ACS Applied Materials & Interfaces

Protein cages are versatile platforms capable of encapsulating a wide range of nanoparticle cargo within biocompatible protein shells while providing tunable functionalities. Here, we investigated a self-assembly system that forms vesicle-like protein cages while simultaneously encapsulating nanoparticles at high density, yielding pomegranate-like protein- nanoparticle hybrid materials. Amphiphilic recombinant fusion protein building blocks based on elastin-like polypeptides, leucin zippers, and fluorescent proteins were employed to assemble vesicle-like protein cages via temperature-triggered liquid-liquid phase separation in the presence of fluorescent polystyrene nanoparticles. Analysis of nanoparticle encapsulation density and protein cage size indicates cooperative interactions between protein building blocks and nanoparticles that mediate the formation of protein-nanoparticle coacervate intermediates, which subsequently convert into core-shell hybrid protein cages, as further supported by kinetics studies. We demonstrate the self-assembly hybrid protein cages incorporating a fluorescent calcium sensor protein and titanium oxide nanoparticles, which exhibit a drastic enhancement in their calcium-sensing capability as a result of nanoparticle encapsulation. This platform offers a broadly applicable strategy that integrates protein biofunctionality with diverse nanoparticle properties for development of advanced hybrid materials.

Cotton-based antimicrobial textiles are attractive for applications requiring improved microbiological control, but their performance depends on effective surface functionalization and retention of the active materials after use and washing. In this work, cotton fabrics were functionalized with hydroxyl-rich graphene oxide (HGO), hydroxyl-rich reduced graphene oxide (H-rGO), and silver nanowires (AgNWs), either individually or in combined treatments, to investigate their deposition onto the textile surface, washing resistance, and preliminary antibacterial activity. The treated fabrics were prepared by immersion-based coating procedures, and the persistence of the deposited materials after repeated washing was evaluated by UV-Vis analysis of the residual wash solutions. Surface morphology before and after washing was examined by scanning electron microscopy. The results showed that graphene-based coatings, particularly HGO, exhibited stronger retention on cotton fibers, while AgNWs were partially retained after repeated washing cycles. SEM images confirmed the deposition of AgNWs on the cotton surface and showed that part of the coating remained associated with the fibers after washing. A preliminary antibacterial assay against Escherichia coli indicated that nanomaterial-treated fabrics inhibited bacterial growth relative to untreated controls, with the combined HGO/AgNWs treatment showing the most promising inhibitory trend under the tested conditions. These findings demonstrate the feasibility of producing cotton fabrics functionalized with hydroxyl-rich graphene derivatives and silver nanowires, supporting their potential as proof-of-concept antibacterial textiles with partial washing resistance.

Under acidic conditions, polycationic polymer coatings function as protective immobilization supports through protonation-mediated local pH buffering. However, it remains unclear how polymer support design parameters, such as film thickness and charge density, govern that vital protonation process. Leveraging the precise control of film thickness and copolymer composition enabled by initiated chemical vapor deposition (iCVD), we systematically investigated how these parameters govern the protonation behavior of poly[glycidyl methacrylate-co-2-(dimethylamino)ethyl methacrylate] (pGD) thin films and, in turn, the activity of immobilized {beta}-galactosidase (LacZ). Infrared spectroscopy suggests that proton penetration was capped at a depth of [~]250 nm in pGD with 65% DMAEMA, limiting the polycationic thickness in pGD films thicker than this value. Consistent with this limit, immobilized LacZ activity under acidic stress (pH 4) increased with protonated thickness up to [~]250 nm and then plateaued. Raising the polycationic monomer content from 25 to 65 mol% increased LacZ activity at pH 4 by up to 83%, consistent with a higher positive charge density providing stronger local pH buffering. To test whether this behavior depends on immobilization sites, we evaluated two approaches: random immobilization (via amine-epoxy ring-opening reactions) and site-directed immobilization (via SpyCatcher/SpyTag binding). Directed immobilization preserved higher LacZ activity than random immobilization, but the protonation-dependent protection trend remained consistent for both strategies. These findings establish protonation depth and charge density as tunable design parameters for polycationic immobilization supports that stabilize enzymes under acidic conditions.

Silica-encapsulated gold nanostars (AuNStar-SiO2) are a widely used plasmonic nanoparticle platform for surface-enhanced resonance Raman scattering (SERRS) bio-applications. In this paper, we demonstrate that coupled nanostar subpopulations can dominate the ensemble-average SERRS response of the suspension and that near-neutral standard cell culture conditions are sufficient to hydrolyze the silica nanoshell and introduce variability in signal intensity following in vitro endocytosis. Monomeric and oligomeric AuNStar-SiO2 fractions were isolated using continuous density-gradient centrifugation and monomeric populations were found to exhibit significantly weaker SERRS compared to their oligomeric counterparts. Using monomer-enriched AuNStar-SiO2, we investigated the stability of the silica nanoshell under conditions representative of sequential acidification during endocytosis and characterized the subsequent changes to nanoparticle optical properties. In acidic environments, reflecting lysosomal pH, the silica shell was stable, whereas near-neutral and alkaline conditions in cell culture medium induced silica-shell hydrolysis, nanostar release, and interparticle aggregation, leading to transient SERS amplification. When cells were treated with AuNStar-SiO2 under near-neutral and acidic conditions, we observed the opposite trend in SERS signal strength. At pH 7.4, the SERRS signal was suppressed even though transmission electron microscopy (TEM) images of intracellular nanoparticles showed progressive extents of silica hydrolysis, while at pH 6.4 SERS signal was strong and the silica shell of intracellular nanoparticles remained intact. Together, these findings show how SERRS output can differ between control conditions and biological applications, highlighting the role that local environmental factors play in nanoparticle stability and performance. Our results highlight the previously overlooked role of silica nanoshell instability on SERRS signal output in physiological environments and describe opportunities to harness silica nanoshell hydrolysis to improve the biomedical application of silica-coated plasmonic probes.

The design of multifunctional nanomaterials that combine chemotherapy with photothermal therapy (PTT) has emerged as a promising strategy to overcome the limitations of conventional cancer treatments. Here, we report the fabrication of a novel therapeutic hydrogel system composed of Folic Acid-functionalized iron oxide nanoparticles (IO NPs) synthesized via an arc-discharge method, loaded with doxorubicin (DOX), and embedded within a bacterial cellulose/polyvinyl alcohol (BC/PVA) matrix. The arc-discharge technique produced crystalline FeNPs with high purity and narrow size distribution. Folic acid conjugation enabled tumor-targeted delivery, while DOX was efficiently incorporated via electrostatic and {pi}-{pi} stacking interactions. Embedding in the BC/PVA hydrogel facilitated sustained drug release and improved biocompatibility. Structural and functional characterization was conducted using X-ray diffraction (XRD), scanning electron microscopy (SEM), UV-Vis spectroscopy, magnetization studies, swelling and rheological analysis, and photothermal heating experiments. In vitro cancer cell studies demonstrated enhanced therapeutic efficacy of the hydrogel system under near-infrared (NIR) irradiation, where synergistic chemo-photothermal effects resulted in significant reduction in cell viability compared to single-mode treatments. This study highlights a multifunctional nanoplatform that integrates targeted delivery, controlled release, and dual therapeutic modalities for effective cancer treatment.

Nanoplastics generated from plastic waste in our ecosystems are becoming increasingly prevalent as bulk plastics exposed to natural factors like water and sunlight fragment to the nanoscale over time. These incidental nanoplastics span a wide range of physicochemical properties, which makes studying nanoplastic interactions in biological systems difficult. Here, we characterized the behavior of incidental nanoplastics generated through mechanical abrasion within coacervate droplets to probe the surface properties of the nanoplastics. We used elastin-like polypeptides (ELPs) to create hydrophobic or charged coacervate microenvironments. Using optical microscopy and fluorescence quantification, we observed that nanoplastics made from polyethylene terephthalate (nPET), nylon 6 (nPA), and polystyrene (nPS) exhibited distinct partitioning behavior with more favorable interactions with hydrophobic droplets. This indicated that the hydrophobic polymer backbone was the predominate surface feature despite exposed functional groups of the incidental nanoplastics, in contrast to findings with model carboxylated latex nanospheres (nPS-COOH). Furthermore, the selective partitioning of incidental nanoplastics into the hydrophobic droplets was able to capture over 80% of nPET in solution, and after recovery of the protein droplet, was able to cumulatively capture over 75% of the nPET feedstock across multiple cycles. This work explores the nuanced surface characteristics of incidental nanoplastics, expands the application of coacervates as chemical probes, and demonstrates a biopolymer approach for effective nanoplastic removal.

Three-dimensional (3D) in vitro tumor models are critical for studying transport-limited drug efficacy in solid tumors; however, many existing platforms are technically complex and remain difficult to access. Stacked paper-based tumor models ("cells-in-gel-in-paper", CiGiP) address this challenge by enabling formation of diffusion-limited microenvironments while allowing direct access to cells from distinct tissue depths. Nevertheless, current CiGiP implementations rely on wax or hydrophobic barrier patterning of paper, which has become increasingly inaccessible. Here, we present a wax-printing-free approach for fabricating stacked paper-supported 3D tumor tissues using a simple 3D-printed press-fit enclosure that holds circular paper layers snugly together, thereby enforcing one-dimensional transport without lateral leakage. Using MDA-MB-231 breast cancer cells embedded in Matrigel, we demonstrate the formation of nutrient-limited microenvironments across the tissue depth, as evidenced by layer-dependent cell viability. The platform enables direct quantification of spatial and temporal drug responses, demonstrated using doxorubicin and paclitaxel, both individually and in combination. Layer-dependent cytotoxicity was measured, and combination treatment analysis revealed antagonistic interactions consistent with prior reports. By eliminating the need for hydrophobic patterning, this approach substantially lowers the technical barriers to constructing stacked paper tumor models and is expected to facilitate broader adoption of paper-supported 3D tissues for drug screening and mechanistic studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/705119v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@55f56aorg.highwire.dtl.DTLVardef@1633288org.highwire.dtl.DTLVardef@18aae9eorg.highwire.dtl.DTLVardef@1ce1532_HPS_FORMAT_FIGEXP M_FIG C_FIG

Developing wound dressings that support healing and allow real-time monitoring is a key priority in modern wound care. In this study, we designed a curcumin-loaded carboxymethyl cellulose (CMC)/polyvinyl alcohol (PVA) composite dressing with integrated pH-responsive colorimetric sensing. The films were made by solution blending and freeze-drying. They formed porous, absorbent structures that quickly absorbed fluid and managed wound exudates effectively. Curcumin served as both a therapeutic agent--delivering antioxidant, anti-inflammatory, and antibacterial effects--and a natural colorimetric indicator through its keto-enol tautomerism, enabling reversible pH-dependent transitions visible to the naked eye. UV-Vis spectroscopy confirmed absorbance shifts under acidic and alkaline conditions. It also showed that curcumin remained [~]80% stable after 14 days in the polymer matrix FTIR and SEM confirmed successful incorporation and uniform distribution of curcumin within the polymer network. Cytotoxicity assays demonstrated excellent biocompatibility, while disc diffusion and MIC assays revealed significant antibacterial activity of the curcumin-loaded films against Pseudomonas aeruginosa, confirming their potential to reduce bacterial growth. Smartphone-based RGB analysis showed a strong correlation with pH (R2 {approx} 0.99), highlighting the feasibility of low-cost digital wound monitoring. Mechanical testing indicated sufficient tensile strength and flexibility for practical wound application. Quantitative antibacterial data (inhibition zone diameter and MIC) supported strong antimicrobial performance. The primary objective of this study was to develop a multifunctional wound dressing capable of both protecting and monitoring wounds in real-time. The proposed system is specifically designed for chronic and infected wounds where pH imbalance delays healing. In addition to antimicrobial activity, the fabricated films demonstrated desirable swelling capacity and sustained curcumin release, further highlighting the practical applicability of the dressing in wound care. Cost- benefit analysis demonstrated clear economic advantages over commercial gauze-based and hydrocolloid dressings. The fabrication method is compatible with industrial scale-up, although process optimization is required. Overall, the curcumin-loaded CMC/PVA dressing provides a multifunctional platform that combines biocompatibility, antibacterial activity, pH-responsive biosensing, and cost-effectiveness for next-generation wound care. Future studies will investigate in vivo performance, long-term stability, and clinical translation potential to validate its effectiveness in real-world conditions. Overall, the curcumin-loaded CMC/PVA dressing provides a multifunctional platform that combines biocompatibility, antibacterial activity, pH-responsive biosensing, mechanical stability, and cost-effectiveness for next-generation wound care. Future studies will investigate in vivo performance, long-term stability, and clinical translation potential.

The global rise of antimicrobial resistance has significantly reduced the effectiveness of conventional antibiotics, highlighting the urgent need for alternative and complementary therapeutic strategies. Nanotechnology-based drug delivery systems, particularly lipid nanoparticles, have emerged as promising tools to enhance antibiotic efficacy while limiting toxicity and resistance development. In this study, we evaluated the antimicrobial activity and drug carrier potential of Ohmline, a novel alkyl-ether glycolipid capable of self-assembling into nanotubes and lipid nanoparticles. First, a wide range of Gram-positive and Gram-negative bacteria were used to test Ohmline nanotubes antibacterial activity. All examined strains were partially inhibited, with a more noticeable effect on Gram-positive bacteria. Then, the synergistic potential of Ohmline combined with commercially available antibiotics (ampicillin, ceftriaxone, and ciprofloxacin) was evaluated using two different approaches: binary mixtures of Ohmline nanotubes and antibiotics and microfluidically produced Ohmline:DMPC (75:25) nanoparticles with the antibiotics encapsulated. Binary formulations demonstrated strong, strain-dependent synergistic effects at sub-MIC antibiotic concentrations, particularly against Enterococcus faecalis and Citrobacter braakii. Notably, antibiotic encapsulation within Ohmline nanoparticles further enhanced antimicrobial efficacy compared to non-encapsulated combinations, achieving near-complete growth inhibition in E. faecalis and significant inhibition in Klebsiella pneumoniae and C. braakii. Overall, our findings demonstrate that Ohmline possesses intrinsic antibacterial activity and acts as an effective lipid nanocarrier that potentiates antibiotic action. The dual functionality of Ohmline supports its potential as a versatile building block for next-generation antimicrobial formulations.

Engineering functional tissue constructs requires not only replicating their 3D architecture but also capturing their dynamic biochemical and mechanical environments. While 3D bioprinting technologies enable spatial control over cell and biomaterial deposition, post-fabrication modulation of material properties remains limited. Photografting approaches allow for spatiotemporal functionalization of certain 3D matrices by chemically binding bioactive factors onto spatially determined regions of a material, but current methods often rely on specialized chemistries with narrow material compatibility. Here, we introduce AddGraft, a biocompatible, off-the-shelf additive designed for semi-orthogonal thiol-ene photografting in vinyl-functionalized hydrogels. AddGraft, a heterobifunctional polyethylene glycol, carries an acrylate moiety for network incorporation during photocrosslinking and a norbornene group for post-crosslinking functionalization. AddGraft integrates into the polymer network during gel crosslinking without altering bulk mechanics, enabling precise modification at any time post-fabrication. We demonstrate compatibility with multiple acrylated biomaterial platforms and light-based volumetric photopatterning technology. Photopatterning achieves high spatial resolution and gradient formation in 3D, while grafting of multi-thiolated crosslinkers allows localized stiffening of hydrogels. Encapsulated human mesenchymal stromal cells exhibit high viability and undergo morphological changes in response to the dynamic tuning of their microenvironment. By decoupling structural and functional roles, AddGraft enables on-demand spatial and temporal control over hydrogel properties. This approach expands the biofabrication toolkit for engineering cell-instructive, 4D tissue environments with translational relevance in regenerative medicine.

Multidrug resistant (MDR) bacterial wound infections are an increasing clinical challenge and require alternatives to conventional antibiotics. Although antimicrobial proteins offer promise, their therapeutic use is limited by poor stability, proteolytic degradation, reduced activity under physiological conditions, and potential toxicity. This work reports pH-sensitive lipid nanocarriers composed of granulysin (GNLY) and oleic acid (OA) for antimicrobial delivery to infected tissues. At neutral pH, GNLY is retained within OA-based nanocarriers and protected from proteolytic degradation. At pH 5.0, such as in infected wounds, the carriers undergo structural reorganization and release GNLY, restoring antimicrobial activity. OAGNLY (32 {micro}g/mL) achieved >3-log reductions in Staphylococcus aureus and Escherichia coli within 1 hour, and up to 4-log reductions in Pseudomonas aeruginosa and Acinetobacter baumannii, at physiological salt concentrations where free GNLY was largely inactive. Minimum inhibitory concentrations were 16 {micro}g/mL for MRSA and 32 {micro}g/mL for colistin-resistant E. coli. Ultrastructural analysis using transmission electron microscopy revealed disruptions of bacterial membranes and intracellular structures following OAGNLY treatment. In a murine surgical wound infection model, topical application of OAGNLY for 4 hours reduced bacterial burden by >5 logs and significantly decreased inflammation, as confirmed by histological analysis. In parallel, OAGNLY demonstrated minimal cytotoxicity to mammalian cells at active concentrations. These findings identify OAGNLY nanocarriers as a promising platform for pH-responsive delivery of GNLY and highlight their potential application for treating MDR skin and soft tissue infections..

Biotherapeutics are required to achieve high remission rates in patients with severe ulcerative colitis (UC); however, adverse effects, complex dosing regimens, administration routes, and low patient compliance may limit their widespread clinical use. Given the localized nature of UC, this study aimed to develop and evaluate a localized delivery strategy for infliximab (IFX), an anti-tumor necrosis factor- (TNF-) monoclonal antibody (mAb) recommended by the European Crohns and Colitis Organization (ECCO) and the American Crohns & Colitis Foundation for moderately-to-severely active UC. Exploiting the intrinsic biocompatibility, mucoadhesivity, and protein-entrapment capacity of lipid mesophases (LMPs), IFX was encapsulated within the gel matrix, providing protection against enzymatic and environmental degradation. IFX-loaded LMPs were designed for targeted delivery to inflamed colonic tissues via rectal or oral administration, with patient-centric oral dosage forms manufactured using a 3D printing approach. A comprehensive physicochemical characterization was performed to elucidate mesophase self-assembly and its relationship with IFX release profiles in biorelevant fluids. Therapeutic efficacy was evaluated in vivo using a dextran sulfate sodium (DSS)-induced colitis rat model, which demonstrated rectal gel retention for at least 8 h and colonic targeting of the oral formulation within 6 h. Under severe inflammatory conditions, LMP-based formulations reduced disease activity, inflammatory biomarkers (TNF- and fecal lactoferrin), and colon shortening to values comparable to those of healthy controls, outperforming the therapeutic efficacy of subcutaneous IFX. Overall, this study establishes a biocompatible delivery platform that enables targeted colonic IFX release and suppresses systemic absorption, representing a promising advancement in the biotherapeutic treatment of UC.

Nanotechnology is rapidly transforming medicine by enabling versatile platforms for targeted delivery, controlled release, and intracellular transport of therapeutic payloads. Polymeric mesoscale nanoparticles (MNPs) are 300 to 500 nm in diameter with a PEGylated surface that exhibit unique renal tropism, specifically toward renal tubular epithelial cells. Despite their well-described therapeutic applications and route of localization to the tubules, we do not yet understand their physicochemical stability and cellular internalization mechanisms. In this study, we investigated the stability of MNPs under stress conditions by subjecting them to repeated freeze-thaw cycles and varying storage conditions to evaluate the effects on particle size and polydispersity index. MNPs demonstrated negligible changes in size and PDI up to 4 freeze-thaw cycles. We found that both empty and dye-loaded MNPs demonstrated negligible change in size under standard -20{degrees}C storage conditions. While empty MNPs were only stable at room temperature for one day, and not at 37{degrees}C, dye-loaded nanoparticles were stable for at least eight days under both storage conditions. We then performed in vitro studies to evaluate MNP cellular uptake mechanisms using the human renal cell carcinoma cell line 786-O treated with pharmacological inhibitors of uptake pathways. We found that MNP internalization is almost entirely prevented by dynamin inhibitors, while macropinocytosis inhibition also reduced uptake, suggesting that such standard nanoparticle uptake pathways are robust to the mesoscale size range. These findings provide key insights into the stability profile and endocytosis mechanisms of MNPs, which are critical for materials scale-up and translation of novel kidney-targeted drug and gene therapies.

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

To date, a panel of different biological models have been used in skin research, ranging from in vivo testing to 2D and 3D cultures. Among these, organotypic skin models represent the current gold standard for preclinical dermatology and toxicology studies. However, they are variable in quality and require long maturation times and a lot of work and cells, the latter often of primary origin. Here, we propose dermal-epidermal spheroids as an alternative model that balances physiological relevance and throughput. Next to corresponding full thickness skin models, different fibroblast/keratinocyte coculture spheroids were generated. These used the commonly employed HaCaT cells as well as two recently immortalized keratinocyte cell lines, NHK-SV/TERT and NHK-E6/E7. To investigate their differentiation with detailed spatio-temporal resolution, a deep-learning segmentation-based pipeline, capable of revealing nuclear morphology and positioning as well as marker expression with single-cell precision, was developed and applied. Moreover, the formation of a functional barrier was assessed by live-imaging of Lucifer yellow diffusion. These experiments identified the NHK-E6/E7 cell line as the most and HaCaT cells as the least suitable alternative to primary keratinocytes in both spheroids and full thickness models. Furthermore, NHK-based coculture spheroids displayed functional maturation, including stratification, cornification, and barrier formation, closely recapitulating these features of corresponding full thickness models. Given the scalability and compatibility with automation, these micro-skin fibroblast/NHK-based 3D coculture spheroids might represent a promising new platform for pharmaceutical, cosmetic, and toxicological testing.

Buccal delivery offers a promising alternative to oral drug administration by enabling direct systemic absorption and avoiding first-pass metabolism. Multilayer polymeric films represent a promising strategy for the sequential delivery of drug and absorption enhancer in the oral cavity. Here, dual- and triple-layer films were fabricated via slot-die coating, incorporating a GLP-1 receptor agonist (GLP-1-RA) and the penetration enhancer sodium glycodeoxycholate (GDC). These were co-loaded in dual-layer films or compartmentalized in triple-layer films. Scanning electron microscopy and optical coherence tomography confirmed well-defined, distinct layers with thicknesses suitable for buccal administration (339 {+/-} 10.24 {micro}m and 487 {+/-} 36.5 {micro}m for dual- and triple-layer films, respectively). Both designs exhibited good mucoadhesion and mucosal compatibility, and preserved the secondary structure of GLP-1-RA. In vitro release studies showed rapid diffusion of GDC and GLP-1-RA from dual-layer films, whereas triple-layer films enabled sustained, sequential release of GDC and GLP-1-RA. Ex vivo porcine buccal mucosa studies showed higher GLP-1-RA and GDC flux from triple-layer films compared to dual-layer films. The films also did not compromise epithelial integrity, in contrast to the direct application of GLP-1-RA and GDC, which caused significant epithelial disruption. These results demonstrate that multilayer film architecture and spatial layering can be harnessed to control release kinetics, maximize peptide penetration, and minimize tissue stress, offering a versatile platform for safe and effective peptide delivery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/700335v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@76858borg.highwire.dtl.DTLVardef@1397e74org.highwire.dtl.DTLVardef@19d1841org.highwire.dtl.DTLVardef@a369c5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Inflammatory bowel diseases (IBDs) are frequently accompanied by anxiety and depression, largely driven by perturbed gut-brain axis signaling. However, current oral therapies remain constrained by the spatial and functional separation between intestinal inflammation and central nervous system dysfunction. Here, we present a comprehensive gut-brain dual region integrated therapeutic strategy based on functionalized Bifidobacterium longum hydrogel (INPs@BL@Gel), in which baicalin and tyrosine are coordinated with Fe(III) to form infinite coordination polymers (ICPs), coated with inulin, assembled onto Bifidobacterium longum (BL), and subsequently encapsulated within a pH- and matrix metalloproteinase-responsive silk fibroin-gelatin hydrogel. INPs@BL@Gel exhibits high drug-loading, effective gastric protection, inflammation-triggered release, and long-term intestinal colonization. Within the inflamed intestine, BL and components synergistically suppress inflammatory responses, restore gut microbiota homeostasis, and promote intestinal barrier repair through multi-target integrated therapy. Importantly, BL combined with components markedly enhances the production of beneficial neuroactive metabolites such as homovanillic acid and short-chain fatty acids, which integrated regulate neuroinflammation, preserve synaptic function, and facilitate blood-brain barrier repair via the gut-brain axis. In vivo studies demonstrate that INPs@BL@Gel not only exert potent therapeutic efficacy against colitis and effectively alleviate associated depression, but also reshape the gut microbiota and restore barrier integrity, achieving an remarkable comprehensive therapeutic effect. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/710940v1_fig1a.gif" ALT="Figure 11"> View larger version (59K): org.highwire.dtl.DTLVardef@1ceb3ceorg.highwire.dtl.DTLVardef@17ed1b4org.highwire.dtl.DTLVardef@f98f8corg.highwire.dtl.DTLVardef@3f6a46_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO (a) Schematic diagram of the design and preparation of functionalized Bifidobacterium longum hydrogel. (b) Exploration of the mechanism of INPs@BL@Gel in treating colitis-associated anxiety and depression through a dual-site multi-target synergistic strategy. C_FIG

Metallic nanoparticles have emerged as novel therapeutic agents due to their distinctive physicochemical properties and broad-spectrum activity, with applications in antimicrobial therapy, drug delivery and bioremediation. Conventional methods for metallic nanoparticle synthesis often utilize toxic chemicals and energy intensive processes that are expensive. Green synthesis offers a sustainable and cost-effective alternative by using biomolecules from plants and microorganisms. In this study, gold (AuNPs), silver (AgNPs), and copper oxide (CuO NPs) nanoparticles were biosynthesized using leaf extracts of Aloe africana Mill., a South African medicinal plant rich in phytochemicals, and the magnetotactic bacterium Magnetospirillum magnetotacticum that naturally produces intracellular nanoparticles. GC-MS analysis revealed 13 known phytochemicals in the A. africana extract including esters, terpenoids, monoglycerides, and fatty acids which served as reducing and capping agents for nanoparticle synthesis. A. africana-derived AgNPs were spherical (11-30 nm) in shape, capped with dihydrosqualene, a known antibacterial compound; and was found to display activity against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Enterococcus faecalis and Staphylococcus aureus) bacteria. These AgNPs however exhibited cytotoxicity to HEK293 and HeLa cell lines. A. africana AuNPs (17-62 nm) displayed diverse morphologies and CuO NPs (55-115 nm) were irregular shaped, and both nanoparticles exhibited limited antibacterial activity and low cytotoxicity. M. magnetotacticum-derived AuNPs (12-21 nm) and AgNPs (51-126 nm) were spherical, with the CuO NPs (42-66 nm) having irregular shapes. Except for A. africana-derived AgNPs, all other metallic nanoparticles displayed poor antibacterial activity. These findings are novel and highlight a dual-function green synthesis platform where A. africana phytochemicals contribute to both nanoparticle synthesis and bioactivity, positioning A. africana AgNPs as promising antibacterial agents.

Antimicrobial resistance has heightened the risk of device-associated infections, in which Staphylococcus aureus (S. aureus) biofilms persist through extracellular-matrix protection and marked physiological heterogeneity. Gold nanoclusters (AuNCs) can disrupt biofilms via redox-associated stress, but limited in vivo stability and poor local retention constrain their translational potential. Here, we synthesized chiral histidine-modified AuNCs (D-, L-, and DL-AuNCs) and formulated them into mildly cationic lipid nanoparticles (AuNCs@LNP) by microfluidic assembly to enhance biofilm engagement while preserving nanocluster identity. The resulting particles were [~]100-120 nm with high gold loading and a modest charge reversal. In established S. aureus USA300 biofilms, free DL-AuNCs reduced viable burden by up to 5.6 log10 CFU/mL, suppressed metabolic activity, and increased ROS signaling. Notably, LNP encapsulation maintained bactericidal activity and further improved killing by [~]1 log10 CFU at matched Au dose, while substantially enhancing biomass removal (crystal violet residual biomass: 63.77% vs 45.17%), consistent with carrier-mediated matrix destabilization. In a subcutaneous implant infection model, a single perilesional dose of DL-AuNCs@LNP reduced implant-associated bacterial burden by 2.6 log10 CFU per implant versus PBS. These results establish a modular antibiofilm platform that couples nanocluster-driven killing with lipid-facilitated biofilm disruption and defines an efficacy-tolerability window to guide optimization of locally delivered antibiofilm nanomedicines. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/707929v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@119134forg.highwire.dtl.DTLVardef@142e7b2org.highwire.dtl.DTLVardef@1799550org.highwire.dtl.DTLVardef@139c33a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Advanced oral squamous cell carcinoma (OSCC) patients with cisplatin-refractory tumors face a poor prognosis and limited therapeutic options. Cisplatin-based systemic chemotherapy has long been the gold standard despite producing unmanageable adverse side effects and toxicity. Compared to Pt(II)-based analogs, octahedral Pt(IV)-based compounds have demonstrated remarkable potential as antitumor prodrugs. Pt(VI) derivates are chemically inert remaining intact until internalized within cells, demonstrating higher tolerability and selectivity towards cancer cells. In this study we compare antitumoral response implementing primary patient-derived 2D and 3D OSCC in vitro models treated with both cisplatin and a novel Pt(IV) compound. We also test the delivery and efficacy of these anticancer drugs via novel encapsulation of this Pt(IV) prodrug in the framework of mesoporous silica nanoparticles (Pt(IV)-cov@MSN). Our results show a significant improvement of delivery and cytotoxicity of Pt(IV)-cov@MSN in both cisplatin-responsive and cisplatin-resistant primary patient-derived in vitro models. We also show how Pt(IV)-cov@MSN elicits p53-dependent apoptotic cell death superior to that obtained with cisplatin treatment in OSCCs. These findings highlight 3D-primary models as key tools for drug and nanocarrier testing, as well as potential targeted and selective delivery strategies for novel chemotherapeutic agents.