ACS Applied Materials & Interfaces

Synthetic optical skin models offer reproducible, tunable optical properties but lack biological responsiveness, while tissue engineered skin models provide cellular authenticity but suffer from optical variability and limited controllability. The growing demand for alternatives to animal models in the development and validation of optical biomedical technologies highlights the need for a new class of test system that combines the strengths of both approaches while addressing their respective limitations. Here, we introduce the concept of a modular biohybrid skin model, a new testing concept that integrates an optically defined artificial epidermal layer, incorporating polydopamine nanoparticles for changes in skin tone, with living human keratinocytes in two and three-dimensional configurations. In the Optical Protection Model, UV-B-induced apoptosis in primary keratinocytes is quantitatively modulated by model pigmentation level, demonstrating a relationship between optical attenuation and caspase 3/7 activity across three artificial skin tone conditions. In a Structured Dermal Model, keratinocytes seeded onto a hydrogel scaffold localize within follicle-like microcavities, as confirmed by live/dead staining and confocal z-stack imaging. Together, these experiments lead to a new category of test system in the space between inert optical models and variable tissue models that may contribute to reducing the reliance on animal models in biomedical optics.

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.

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.

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.

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.

Layered double hydroxides (LDHs) are increasingly explored for agricultural, environmental, and biodelivery applications, but their composition-dependent effects on mammalian cells remain insufficiently defined. Here, we synthesized Al-Ni, Al-Co, and Al-Cu LDH nanoparticles and evaluated their physicochemical properties and biological responses across exposure-relevant mammalian cell models. The formulations showed hydrodynamic diameters of approximately 200-300 nm, moderate dispersity, strongly positive surface charge, and characteristic lamellar LDH features. Cytotoxicity was assessed using MTT, Calcein-AM, and Hoechst-PI assays in HaCaT, A549, and HT-29 cells, representing dermal, pulmonary, and intestinal exposure contexts, together with NIH/3T3 fibroblasts as a sensitive comparative model. LDH toxicity was strongly dependent on metal composition and cell type, with an overall trend of Al-Cu > Al-Co > Al-Ni and more pronounced cytotoxic effects in A549 and HT-29 cells. To detect cellular perturbations beyond overt viability loss, we applied high-content imaging using Live Cell Painting. Multiparametric single-cell profiling revealed composition- and dose-dependent alterations in acidic vesicle organization, nuclear texture, and cytoplasmic granularity. Notably, phenotypic deviations were detected at concentrations below those producing measurable effects in conventional viability assays, and linear discriminant analysis separated the phenotypic signatures induced by the three LDH formulations. Together, these findings show that LDH biological activity cannot be generalized across metal compositions and that high-content phenotypic profiling provides added sensitivity for detecting early cellular perturbations. This integrated approach supports composition-aware nanosafety evaluation and may inform the safer development of LDH-based technologies for agricultural and biotechnological applications.

Most antimicrobial drug candidates currently in development are derivatives of established antibiotic classes. In contrast, antimicrobial heteroatom-doped carbon quantum dot (CQD) nanoparticles vastly differ from their chemical antibiotic counterparts and exhibit potent antibacterial activity and favourable biocompatibility, representing a promising alternative strategy, particularly for topical applications. Here, we report the incorporation of cobalt-doped carbon quantum dots (Co-CQDs) into injectable, biocompatible hydrogels capable of both sensing pH and eliminating bacteria. Ultrasmall Co-CQDs demonstrated broad-spectrum activity against gram-positive Methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative Pseudomonas aeruginosa (PAO1), mediated by membrane hyperpolarisation and reactive oxygen species (ROS) induced membrane damage. The particles showed negligible effect on primary fibroblast and endothelial cell viability at concentrations that were bactericidal to MRSA. Polymeric hydrogels were fabricated via electrospinning of chitosan, polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA) polymer blends incorporating Co-CQD and pH-responsive HPTS particles. This approach provided accurate measurement of environmental pH within the physiological range observed across healthy and chronic wounds. In vivo, the injectable hydrogels exhibited robust antimicrobial efficacy against MRSA without impairing wound closure relative to untreated controls, while also reducing inflammatory immune responses in infected tissues. Collectively, these findings demonstrate the potential of ultrasmall metal-doped CQDs for infection control and their integration into 3D matrices as multifunctional theragnostic platforms.

Amyloid fibrils are traditionally associated with protein misfolding disorders; however, increasing evidence indicates that they can also perform beneficial biological functions, including antimicrobial defense. Here, we investigated whether structurally distinct amyloid polymorphs of hen egg white lysozyme (HEWL) exhibit enhanced antibacterial activity compared with the native protein. HEWL was converted into two amyloid polymorphs, flexible fibrils (FFs) and rigid fibrils (RFs), and their antibacterial activities were evaluated against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli (Top10) and Salmonella Typhimurium. Fibril formation was confirmed by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence, and transmission electron microscopy (TEM), demonstrating morphologically distinct amyloid assemblies with different secondary-structure organizations. Fluorescence-based bacterial growth assays showed that native HEWL exhibited only moderate antibacterial activity, whereas both amyloid polymorphs produced potent, concentration-dependent bacterial growth inhibition. FFs and RFs consistently displayed greater antibacterial efficacy than native HEWL across all tested strains, with FFs exhibiting slightly stronger activity against S. Typhimurium. At concentrations of 600-800 M, FFs achieved >90% growth inhibition for all bacterial species examined. Cytotoxicity studies using SH-SY5Y human neuroblastoma cells demonstrated minimal toxicity for native HEWL, modest effects for FFs, and substantially greater toxicity for RFs, indicating that amyloid polymorphism influences both antimicrobial activity and mammalian cell compatibility. Collectively, these findings establish a direct relationship between amyloid structure, antibacterial efficacy, and cytotoxicity. The combination of potent antibacterial activity and relatively low cytotoxicity identifies FFs as a promising functional amyloid biomaterial for the development of next-generation antimicrobial materials.

Tryptophan is a biologically important redox-active amino acid whose functions in proteins, including long-range electron transfer, protection against oxidative damage, and environmental sensing, are governed not only by its chemical identity but also by its precise structural organization. Inspired by this biological principle, we investigated whether controlling the organization of tryptophan within crystalline materials could provide a strategy for modulating its physicochemical properties and molecular accessibility. Using identical molecular components but distinct assembly pathways, tryptophan was organized either as a confined guest within a preformed Zn-imidazolate framework, yielding a star-shaped crystalline architecture, or as an integral coordination component during framework growth, producing a distinct layered Zn- tryptophan crystalline framework. Although assembled from the same building blocks, these two organization modes generated fundamentally different crystal structures, morphologies, and mechanisms of biomolecule incorporation. In both architectures, incorporation of tryptophan into the crystalline environment preserved its intrinsic fluorescence while producing robust fluorescence under multiple excitation wavelengths, highlighting the strong influence of molecular organization on its optical response. The structural modes also exhibited distinct encapsulation efficiencies and pH-dependent molecular accessibility, while secondary calcium-alginate fixation provided an additional level of control over guest retention without disrupting the underlying crystalline architecture. These results demonstrate that engineering the structural organization of tryptophan provides a versatile strategy for tuning the optical behavior, molecular accessibility, and functional integration of a biologically important redox-active amino acid in crystalline materials, establishing a foundation for future biomimetic redox architectures, responsive sensing platforms, and controlled molecular delivery.

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

Implant-associated infections are driven by bacterial biofilm formation and remain difficult to eradicate using conventional antibiotic-based strategies. Here, we present a dynamically actuated reconfigurable topographical surface (DARTS) that integrates intrinsically bactericidal nanoscale surface topography with programmable mechanical actuation to achieve durable, antibiotic-free infection control. Using a scalable bottom-up nanofabrication strategy, we generate tunable wrinkled MXene topographies that exhibit contact-mediated bactericidal activity against both Gram-positive and Gram-negative bacteria without chemical leaching. Integration with a soft robotic actuator enables reversible modulation of surface geometry, which synergistically enhances bacterial removal and killing, resulting in near-complete disruption of mature biofilms. Dynamic actuation further sensitizes released bacteria to antibiotic treatment. In a mouse subcutaneous implant infection model, DARTS with actuation achieves sustained suppression of bacterial burden and markedly improves host tissue outcomes. Remote, noninvasive actuation using near-infrared laser stimulation further highlights the translational potential of this platform for implantable antibacterial applications.

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

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.

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

The first and critical reaction in magnetic hyperthermia to induce the death of cancer cells is the production of ROS (reactive oxygen species). We previously showed that it is possible to specifically deliver iron oxide magnetic nanoparticles (IONPs) in the lysosomes of cancer cells and eradicate them by targeted magnetic intra-lysosomal hyperthermia (MILH) via the application of a high frequency alternating magnetic field (AMF) without macroscopic temperature elevation. The mechanism involves a local temperature elevation at the IONPs surface which enhances the ROS production through the Fenton reaction; ROS then peroxide the proteins and lipids of the lysosomal membrane, inducing its permeabilization and leading to lysosomal enzymes release and cell death. Fe ions, critical to produce ROS in MILH, were assumed to be released by IONPs. We thus developed PEGylated multi-cores IONPs called NanoFlowers (NF@PEG) presenting or not a SiO2 shell (NF@SiO2@PEG), the later preventing the Fe3+ release from IONPs. NF@PEG released Fe ions and produced ROS production in vitro, in acidic medium mimicking lysosome upon AMF exposure, whereas NF@SiO2@PEG did not. Surprisingly, both nanoparticles increased the ROS production in cells, induced lysosome permeabilization and cell death, and slowed down the proliferation of cancer cells with the same efficacy, upon AMF application, indicating that MILH was efficient in absence of Fe3+ release from IONPs. In contrast, Ferristatin-II, an iron uptake inhibitor, prevented the ROS production and cell death in MILH induced by both IONPs, elucidating the role of endogenous iron cations responsible for the ROS production ROS in MILH to kill cancer cells.

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.

Atomically precise gold nanoclusters (AuNCs) are ultra-small particles composed of ten to hundreds gold atoms and exhibit unique photophysical properties. Significant progress has been made in tuning and extending their luminescence in the near-infrared window through the design of AuNC assemblies. Herein, we report a straightforward method for synthesizing highly pure, programmable DNA tetrahedra functionalized with a controlled number of AuNCs (from one up to four AuNCs). Using ligand exchange chemistry, AuNCs bearing a single grafted ssDNA onto them were produced. These constructs then served as building blocks for synthesizing tetrahedra through DNA hybridization. Products obtained at each stage of the synthesis were thoroughly characterized using a range of complementary technics. Notably, mass spectrometry in native mode provided novel insights into the accurate composition and stoichiometry of these architectures. This study paves the way for the synthesis and the characterization of a variety of new three-dimensional, DNA-guided AuNC assemblies that may serve as powerful theranostics and biophotonic tools.

This study presents the development and spectroscopic characterization of an erlotinib-functionalized gold nanoparticle (erlotinib:AuNP) nanosystem designed for targeted delivery to metastatic non-small cell lung cancer H1299 cells. Initial MTS assays demonstrated that free erlotinib induced a concentration-dependent reduction in cell viability, while 0.1 {micro}M erlotinib exhibited negligible cytotoxicity and was therefore selected for nanosystem fabrication. AuNPs alone showed minimal toxicity toward H1299 cells over the investigated concentration range. Following conjugation of erlotinib with AuNPs, the resulting nanosystems reduced cell viability to approximately 60%, indicating enhanced biological activity of the drug after nanoparticle-assisted delivery. Fluorescence microscopy confirmed the intracellular internalization of the nanosystems in H1299 cells, with nanoparticle aggregates predominantly localized in the perinuclear and perimitochondrial regions. Three-dimensional Raman spectroscopy (3D RS) mapping further verified the intracellular localization of the conjugates through characteristic Raman signatures of erlotinib:AuNPs. Importantly, 3D RS enabled detection of nanosystems at concentrations below the sensitivity limit of fluorescence imaging, demonstrating superior analytical performance for intracellular nanosystem tracking. Atomic force microscopy-infrared (AFM-IR) spectroscopy coupled with principal component analysis (PCA) demonstrated substantial biochemical modifications induced by the erlotinib:AuNP nanosystems, including enhanced lipid-related spectral features and significant alterations in protein secondary structure, particularly the increased contribution of unordered and antiparallel {beta}-turn conformations. The obtained results demonstrate that combining plasmonic nanocarriers with advanced vibrational spectroscopy enables highly sensitive monitoring of intracellular drug delivery and nanosystem-induced biochemical responses in cancer cells.

Triple-negative breast cancer (TNBC) remains a major therapeutic challenge due to the lack of effective molecular targets and the dose-limiting off-target toxicity of conventional chemotherapy. Here, we design and construct a mirror-image DNA (L-DNA) nanostructure functionalized with an epithelial cell adhesion molecule (EpCAM)-specific aptamer for targeted delivery of doxorubicin (DOX) to TNBC cells. The L-DNA nanostructure retains thermodynamic properties comparable to natural D-DNA while exhibiting substantially enhanced resistance to nuclease and serum-mediated degradation due to its mirror-image chirality. Thermal melting and serum stability assays confirmed superior structural stability of the L-DNA nanostructure compared to D-DNA counterparts. In vitro cytotoxicity studies demonstrated that the EpCAM-targeted L-DNA nanostructure has the potential to selectively inhibit the growth of EpCAM-positive TNBC cells while reducing cytotoxicity in normal cells. These findings demonstrate that combining aptamer targeting with mirror-image DNA nanotechnology provides a stable and selective nanoplatform for chemotherapeutic delivery, which can potentially improve the precision and therapeutic efficacy of treatment for aggressive breast cancers.

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