ACS Applied Materials & Interfaces

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.

Sustainable synthesis of photoluminescent nanomaterials with tuneable surface chemistry and defined biological activity remains a central challenge in green nanoscience. Here we show that the energy-input route used to carbonise a single bearberry (Arctostaphylos uva-ursi) extract precursor system exerts a decisive and mechanistically coherent influence over the surface chemistry, optical performance, and bioactivity of the resulting carbon quantum dots (CQDs). Hydrothermal processing (160 {degrees}C, 6 h) yields particles of 7.13 nm hydrodynamic diameter enriched in surface hydroxyl and carbonyl groups, a higher graphitic sp{superscript 2} carbon fraction (43.06%), and potent DPPH radical scavenging activity. In contrast, microwave-assisted synthesis yields 9.65 nm particles with a higher surface carboxylate content (O-C=O: 19.06%), enhanced fluorescence quantum yield, and increased intracellular uptake. Uptake is statistically significant in retinal epithelial cells at 200 {micro}g/mL (p < 0.001) and shows concentration-dependent accumulation in zebrafish larvae from 100 {micro}g/mL (p < 0.05). Combined XPS C 1s deconvolution and FTIR difference spectroscopy indicate that incomplete decarboxylation under microwave conditions underlies these distinct properties. Both formulations maintained full cytocompatibility across 10-250 {micro}g/mL in both RPE-1 and HeLa cells, with no statistically significant reduction in viability at any tested concentration. These findings define a synthesis-route-encoded structure property relationship that enables rational selection between antioxidant-optimised and imaging-optimised CQD formulations from an identical green precursor system.

Although persistent infection from chronic wound lowers the efficacy of single therapy; But combination therapy with prolonged drug release have shown promising effect, such as elimination of heavy bacterial film and multi/single drug resistance with minimal side effects. One such breakthrough is multilayered scaffold that are chemically and physiologically stable. To achieve this objective, we demonstrated construction of layer-by-layer (LBL) assembly aligned by alternate deposition of (PEI/PSS and PAA/PSS) based on electrostatic force. These films exhibited loading of Ibu and gen which can be altered depending on different parameter such dipping time, pH and number of layers. Briefly, gen was encapsulated onto PPGNRs, as GNRs have good stability and their antibacterial properties can be enhanced by tunning surface upon adding chemical drugs. Such functionalized GNRs showed excellent photo thermal property which assisted controlled release of gen via deconstruction of thin layers, whereas burst release of ibu. Furthermore, complete disruption of bacterial colonies when combined with near infrared irradiation (NIR). The formed LBL endowed the great healing capability, controlled antibiotic release solve the problems of bacterial resistance due to synergistic effect. Taken together, the antibacterial, cytocompatibility, and stimuli responsive characteristics of this robust multilayer assembly can be promising multifunctional drug delivery system in different medical aliments.

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

Bacterial biofilms on indwelling medical devices is a major driver of healthcare-associated infection despite significant advances in antifouling surface engineering, suggesting that laboratory antibacterial performance does not fully capture the determinants of clinical colonization. Here we show that adherent immune cells constitute a previously underappreciated conditioning layer that promotes biofilm formation on otherwise antifouling biomaterials. Pre-exposure of clinically used substrates to macrophages, monocytes, neutrophils or human peripheral blood cells markedly increased Staphylococcus aureus and Escherichia coli adhesion and aggregation. These studies also reveal that immune cells promote biofilms even after cell death with cellular debris acting as a conditioning agent. We demonstrate that reactive-oxygen-species amplification by incorporating bismuth telluride into a silicone composite converts adherent immune cells from passive conditioning agents into active bactericidal effectors. We note that this antimicrobial composite confers durable antibacterial protection across early, delayed and late infection time points in a murine implantation model. Together, these findings introduce a class of immune-coupled antibacterial materials as an alternative to the current antifouling paradigm.

Biofilm extracellular matrix (ECM) varies with environmental conditions and substrate properties. Understanding the surface-biofilm relationship helps to perfect antibacterial strategies and to design new engineered living materials (ELMs). In this work, we studied how cationic and anionic polyelectrolyte coatings affect macroscopic features of Escherichia coli curli-producing biofilms, as well as the properties of their curli amyloid fibers. Cationic coatings limited biofilm spreading, increased their surface density and water absorption, which correlated with a higher yield of curli amyloid fibers with looser structure. In contrast, anionic surfaces allowed for standard biofilm spreading, with a lower fiber yield but a more compact and chemically stable fiber structure. Higher biofilm rigidity and adhesion were measured on both types of charged surfaces. Thus, we propose that the differences in biofilm macroscopic properties result from a trade-off between curli quantity and quality in the ECM, namely fiber density and molecular packing, as well as their interaction with water. Our findings provide insights on how the biophysical properties of the ECM can be controlled by tuning the substrate physico-chemical characteristics with charged coatings. This work opens up new avenues for developing antimicrobial strategies, as well as tailoring the properties of amyloid-based ELMs. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/721109v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@191cd79org.highwire.dtl.DTLVardef@148f914org.highwire.dtl.DTLVardef@1d8c2f8org.highwire.dtl.DTLVardef@1e84eaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

This study presents the first investigation of the adsorption behaviour of Afatinib on gold nanoparticle (AuNP) monolayers, employing a combination of AFM-SEIRA and SERS techniques. Two types of AuNPs with distinct sizes, synthesized using different reagents, were employed to elucidate the influence of surface type on drug adsorption. The first type of AuNPs was synthesized using sodium borohydride (SB), whereas the second type was obtained using hydroxylamine hydrochloride (HH) as the reducing agent. AFM-SEIRA revealed that Afatinib interacts to the AuNPs primarily through the quinazoline ring, amide group, and amino moiety, with adsorption geometry strongly dependent on nanoparticle type. Contributions from CH3 and CH2 moieties were also identified, indicating their role in stabilizing the molecule/metal interface. Time-resolved SERS studies demonstrated that the adsorption process is dynamic and involves molecular reorientation, followed by gradual desorption, which is accelerated at physiological temperature (37 {degrees}C). Competitive adsorption experiments with phenylboronic acid (PBA) showed that Afatinib exhibits higher affinity toward AuNPs, however, co-adsorption leads to reduced stability of both species on the surface. The results reveal molecular insights into drug/nanoparticle interactions and emphasize the role of surface functionalization in efficient nanocarrier design. This work deepens understanding of adsorption at plasmonic interfaces for biomedical use.

Foreign body response is a common yet serious challenge for biomedical implants. It can trigger inflammation and eventually lead to the formation of a fibrotic capsule, which compromises device function. Although significant efforts have been made to develop antifibrotic surface coatings for implantable materials, developing broadly applicable solutions remains challenging due to the diversity of materials used in biomedical implants. Here, we propose a simple and versatile strategy to develop antifibrotic coatings for biomedical implants. Photoreactive benzophenone groups are incorporated into designer polymers to enable covalent attachment to various substrates. The effect of benzophenone group density within polymer chains on surface coating efficiency was investigated, and an optimal BP incorporation ratio was identified. Polymers incorporating varying ratios of an anti-fibrotic small molecule and anti-fouling zwitterionic moieties were synthesized and successfully attached to silicone implants. In vivo evaluation of these implants in C57BL/6 mice identified an optimized polymer composition that reduced fibrotic capsule thickness by around 60%. Coating of commercial medical catheters with this optimized polymer reduced collagen deposition by over 3.5-fold following 4 weeks of implantation in the peritoneal space of C57BL/6 mice. Finally, we demonstrated that the optimized polymer coating can be readily applied to a variety of commonly used biomedical materials using this straightforward method, highlighting the versatility of the approach. This work provides a facile and broadly applicable strategy for developing antifibrotic coatings, which has the potential to expand the design of surface modifications aimed at improving the performance of biomedical implants.

Silicones are elastomers that have a wide variety of uses, including biomedical applications such as the coating of biomedical devices and as implants. Soft silicones with mechanical properties similar to those of biological tissues have particularly gained use as substrates for cell culture in mechanobiology studies. In this context, it would be desirable to be able to alter their surface mechanical properties with a relatively simple physical treatment. While deep ultraviolet (deep UV) or ultraviolet C (UV-C) treatment has been previously used as a surface treatment method for stiffer silicones formulations, the effect of this treatment on soft silicones relevant for mechanobiology applications is still uncharacterized. We first used nanoindentation to determine the Youngs modulus of two types of soft silicones, Qgel and GEL-8100/Syl (GEL-8100 with Sylgard-184 crosslinker), both with initial moduli in the kilopascal range. We show that nanoindentation in the presence of 1% sodium dodecyl sulfate avoids adhesion between the nanoindentation glass probe and the soft silicones. After deep UV exposure in the presence of air, nanoindentation revealed that the apparent Youngs moduli of the soft silicones Qgel and GEL-8100/Syl increased by 70% and 33%, respectively. The bulk rheology of the soft silicones were not affected, suggesting that this corresponds to a surface stiffening effect with a topical stiffening of at least several hundred kilopascals. Energy-dispersive X-ray spectroscopy results show an increase in the mole fraction of oxygen, consistent with oxidation of the surface. Attenuated Total Reflectance Fourier-Transform Infrared spectra show evidence of Si-OH group formation in GEL-8100/Syl and silicon sub-oxide formation in Qgel. Consistent with this, water contact angle measurements show enhanced hydrophilicity after deep UV treatment. Our results have implications for using soft silicones as substrates in mechanobiology studies and in processes where deep UV light is used in the surface treatment of soft silicones.

Bioxolography enables high-resolution fabrication of geometrically complex, cell-laden constructs for tissue engineering. However, tissue-relevant cell densities conflict with the optical transparency required for efficient dual-color volumetric printing. In this work, we extend the Bioxolography toolbox to include refractive index (RI) matching for cell-laden bioresins using iodixanol (IDX). Remarkably, IDX enhances optical transparency and boosts reactivity -- a phenomenon unique to Xolography. Yet, excessive IDX compromises dual-color efficiency through increased absorption and undesired UV-only curing, underscoring a central trade-off between optical clarity and photochemical performance. Systematic tuning of resin compositions along an iso-refractive index line demonstrated the versatility of Bioxolography, with IDX enhancing polymerization and 4-Hydroxy-TEMPO providing biocompatible inhibition. Optimizing composition and printing parameters yielded GelMA hydrogels with cell densities up to 5{middle dot}106 cells{middle dot}mL-1. Cell-laden prints achieved sub-100 {micro}m resolution and complex geometries such as channels and gyroids. Using skeletal muscle tissue as a model, we validated RI matched Bioxolography as a promising strategy for tissue engineering by demonstrating cell alignment along printed grooves and formation of mature muscle fibers characterized by MyoHC+ staining and fusion index. By integrating physical, chemical, and biological perspectives, this work advances Xolography toward biomaterials development and reinforces its position as an emerging volumetric (bio)printing technology. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/729865v1_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@407d86org.highwire.dtl.DTLVardef@1f116b4org.highwire.dtl.DTLVardef@109a437org.highwire.dtl.DTLVardef@1fc4ece_HPS_FORMAT_FIGEXP M_FIG C_FIG For printing higher cell density bioresins with Xolography, iodixanol (IDX) is added for refractive index-matching. The addition leads to an unexpected additional effect with increased reactivity in the dual-color photopolymerization. With careful adjustment of the resin composition and the printing parameters, Bioxolography is proven as a viable tool for tissue engineering.

Traditional bioelectronic devices are limited by poor biointerfacing due to their substantial mismatch in mechanical and biochemical properties. In tissue engineering, soft and bioactive materials support biointegration by harnessing or mimicking the natural extracellular matrix (ECM). Building bioelectronic devices from ECM should improve their biointegration, yet there are limited methods to fabricate them due to current manufacturing approaches. An additive manufacturing strategy is presented here for collagen-based bioelectronic interfaces that integrates conducting polymer electrodes with ECM-based substrates or encapsulation layers. Addition of poly(ethylene glycol) diglycidyl ether (PEGDE) to poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) colloidal dispersions enables direct extrusion-based patterning under mild conditions compatible with collagen substrates, and forms aqueous stable and highly conducting printed patterns (2788 S m-{superscript 1}). The resulting interfaces maintain stable electrochemical performance over 7 days in physiological environments, and support primary human cell adhesion, viability, and proliferation across both material regions. A sacrificial patterning strategy using 3D printed cacao butter further enables spatial control of collagen encapsulation. This approach establishes a framework for fabricating functional bioelectronic devices based on ECM to further enhance device biointerfaces for tissue models and implantable systems.

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

Resin three-dimensional (3D) printing is an increasingly popular manufacturing and prototyping method used to create microphysiological systems (MPS), but resin cytotoxicity significantly hinders its adoption, especially when sensitive cell models are incorporated. The mammalian oocyte and early preimplantation embryo consist of cells that are highly sensitive to toxicants and thus represent stringent cell-based models for biocompatibility testing. We developed a Multi-Endpoint Oocyte Safety Assay (MEIOSA) to evaluate the biocompatibility of four ISO 10993 biocompatible BioMed resins (Clear, Durable, Elastic 50A, and Flex 80A). MEIOSA assesses the viability, morphology, meiotic stage, and meiotic spindle morphology of the oocyte after in vitro maturation (IVM). Oocytes were in vitro matured in plate inserts 3D printed with the four BioMed resins. Oocytes cultured in rigid resins (Clear and Durable) or elastomeric resins (Elastic 50A, and Flex 80A) exhibited impaired meiotic progression and complete oocyte degeneration, respectively, relative to controls cultured in polystyrene which matured normally. To determine whether such cytotoxicity could be prevented, we coated the resin inserts with a 5 {micro}m impermeable Parylene-C (PC) barrier. PC coating completely rescued the degeneration and meiotic maturation defect phenotypes for all resins. Remarkably, when the most cytotoxic material (Flex 80A) was coated with PC, the resulting eggs were fertilization-competent and produced embryos capable of normal preimplantation development via in vitro fertilization. Our findings demonstrate that standardized viability-based biocompatibility tests do not identify cytotoxic effects for all cell types and establish MEIOSA as a high sensitivity test model to robustly evaluate biomaterial biocompatibility. Furthermore, PC coating prevents the toxic effects of all resin-3D-printed materials tested, opening up a new toolbox to create MPS compatible with reproductive, and by extension, other sensitive cell cultures. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/730268v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@18fab44org.highwire.dtl.DTLVardef@1352e5forg.highwire.dtl.DTLVardef@77889forg.highwire.dtl.DTLVardef@1aabd89_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

The multivalent display of surface glycoprotein hemagglutinin (HA) on Influenza A viruses (IAVs) enhances the overall binding avidity to sialylated glycans on host cell surfaces. While precomplexing HA trimers with antibodies increases multivalency and avidity, this method does not replicate the virions geometry and limits insights into the multivalent binding process. Here, we use perfectly controllable icosahedral protein nanoparticles to examine the multivalent HA receptor-binding properties. We compare three HA presentation systems with varying degrees of multivalency: single HA trimers, antibody-precomplexed HA trimers, and HA trimers on nanoparticles. Our results indicate that increasing HA valency enhances binding avidity across various glycan surfaces, including erythrocytes, cells, and lipid bilayers with varying glycan densities, while maintaining receptor specificity. By combining functional and non-functional HA trimers during nanoparticle formation, we create statistical mixtures of nanoparticles with varying valencies. At high receptor densities, nanoparticles with few functional trimers still bind strongly, whereas at low receptor densities, a patch of five HA trimers appears necessary for binding. As a key finding, we observe that such a statistical mixture of nanoparticles with functional and nonfunctional HAs binds to glycan surfaces in a stronger density-dependent manner than fully functional particles. We also observe differences in binding modes that correlate with the number of functional trimers, the glycan structure (linear vs branched), and the densities achievable with these glycans. Overall, our findings demonstrate that the presentation of multivalent HA plays an enormous role in the response to glycan receptor type and density, with implications for the future design of virus monitoring, viral inhibitors, and targeting vectors.

DNA origami nanoparticles (DONs) are attractive nanocarriers of controllable size, shape and addressability that have potential for treating eye diseases by overcoming ocular barriers. However, suboptimal physiological stability and poor cell uptake due to the negative charge may limit their use. Previous reports show that electrostatic complexation of DONs with cationic PEG-oligolysine block-copolymers like PEG5K-K10 can improve structural integrity and promote cell internalization. Here, we investigated a dual approach of PEG5K-K10 coatings and PL3 targeting peptides to improve uptake of 24-helix bundle (24HB) DONs into Y-79 retinoblastoma cells. Uptake studies revealed that PEG5K-K10 was essential for DON uptake in Y-79 cells, as uptake only occurred upon exceeding a distinct PEG5K-K10 amount. Longer exposure times or increased polymer amounts improved cell association. However, no beneficial effect of PL3 was observed. While free PEG5K-K10 reduced cell viability at higher concentrations (IC50 36.8 {micro}M), coated DONs were well-tolerated. Furthermore, single particle tracking in ex vivo porcine eyes revealed comparable vitreal mobility for uncoated and coated 24HB, with a slight decrease at higher coating amounts. Our findings highlight that PEG5K-K10 can enhance ocular cell uptake without limiting nanoparticle diffusivity in the vitreous, and support further optimization of DONs for ocular drug delivery.

Optical fiber-assisted printing (OFAP) was recently introduced as a straightforward light-based platform for the spatially controlled photopolymerization of hydrogel-based resins. Here, we extend this concept toward optical fiber-assisted bioprinting (OFAB) by processing cell-laden GelMA- and GelMA/PEGDA-based bioresins in a freeform embedded printing configuration. The system relies on a 405 nm LED-coupled optical fiber mounted on an automated 3D motion platform, enabling localized photocrosslinking directly within a resin bath. First, GelMA and GelMA/PEGDA formulations containing LAP and tartrazine were screened to evaluate the influence of light intensity, printing velocity, and material composition at the line width and curing depth. Single-line features with widths down to 70 {+/-} 20 {micro}m were obtained under optimized conditions, while more robust printing conditions yielded reproducible features in the range of 200-300 {micro}m. Photorheological and rotational rheology measurements confirmed that the formulations provide both thermoresponsive support during printing and photocrosslinked stability after processing. The incorporation of L929 cells demonstrated high cytocompatibility for GelMA and GelMA/PEGDA 6000 Da formulations, with viabilities above 90% after 7 days for selected printed constructs. Importantly, increasing the cell concentration up to 1 x 107 cells mL-1 did not prevent printing and reduced the extent of overcuring, suggesting that cell-induced turbidity can improve spatial confinement of polymerization in OFAB. Finally, a customized OFAB printer was developed to enable temperature-controlled processing and the fabrication of centimeter-scale 3D structures, including cell-laden constructs. Overall, this work establishes OFAB as an accessible and modular bioprinting strategy for cell-laden and optically turbid hydrogel resins, complementing existing light-based biofabrication approaches.

Oral routes of delivery are logistically simple and enables easy administration of therapeutics. However, oral delivery of proteins is still challenging due to the proteolytic environment within the gastrointestinal (GI) tract. To protect protein cargo from degradation, polymer encapsulation is commonly used, and when it is combined with cell-free gene expression (CFE) approaches that enable the rapid and flexible production of proteins, it potentially allows for on-demand production of protein therapeutics. Here, we investigated the suitability of chitosan coated alginate (Alg/Cht) microcapsules for encapsulation of proteins and CFE lysates for oral delivery. We show that CFE lysates can produce functional mCherry, a model fluorescent protein, in the presence of alginate polymers, although direct contact with chitosan did inhibit protein synthesis. We encapsulated CFE lysates or purified mCherry protein into alginate cores before crosslinking them using internal gelation techniques and coating with chitosan to test their protective capacity for oral delivery. Alg/Cht microcapsules protected mCherry protein cargo from degradation in simulated human gastric fluids and mouse gastric extracts and facilitated controlled cargo release upon exposure to conditions that simulate the intestinal environment. None of the individual CFE or encapsulation components induced inflammation in mouse GI tracts when administered via oral gavage. We also observed a delayed release of fluorescent bead cargo from Alg/Cht microcapsules in mouse intestines following oral gavage. Together, our data suggest that CFE lysate-loaded Alg/Cht formulations can be flexibly used to produce proteins and safely deliver them to the GI tract for potential therapeutic applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=40 SRC="FIGDIR/small/730178v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@d3e4c3org.highwire.dtl.DTLVardef@14fbea1org.highwire.dtl.DTLVardef@6c68cforg.highwire.dtl.DTLVardef@15508ec_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LICell-free gene expression lysates are active in chitosan coated alginate (Alg/Cht) microcapsules. C_LIO_LIAlg/Cht microcapsules exhibit controlled release in vitro in simulated intestinal-like conditions. C_LIO_LICell-free and encapsulation components do not induce inflammation in the gastrointestinal tracts of male or female mice. C_LIO_LIAlg/Cht microcapsules show controlled delayed cargo release in vivo when orally gavaged in mice. C_LI

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.

Recent advances in neuroscience have highlighted the central role of glial cells, particularly astrocytes, in regulating neural network activity through calcium-dependent neuron-glia communication. In parallel, nanostructured cluster-assembled materials have emerged as promising candidates for developing brain-machine interfaces, because of their biomimetic morphology, mechanotransductive properties and neuromorphic behavior. Among these, nanostructured zirconium oxide (ns-ZrOx) thin films have recently demonstrated memristive and signal-processing capabilities compatible with biohybrid neural systems, yet their interaction with heterogeneous neuroglial networks remains poorly understood. Here, we investigate the biocompatibility and functional effects of ns-ZrOx interfaces on primary astrocytes and dorsal root ganglion (DRG) neuron-glia co-cultures, comparing nanostructured and flat zirconia substrates. Both substrates supported cellular adhesion, survival, and differentiation. However, ns-ZrOx selectively enhanced glial calcium signaling, increasing transient amplitude and accelerating response kinetics in both central and peripheral glial populations. Our findings identify ns-ZrOx as an active neurogliomorphic interface capable of modulating neuron-glia communication through nanoscale material properties. By bridging glial physiology with neuromorphic nanomaterials, this work supports the development of hybrid bioelectronic platforms integrating living neural networks with adaptive functional materials for brain-inspired computing and advanced neural interfaces.