ACS Applied Bio Materials

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.

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.

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.

Dendrimers are nanosized molecules with potential antimicrobial applications. This study evaluates the antibacterial and anti-biofilm properties of two cationic dendrimers, NVX-G6 (G6) and NVX-G9 (G9), against clinically relevant bacterial pathogens. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC99) were determined for Escherichia coli, Pseudomonas aeruginosa, methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus aureus (MRSA). The synergy of dendrimers with ceftazidime and vancomycin was evaluated using checkerboard assays. Furthermore, biofilm formation inhibition assays and fluorescent microscopy were performed to assess dendrimer interactions with bacterial biofilms. The results indicate that G6 and G9 exhibit limited direct antibacterial activity at high concentrations (MIC > 1024 {micro}g/mL) but demonstrate synergistic effects when combined with ceftazidime against E. coli and P. aeruginosa (FIC < 0.5). Notably, both dendrimers penetrated and colocalized within established biofilms, with time-dependent reductions in biomass observed after extended incubation, suggesting a role in progressive biofilm disruption rather than acute inhibition of formation, although significant biomass reduction was not observed under standard assay conditions. These findings contribute to the understanding of dendrimer-antibiotic interactions and their implications in antimicrobial and nanomedicine therapy.

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.

This study investigates a novel light-activated drug delivery system designed to produce on-demand drug release. The light-activated system was developed by incorporating a photostable photothermal agent, croconium dye, into liposomes to enable thermally triggered drug release. The drug release from the liposomes was determined at three powers of 210, 295, and 380 mW under 0-, 1-, and 2-minute light irradiation. A continuous wave 808 nm laser was used as the light source. Dexamethasone sodium phosphate (DSP) released from the liposomes was tunable depending on the power and irradiation time with a range of 1 -19 g released depending on irradiation power and time. For local temperature measurement during the photothermal activation, polymerized 10, 12 - Pentacosadiynoic acid (PCDA) was incorporated in the lipid bilayer. Under heating polymerized PCDA undergoes a transition into a red phase from a blue phase. Utilizing the spectrum changes under known temperatures a regression model was developed to calculate the local temperature of the liposomes under irradiation. The ability of the liposomes to release DSP under irradiation in the presence of a phantom tissue was tested under different attenuation coefficients to match various common biological tissues. The liposomes were still able to release DSP in the presence of tissue phantoms for a certain thickness of the tissue. Finally, the cytotoxicity of the liposomes with the croconium dye for chemical and thermal toxicity was determined. The liposomes displayed good biocompatibility with Human Microvascular Endothelial Cell line-1 (HMEC-1). The results support the use of croconium dye as a potential alternative to commonly photothermal agents used in drug delivery such as metal nanoparticles. Future work will focus on optimization of absorbance spectrum for drug release, and in vivo studies for efficacy and safety.

Membrane models of scaffolded discoidal lipid bilayers called nanodiscs have proven to be a valuable tool for the study of membrane proteins in a native environment. DNA-scaffolded membrane model has emerged as an alternative tool for membrane protein studies. Taking advantage of the designability of DNA nanostructure, we created a double-decker double-stranded DNA ring (DDring) to self-assemble DNA-based nanodiscs (DNA-ND). The DDring is 17 nm wide and 4 nm high, and equipped with 28 alkyl chains on the inside that can interact with each hydrophobic leaflet of the lipid bilayer. We further demonstrate the functionality of DNA-ND membrane model with the assembly of membrane proteins. DDrings are suited to neutral or cationic charged phospholipids and detergents. This study provides more insights into the potential use of DNA- assisted nanodiscs for membrane protein characterization.

BackgroundEarly enamel demineralization corresponding to ICDAS 0-1 is difficult to detect through routine visual-tactile examination, as initial mineral loss often precedes visible surface change. Existing optical adjuncts improve detection but frequently require specialized equipment, high costs, or ionizing radiation, limiting widespread clinical use. ObjectiveTo develop a low-cost, luciferin-inspired fluorogenic peptide biosensor capable of selectively binding early enamel porosity and producing a quantifiable green luminescent signal under standard dental blue-light activation. MethodsA calcium-affinitive peptide (P-Ca) was synthesized and functionalized with an inexpensive fluorogenic ester-quencher pair that becomes fluorescent upon conformational stabilization on partially demineralized enamel. Thirty extracted molars, collected as anonymized biowaste from orthodontic procedures, were sectioned and assigned to sound enamel, mild demineralization (pH-cycling, 48 h), or moderate demineralization (96 h). After 60 s incubation with P-Ca, specimens were illuminated using a dental curing light (450-470 nm). Emission spectra ({lambda}_max 515 {+/-} 5 nm) and fluorescence intensities were quantified and compared with quantitative light-induced fluorescence (QLF). Cytocompatibility was evaluated using an immortalized human gingival fibroblasts cell line (HGF-1). ResultsFluorescence intensity increased in accordance with demineralization severity (p < 0.001), and luminescent output strongly correlated with QLF {Delta}F values (p < 0.001). HGF-1 viability remained above 95% after 24 h exposure. ConclusionThis in vitro study supports the feasibility of a fluorogenic peptide biosensor as an inexpensive, non-radiographic adjunct for early enamel demineralization detection, with clear potential for future chairside translation.

Cancer of the uterine corpus is the fourth leading cancer and the fifth leading cause of cancer-related death in women in the United States. Chemotherapeutic resistance, specifically platinum-resistance, contributes to this problem. Therefore, an alternative treatment regimen is required. Using inorganic copper oxide nanoparticles (CuO NPs), we evaluated cancer cell responses indicative of anti-neoplastic activity. CuO NPs were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), dynamic light scattering (DLS) and laser Doppler velocimetry (LDV). The nanoparticles were rod-like and had a diameter of 70 {+/-} 30 nm and a copper content ranging from 77% - 82.6%. The hydrodynamic diameter and the zeta potential significantly decreased with more particles in solution. These materials were also used in four endometrial cancer cell lines and one cervical cancer cell line to evaluate cell viability, apoptosis, migration, and reactive oxygen species. In endometrial cancer cell lines, the IC50 values ranged from 1.028 ug/mL in HEC-1A cells to 73.62 ug/mL in Ishikawa cells, indicating that different cells have vastly different responses to CuO NPs. The results also indicated cell line-dependent differences in apoptosis, oxidation potential, and migration. Further, the cervical cancer cell line was modified using CRISPR technology to highlight a common germline mutation that causes earlier onset and more aggressive cancer progression. These genetic mutations resulted in differences in a loss of redox potential without observable changes in apoptosis or migration. The results of these studies indicate that CuO NPs elicit effects dependent upon the stage of cancer. Anticipated long-term applications of these studies includes the potential as a target-specific anti-cancer agent, designed using knowledge at the interface of colloids and the tumor environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/729888v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e0fe68org.highwire.dtl.DTLVardef@5e776dorg.highwire.dtl.DTLVardef@1f2aad6org.highwire.dtl.DTLVardef@add7fc_HPS_FORMAT_FIGEXP M_FIG C_FIG Copper oxide nanoparticles (CuO NPs) were characterized upon receipt using electron microscopy, elemental analysis, dynamic light scattering, laser Doppler velocimetry, and Fourier-transform infrared spectroscopy. These CuO NPs were exposed to HeLa cells with and without DNA mismatch repair deficiencies to assess the impacts on cancer cell migration, apoptosis, and redox potential.

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.

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.

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.

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.

Chronic wound healing is often impaired in conditions such as metabolic syndrome, requiring effective therapeutic interventions to promote tissue regeneration and repair. In this study, we evaluated the wound healing potential of petroleum jelly (P Jelly)-based bioactive glass ointments (PBGCu) with varying copper concentrations (0, 1, and 3 wt%) in both in vitro and in vivo models of wound healing. PBGCu formulations demonstrated high biocompatibility with human dermal fibroblasts (HDF) and human umbilical vein endothelial cells (HUVEC). Additionally, PBGCu ointments exhibited strong antibacterial activity against Staphylococcus aureus, suggesting their utility for the care of chronic wounds. In both metabolic syndrome mouse and pig models, PBGCu3-treated wounds showed significantly faster wound closure, enhanced epithelial regeneration, and increased dermal thickness compared to saline and P Jelly controls. Histological analysis also revealed 50% increased vascularization (p < 0.0001) and a 90% reduction in scar formation (p < 0.0001) in PBGCu3-treated wounds. These findings show that PBGCu formulations, especially at 3 wt% copper concentration, significantly improve wound healing by promoting epithelial regeneration, dermal tissue formation, and vascularization, while also offering antibacterial protection. The sustained Cu2+ ions release from PBGCu ointments provides long-term support for tissue regeneration, positioning this ointment composition as a promising therapeutic tool for chronic wound management. Future studies will focus on elucidating the underlying mechanisms and evaluating the therapeutic efficacy of PBGCu formulations in infected wounds. HighlightsO_LIDeveloped a Petroleum Jelly-based copper-doped bioactive glass ointment (PBGCu) enabling sustained and controlled Cu{superscript 2} ion release. C_LIO_LIPBGCu significantly accelerated wound closure and improved epithelial and dermal tissue regeneration. C_LIO_LIPBGCu enhanced hair follicle regeneration and tissue remodeling in full-thickness wounds. C_LIO_LIValidated therapeutic efficacy in both mouse and pig models that support translational relevance. C_LIO_LIOffers a simple, low-cost, and clinically adaptable topical formulation for metabolic syndrome-related wound complications. C_LI

Innate immune agonists are promising therapeutic agents to induce immune responses against cancer. However, these agents have been limited by toxicity associated with systemic accumulation and activity in off-target cells. In this work, a targeted nanoparticle (NP) platform to encapsulate and protect the Toll-like receptor 3 (TLR3) agonist polyinosinic-polycytidylic acid (poly(I:C)) and promote its specific delivery to antigen presenting cells (APCs), macrophages and dendritic cells, for activation of this cell population was designed. To determine NP physiochemical properties that promote APC delivery, we developed a library of NP surface chemistries formed by electrostatic adsorption of polyanion coatings onto liposomes using layer-by-layer (LbL) assembly and screened the particles on APCs and off-target cells. Dextran sulfate was identified as a promising coating to enhance specific APC delivery. We applied these design parameters to develop a poly(I:C)-loaded NP for an APC-targeted immunotherapy. In a model of metastatic ovarian cancer, the LbL NP prolonged poly(I:C) retention in the peritoneal space--with 2-fold remaining 24-48hr after administration compared to free poly(I:C)--ultimately reducing systemic accumulation and associated toxicities. Compared to free drug, the NP reduced the increase in serum levels of TNF, IL-6, and CXCL10 by 9-, 4-, and 31-fold respectively. NP-treated mice experienced lower weight loss and recovered more quickly at a higher poly(I:C) dose, indicating a widening of the therapeutic window. The NP formulation enhanced accumulation of poly(I:C) in the tumor 2-fold and activation of the target APC population compared to free drug, and ultimately slowed tumor growth and extended survival in combination with doxorubicin chemotherapy. Overall, this work demonstrates a modular NP delivery strategy to improve the delivery, safety, and therapeutic window of a TLR3 agonist.

Controlled drug delivery systems have important implications in many therapeutic applications to improve patient health, but it remains challenging to deliver therapeutics to specific target sites while being released at controlled rates to minimize the off-target accumulation. Here, we report a drug (e.g., vancomycin)-modulating other drug release strategies in human serum albumin (HSA)-based nanoparticles (NPs) to achieve multiple drug encapsulation and controlled release of drugs. Without external stimuli but only with water, the release kinetics of drugs such as sulfasalazine and epidermal growth factor can be modulated by adding vancomycin. The mechanistic study suggests that the release kinetics are related to the secondary structure of albumin through the interactions between drugs and HSA. Our strategy achieved controlled drug delivery through modulating the secondary structure of albumin with a water-soluble drug as a modulator, which provides a promising way to simultaneously deliver multiple drugs while releasing in a predicted and sustained manner.

This work presents a facile and rapid fabrication method for high-performance bioelectrode coatings through electropolymerization of poly(3,4-ethylenedioxythiophene) (PEDOT) and polydopamine (PDA). Expanding on previous work, we develop a cyclic voltammetry electropolymerization process to deposit PEDOT:PDA coatings on 2 mm gold electrode substrates. The coatings exhibit a 40-fold increase in cathodic charge storage capacity ([~] 40mC{middle dot}cm-2) and significant impedance modulus reduction compared to uncoated gold electrodes. Morphological characterization revealed a uniformly porous surface that corroborates the enhancement in electrochemical performance. Our scalable approach offers a promising option to fabricate bioelectrodes for application to neural interfaces and implantable and wearable bioelectronics. Clinical RelevanceThe improved electrochemical performance and scalable fabrication of PEDOT:PDA coatings support their potential to enhance the stability and signal quality of neural and implantable bioelectronic devices.

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.

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

Black extremotolerant fungi form persistent biofilms on a wide range of natural and engineered substrates. Able to weather minerals, affect stone monument surfaces, and colonize solar panels, they demonstrate a strong capacity to interact with and modify material surfaces, even under most extreme conditions. In this study, we establish a methodological workflow for the structural and mechanical characterization of melanized biofilms formed by the black fungus Knufia petricola. This species represents a broader group of resilient surface colonizers and provides a model for in-depth investigation. When grown on solid agar/air interface, this species predominantly forms a compact biofilm, composed of spherical cells, while retaining the capacity for filamentous growth, providing a suitable framework to explore morphology-dependent biomechanical responses. The proposed toolbox combines complementary analytical techniques spanning multiple spatial scales, including shear-rheology to quantify bulk viscoelastic behavior, micro-indentation to resolve local stiffness of the biofilm surface, micro-computed tomography for non-destructive three-dimensional visualization of biofilm architecture, and cryogenic preparation methods and electron microscopy for high-resolution ultrastructural analysis. As a case study, we applied this workflow to compare biofilms grown on two nitrogen sources (NO3- vs. NH4+). Our results reveal that the nitrogen source plays a key role in biofilm morphology across multiple hierarchical levels - ranging from cell division patterns and distribution of extracellular polymeric substances (EPS) to overall mechanical properties, where NO3- leads to budding-dominated growth and increased stiffness, whereas NH4+ promotes meristematic growth and softer biofilms. The successful transfer and integration of methods originally developed for bacterial biofilm research highlights the feasibility of quantitative mechanical analyses in fungal systems. This multiscale toolbox provides a foundation for advancing the mechanistic understanding of fungal biofilms and biofilm-material interactions, with implications for geomicrobiology, material biodeterioration, and the design of bio-inspired functional materials. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/720134v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@7fd08borg.highwire.dtl.DTLVardef@15476c2org.highwire.dtl.DTLVardef@40be50org.highwire.dtl.DTLVardef@8e9410_HPS_FORMAT_FIGEXP M_FIG C_FIG