PEG-acrylate molecular weight, polymer weight percent, and UV crosslinking dose, and related formulation-dependent probe accumulation to particle swelling behavior. Increasing nominal PEG-acrylate molecular weight and decreasing polymer weight percent produced more highly swollen particles and strongly enhanced accumulation of fluorescent streptavidin conjugates, with the largest effects observed for bulky labels such as allophycocyanin and phycoerythrin. Gold nanoparticle accumulation was even more formulation-restricted, with detectable colorimetric signal observed primarily in the most permissive formulations. These findings establish design rules linking PEG hydrogel formulation to size-dependent accumulation and show that formulations suitable for small probes may be inadequate for larger reporters. More broadly, this framework may inform the design of hydrogels for particle-based assays as well as other applications where transport of macromolecules or nanoscale materials is important.

The formation of magnesium pyrophosphate (Mg2P2O7) in nucleic acid amplification and cell-free transcription systems has attracted considerable attention, since Mg2P2O7 serves as a reliable indicator of reaction efficiency. However, real-time monitoring of Mg2P2O7 remains challenging, relying largely on time-consuming analytical techniques or end-point detection methods. Here, we report a Mg2P2O7-driven co-aggregation mechanism involving glutathione-capped gold nanoclusters (GSH-AuNCs) that induces fluorescence enhancement, enabling real-time crystal formation monitoring. The mechanism was first investigated in simplified mixtures containing pyrophosphate (P2O74-) and magnesium (Mg2+) ions. Real-time fluorescence profiles revealed that the GSH-AuNCs/Mg2P2O7 co-aggregation can be correlated with crystal formation/growth/solubilization and solution turbidity, while distinct kinetic patterns can be indicative of the crystal size at the end of the reaction. As a next level of complexity, we examined the effects of common components in an enzymatic amplification reaction, i.e., dithiothreitol (DTT), ammonium sulfate ((NH4)2SO4), deoxynucleotides (dNTPs) and Bst polymerase, on Mg2P2O7 formation through real-time GSH-AuNCs fluorescence variations. Guided by the above results, we studied and selected the experimental parameters for the design of an optimized qualitative (end-point) or quantitative (real-time) genetic test. Finally, the loop-mediated isothermal amplification (LAMP) was used as a platform to demonstrate the quantification of Influenza A RNA within the range of 102-108 copies/reaction. The resulting one-tube, contamination-free assay was shown to have a response time of <25 min even in a crude saliva sample. Beyond diagnostics, this crystallization-activated fluorescence strategy may also support real-time investigation of Mg2P2O7 formation in other biotechnological processes, including in vitro transcription and Mg2P2O7-bioorganic composites synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/744482v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@96dd88org.highwire.dtl.DTLVardef@aa122dorg.highwire.dtl.DTLVardef@18f4abforg.highwire.dtl.DTLVardef@745f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

The recent development of Volumetric Imaging via Photochemical Sectioning (VIPS) has enabled nanoscale imaging of whole-mount tissue samples of virtually any size by embedding intact tissue in a photocleavable, superabsorbent hydrogel. However, the efficacy of sample embedding, imaging, and photochemical sectioning is fundamentally governed by the mechanical stiffness, structural stability, and photodegradation kinetics of the photocleavable hydrogel (PC-gel) polymer network. To elucidate the effect of the photosensitive crosslinker design on these critical properties, we synthesized a set of photocleavable crosslinkers (PCs) with varying polyethylene glycol (PEG) backbone lengths and prepared the corresponding PC-gels under a fixed monomer formulation and polymerization condition. We quantified and compared the viscoelastic properties of the formed PC-gels at their swollen states, and found that the crosslinker length markedly reshaped the PC-gel mechanics. In addition, we evaluated the light-triggered degradation of the PC-gels using both wide-field and spatially-controlled illumination. We found that PC-1000, PC-1500, and PC-2000 gels remained comparably photodegradable, all enabling on-demand, spatially confined decrosslinking under such illuminations. These results provide practical guidelines for modulating the crosslinker architecture of PC-gel polymer networks to achieve optimal physicochemical properties for whole-mount tissue imaging using VIPS.

Existing pooled lipid nanoparticle (LNP) screens lack spatial information on formulation localization, cellular uptake, and associated multicellular tissue responses. Here we introduce NanoSTAMP, a spatially resolved, pooled in vivo screening platform for barcoded LNP libraries that uses fluorescence in situ hybridization (FISH)-based barcode readout and is compatible with spatial omics. NanoSTAMP links LNP formulation to cell-type specific uptake, cargo expression, and nearby multicellular neighborhoods within intact tissue, which enables spatially-informed design of RNA delivery, establishing tissue architecture as a dimension of LNP performance.

Nile Red is widely used for the detection of microplastics because its fluorescence emission is sensitive to local polarity and can distinguish hydrophobic plastics from hydrophilic ones. The fluorescence of the molecular rotor, 9-(dicyanovinyl)-julolidine (DCVJ) is less sensitive to polarity but more to viscosity. DCVJ is less widely used for microplastic analysis, although it has been used to detect polystyrene nanobeads. Here, we compared these dyes with standard samples from the Hawaii Pacific University Polymer Kit 1.0 and confirmed that Nile Red, in general, was better for the detection and identification of microplastics. Fluorescence emission was analyzed using photography, as well as spectroscopy. The color and peak emission wavelength of some stained environmental microplastics were affected by additives. Raman spectroscopy was used to confirm the chemical identity of such samples. Although DCVJ emits green fluorescence on binding to some microplastics, a peak at 620 nm has been reported with polystyrene nanobeads, attributed to dimer/excimer formation. We confirmed this property and directly observed diffraction-limited spots using fluorescence microscopy, attributed to single or just a few nanobeads. Nile Red also stains polystyrene nanobeads and gave stronger signals than with DCVJ, but Nile Red was prone to false positives due to dye aggregation in aqueous solutions.

We present an active mixing toolhead for extrusion bioprinting. The tool enables the programmable fabrication of tumor microenvironment gradient arrays, through controlled deposition of mixed hydrogel precursor formulations into 384-well plates, pre-seeded with tumor cells. It operates on an open-source bioprinter and can actively mix arbitrary ratios of two hydrogel precursors prior to extrusion. These concentration gradient arrays are compatible with quantitative image analysis of cell viability and morphological responses to hydrogels conditioned with drug or extracellular matrix (ECM) proteins. The tools capacity to mix and print hydrogel precursor gradients was demonstrated using alginate and highly concentrated mCherry-conjugated mini-spidroin solutions. Hydrogel precursor stocks contained fluorescent reporters to facilitate quantifications of mixing efficiency, and as proxies for drug and ECM protein concentrations. The tool was applied to generate hydrogel-based gradients of the apoptosis-inducer staurosporine, from which concentration-dependent MDA-MB-231 breast cancer cell death responses were quantified. Gradient arrays of the ECM protein laminin-511, implicated in breast cancer tumorigenesis, were generated and revealed that increasing laminin-511 concentrations potentiated staurosporine-induced cell death. The study demonstrates the utility of this active mixing toolhead for producing hydrogel gradient arrays, and demonstrates the relevance of studying drug-responses in tumor microenvironment models that account for disease-specific ECM components.

Macrophages are highly dynamic cells that maintain tissue homeostasis by regulating both initiation and resolution of inflammation. During efferocytosis, macrophages recognize the eat me signal, phosphatidylserine (PS), exposed at the surface of apoptotic cells, leading to the resolution of inflammation and acquisition of a pro-resolving phenotype. Inspired by this endogenous mechanism, PS-based biomaterials have demonstrated immunomodulatory potential. However, the molecular mechanisms underlying PS-mediated macrophage reprogramming remain poorly understood. Here, submicron PS-exposing polymeric particles (PSPs; [~]300 nm) were developed to improve the suitability of PSP formulations for future systemic administration while preserving their immunomodulatory activity. PSPs were efficiently internalized by macrophages through both actin- and dynamin-dependent pathways. PSP treatment significantly reduced IL-6 and IL-12p70 production in LPS-stimulated macrophages, whereas induction of the classical anti-inflammatory M2 marker CD206 was limited. Transcriptomic analysis revealed coordinated attenuation of inflammatory signaling pathways, including downregulation of Myd88, Nfkb1, Rel, and Irf8, together with activation of NRF2-associated antioxidant pathways characterized by increased expression of Nfe2l2, Hmox1, Prdx1, Gclm, and Gclc. Activation of antioxidant-associated genes together with reduced Irf8 expression suggests that PSP promotes inflammatory resolution through coordinated redox adaptation and selective attenuation of inflammatory signaling. Collectively, these findings provide mechanistic insight into PS-mediated macrophage reprogramming and support the future development of systemically administered therapies for chronic inflammatory diseases, including vascular inflammatory disorders. HighlightsO_LISubmicron PSPs retain immunomodulatory activity of apoptotic cell-mimicking biomaterials. C_LIO_LIPSPs are rapidly internalized through actin- and dynamin-dependent pathways. C_LIO_LIPSPs attenuate inflammatory signaling and selectively suppress IL-6 and IL-12p70 production. C_LIO_LIPSPs induce NRF2-associated antioxidant and glutathione responses. C_LIO_LITranscriptomics reveals an early redox-adaptive macrophage program. C_LI

Chronic wounds such as diabetic foot ulcers are typically more alkaline than healing wounds, making wound pH a valuable diagnostic and prognostic marker. However, point-of-care pH monitoring remains limited by the availability of point-of-care wound pH sensing systems that offer quantitative pH determination, low toxicity, and small portable detectors. Here, we report a colorimetric pH-sensing wound dressing that enables in situ pH detection using a conventional smartphone camera. The anionic pH-sensitive dye HPTS was loaded onto cationic microparticles and embedded within a calcium-crosslinked alginate hydrogel. Across the clinically relevant range of pH 6.0-9.0, increasing pH produced a progressively more intense yellow coloration, quantified through the blue channel of smartphone-acquired RGB images. The dressing displayed a strong, rapid, and reversible signal in vitro with low dye release. In a full-thickness excisional wound model in mice, wound pH changes were detected in vivo. The combination of a pH-sensitive colorimetric hydrogel with a conventional smartphone detector offers accessible wound pH monitoring at the point-of-care.

Online structural characterization during microfluidic lipid self-assembly is important for understanding and controlling the formation of nonlamellar liquid crystalline nanodispersions. Here, we report a 3D-printed, X-ray-compatible hydrodynamic flow-focusing microfluidic chip with variable channel dimensions, integrated with synchrotron small-angle X-ray scattering (SAXS), for position-resolved SAXS-on-chip monitoring of Ca2+-triggered hexosome formation. Hexosomes were produced under continuous flow by mixing ethanolic solutions of docosahexaenoic acid monoglyceride (MAG-DHA), the negatively charged phosphatidylglycerol DOPG, and -tocopherol with Ca2+-containing PIPES buffer. Online SAXS-on-chip measurements detected three Bragg reflections characteristic of the internal inverse hexagonal (H2) phase on a tens-of-milliseconds residence-time scale, revealing rapid structural evolution during microfluidic mixing. Complementary ex situ SAXS identified the DOPG/Ca2+ molar ratio as a key parameter modulating the direct vesicle-to-hexosome transformation and the compactness of the internal H2 nanostructures. Dynamic light scattering showed that the flow-rate ratio modulated nanoparticle size, yielding hexosomes with mean hydrodynamic diameters in the range of approximately 120-175 nm and polydispersity index values down to 0.14 at a total flow rate of 200 {micro}L min-1. Cryo-TEM revealed coexistence of hexosomes and vesicular nanostructures, highlighting morphological heterogeneity, while Coarse-Grained Molecular Dynamics simulations supported a central role of Ca2+-DOPG association in promoting a direct lamellar-H2 phase transition. Overall, this work shows that 3D-printed SAXS-compatible microfluidics can integrate continuous production with online structural characterization, providing a basis for future formulation and process optimization of drug-loaded cubosomes, hexosomes, and related nonlamellar liquid crystalline nanodispersions.

Embedded extrusion printing can process collagen-rich bioinks, but their low viscosity and slow fibrillogenesis compromise print fidelity and post-deposition stability. Here, we developed a collagen fibril-inducing support bath (FIB) that combines mechanical support for embedded printing with biochemical induction of collagen assembly. Microfibrillated or nanofibrillated cellulose was incorporated into a fibril-inducing buffer, and formulations were screened at 37 degrees Celsius for rheological behaviour and optical transparency. The selected FIB was evaluated by printing 1% and 5% (w/v) articular cartilage-derived extracellular matrix (ECM) inks at 10-20 mm/s and compared with a cellulose-only control bath. FIB exhibited yield-stress, shear-thinning and rapid recovery behaviour that supported reproducible filament deposition. Unlike the control bath, FIB enabled intact construct retrieval following stabilisation and promoted the formation of fibrillar collagen within the printed strands. Scanning electron microscopy revealed D-banded collagen fibrils preferentially oriented along the deposition direction, with dominant orientation peaks within +/- 10-15 degrees. The platform supported the fabrication of 15 x 15 x 1.5 mm sheets and 6 x 6 x 6 mm scaffolds whose macroscopic dimensions were retained after processing. Constructs produced from 5% ECM inks exhibited approximately fourfold higher ramp and relaxation moduli than those produced from 1% ECM inks. Extracts from both formulations caused no detectable reduction in cell metabolic activity after 24 h or 72 h. Mesenchymal stem/stromal cells (MSCs) seeded onto printed sheets became markedly elongated and aligned by day 3, with approximately 80% of cells having an aspect ratio exceeding 1.5, significantly greater than cells seeded onto casted ECM controls, with a mean deviation of ~9 degrees from the filament print direction. These findings establish FIB as a bioactive support bath that couples embedded printability with collagen fibrillogenesis, enabling recoverable collagen-rich constructs with aligned fibrillar architecture that directs early cellular organisation.