Langmuir

Surface functionalization of inorganic quantum dot nanoparticles is of great interest in the application of these materials toward a wide range of biological applications where membrane interactions are critical. The use of amphiphilic lipids to functionalize the surfaces of quantum dots represents a promising alternative to produce water-soluble and membrane-active materials with facile tuning of the quantum dots surface properties. Here, we demonstrate an experimental approach that yields lipid-coated quantum dots with highly tunable surface charge by controlling the concentration of cationic lipids during preparation. Through fluorescence-activated cell sorting assays, we show that these cationic lipid-coated quantum dots can enhance membrane interactions and increase membrane labeling density in live HEK293 cells. We further employed coarse-grained molecular dynamics simulations to model the lipid self-assembly process using an implicit solvent force field and subsequently model the adsorption of lipid-coated quantum dots to model membranes. Our simulations show that we can control the effective surface charge of lipid-coated quantum dots and influence the strength of adsorption to oppositely charged lipid membranes, a process that is mediated by the release of counterions at the quantum dot-membrane interface. This work supports the future development of biocompatible and water-soluble inorganic nanoparticles with highly tunable surfaces, and provides mechanistic insight into how different lipids can influence nanoparticle-membrane interactions at a molecular scale.

Liposomes are self-assembled lipid vesicles capable of encapsulating both hydrophilic and hydrophobic therapeutics, making them versatile platforms in drug delivery and biomedical technology. In this study, the limitations of the classical thin-film hydration method were critically evaluated, and a sustainable, systematically optimized strategy was established for generating defined liposomal lamellar phases. Hydration conditions were optimized, and 4 mL of buffer per 10 mg of lipid was determined to be optimal for effective rehydration and improved statistical reliability of vesicle measurements. A refined probe-sonication protocol (20% amplitude, 5 s ON/55 s OFF pulse) enabled controlled transformation of multivesicular vesicles into stable multilamellar and unilamellar vesicles at net ON-times of 90 s and 185 s, respectively, without overheating or contamination. In addition, a Python-based machine-learning tool was developed for vesicle size characterization. Collectively, these optimizations provided a reproducible and sustainable framework for preparing liposomes across different lamellar phases.

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.

Enveloped viruses can enter host cells by fusing their membrane with that of the host cell, a process known as membrane fusion. This process depends on specific fusion proteins located on the viral particle surface, which contain a short, relatively hydrophobic segment called "fusion peptide" that binds to the host membrane. To investigate the fusion efficiency of various fusion peptides, we create simplified non-infectious virus like particles decorated with different fusion peptides and fuse them with an artificial cell membrane. For this purpose, microfluidic devices are used to create supported lipid bilayers while the result of the fusion process is studied by fluorescence microscopy. Our study provides structural insights into the interactions between virus particles and cell membranes, which can facilitate the development of new therapeutic strategies and more effective viral vectors for therapeutic applications.

Manufacturing and storage processes can expose microbes to oxidative stress, reducing viability and limiting their use in biotechnological applications. Here, we evaluate graphene quantum dots (GQDs) containing hydroxyl and carboxyl groups as protective additives that mitigate peroxide-induced oxidative stress in Escherichia coli. GQDs did not adversely affect bacterial growth under basal conditions and restored growth in the presence of hydrogen peroxide. Using the membrane-partitioning fluorescent probe C11-BODIPY, we found that GQDs reduced peroxide-induced oxidation in bacterial membranes. We further used redox-sensitive roGFP2 probes to monitor intracellular oxidative stress and found that GQDs suppressed intracellular hydrogen peroxide accumulation and attenuated disruption of glutathione redox homeostasis. Together, these results show that GQDs protect bacteria by limiting peroxide-driven oxidative damage at both membrane and intracellular levels. This work supports the potential use of GQDs as protective additives for microbial formulations that are susceptible to oxidative stress.

Significant cellular processes, including protein sorting, signal transduction, and pathogen entry, amongst others, are associated with membrane microdomains, also known as lipid rafts. Lipid rafts, due to their unique biophysical properties compared to their surrounding environment, which stem from their distinct lipid and protein profiles, have garnered interest in methods and techniques that tune their coexisting liquid-ordered/liquid-disordered state, aiming to disrupt or destabilize them. Since cholesterol stabilizes the membrane domain, cholesterol-depleting compounds like cyclodextrin can be used to destabilize and disrupt the membrane rafts. Overall, given the membrane rafts importance in biological processes, it is crucial to understand the biophysical factors that influence its stability. In this study, we present a new method for disrupting and dissolving lipid rafts in a model system of phase-separated supported lipid bilayer (SLB) patches composed of DOPC, DPPC, and cholesterol. Using fluorescence microscopy to monitor the liquid ordered (Lo) and liquid disordered (Ld) phases of the SLB patches, we observed that adding DOPC liposomes causes a transformation of the co-existing Ld and Lo phases into a single-phase bilayer. On the other hand, adding liposomes that match the lipid content of the phase-separated SLB patch increase the areas of the existing Ld and Lo phases. This work also offers a new method for redistributing raft-localized molecules, confirmed by tracking the redistribution of cholera toxin bound to GM1 after domain dissolution with DOPC liposomes. The work describes an alternative method for dynamically altering membrane composition and dissolving domains via liposome addition, rather than lipid depletion or exchange.

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionite's ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin-streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied this method to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.

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

Biomolecular condensates (BMCs) formed through liquid-liquid phase separation (LLPS) play key roles in cellular organization, yet their molecular-scale interactions with membranes remain poorly understood. Here, we use surface-enhanced infrared absorption spectroscopy (SEIRAS) to quantify the secondary structure of poly-GR (glycine-arginine) inside condensates in contact supported lipid bilayers. Amide I SEIRAS provides a surface-sensitive measure of the peptide backbone conformation at the membrane-condensate interface. Results show that neutral POPC bilayers preserve the {beta}-sheet rich organization of the bulk condensate, whereas negatively charged POPC/POPS bilayers reduce {beta}-sheet content, and enhanced {beta}-turn formation, accompanied by perturbations of the lipid headgroups. These results demonstrate that membrane charge modulates condensate secondary structure, hydration, and interfacial behavior, providing molecular-level insight into electrostatic regulation of condensate organization at the membrane-water interface. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/726873v1_ufig1.gif" ALT="Figure 1"> View larger version (112K): org.highwire.dtl.DTLVardef@9d9e27org.highwire.dtl.DTLVardef@1fa3fe4org.highwire.dtl.DTLVardef@78bcceorg.highwire.dtl.DTLVardef@b3a2ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

Curcumin-functionalized gold nanoclusters are promising platforms for catalysis and drug delivery, yet the molecular determinants of their stability, morphology, and solvent response remain unclear. Here, microsecond all-atom molecular dynamics simulations are employed to investigate a 2 nm gold nanoparticle noncovalently coated with different curcumin forms, including neutral enol and trans-keto tautomers, the deprotonated enolate, and their mixtures in water-ethanol and water-methanol solvents. Layer-resolved analyses of radius of gyration, density profiles, and surface coverage reveal that neutral enol and trans forms generate compact assemblies with near-complete surface coverage, whereas enolate-rich systems adopt more expanded conformations with solvent-exposed molecules. Mixed systems preserve these intrinsic packing characteristics while improving overall coverage. Solvent substitution from ethanol to methanol reduces {pi}-{pi} stacking, strengthens Au-curcumin interactions, and increases surface coverage, yielding more compact nanostructures. Free energy and potential of mean force calculations indicate that deprotonated curcumin most effectively screens Au-Au interactions and stabilizes dispersed nanoparticles, while neutral tautomers provide moderate stabilization. Curcumin also enhances the loading of anticancer drug doxorubicin (DOX) onto Au nanoparticles, improving biocompatibility. Enolate(An)-containing systems produce extended structures with weaker membrane interactions, whereas neutral curcumin complexes form compact, positively charged assemblies that strongly bind to negatively charged cancer cell membranes. These findings clarify how tautomeric state and solvent environment cooperatively govern interfacial organization and colloidal stability, establish design guidelines for curcumin-based gold nanocarriers in catalysis, sensing, and drug delivery applications.

The internal dynamics of liquid-liquid phase-separated systems are governed primarily by polymer packing, excluded-volume effect, and interactions between polymers and encapsulated macro-molecules. Although one immediate effect of such a constrained microenvironment is diffusion limitation, it remains unclear whether encapsulated macromolecules can also exhibit phase composition-specific functional behaviour that is not observable in a well-mixed aqueous environment. In this regard, different phases in a phase-separated environment can be accessed via a phase diagram that demarcates the region between two-phase (droplets) and one-phase (polymer-rich, no droplets) regimes. While the two-phase region is heterogeneous, most previous work on encapsulating functional macromolecules in phase-separated droplets uses a single point from the phase diagram. This leaves a clear gap in understanding on how the function scales across this landscape of droplets and identifying regions advantageous for the encapsulated macromolecule and its function. Here, using the Spinach light-up RNA aptamer, we show that RNA function does not scale uniformly across the phase diagram. We show that RNA can exhibit phase composition-specific functional behaviour due to constraints imposed by the internal microenvironment of phase-separated droplets. Furthermore, using variants of the Spinach aptamer, we show that fluorescence activity differences among the variants vary differently with phase-separation regimes across the phase map, suggesting that some regions of the phase diagram can confer a selective advantage. Our results highlight the potential of liquid-liquid phase-separated internal microenvironments in guiding the differentiation of functional RNA variants, which could serve as a physical selection pressure in pre-cellular evolution.

Polydimethylsiloxane (PDMS) is an excellent material for the construction of biomimetic leaf replicas which reproduce leaf surfaces with high fidelity. This allows for the study of leaf surface-colonizing bacteria and the impact of the leaf topology on bacterial distributions and behavior. However, their application is limited to short-term experiments, as long term survival of microorganisms on their surface is not possible due to a lack of nutrient replenishment. On living leaves, nutrients diffuse across the cuticle via leaching, a process not yet replicated in biomimetic systems. Here, we explore whether water and fructose can be supplied to microbial colonizers on PDMS membranes by mimicking leaching. We created hybrid membranes by incorporating polymers (Carbopol, Pemulen, cellulose microfibers, cellulose nanocrystals, and polyvinylpyrrolidone) to enhance nutrient transport. We determined that bulk diffusion of water correlated negatively with membrane thickness and positively with polymer concentration. Further, fructose diffusion across hybrid membranes reached similar rates compared to isolated Populus x canescens leaf cuticles. Under high relative humidity, these membranes supported long-term bacterial survival. Our findings represent important steps towards the development of topomimetic leaf surfaces that sustain microbial life, enabling further investigation into the microbe-microbe interactions that take place on leaves.

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

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.

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.

Cell-penetrating peptides (CPPs) can enable intracellular access while avoiding cytotoxicity, yet their behavior is highly sensitive to membrane composition. Here we show that lipid composition and leaflet asymmetry act as a switch that determines whether the amphipathic CPP MAP undergoes non-disruptive translocation or stabilizes membrane pores that drive leakage. Using computational electrophysiology (CompEL) simulations in membranes of increasing physiological relevance, we find that symmetric anionic bilayers favor MAP insertion coupled to transmembrane pore stabilization in POPC:POPG membranes, reproducing progressive dye release in liposome leakage assays. Incorporation of cholesterol reduces the number of inserted peptides yet enhances pore stabilization, consistent with faster leakage kinetics in cholesterol-containing vesicles. In contrast, an asymmetric membrane model mimicking the eukaryotic plasma membrane (POPC outer leaflet; POPC:POPS inner leaflet) supports MAP translocation without sustained membrane disruption. This delivery-relevant mechanism is supported by efficient MAP internalization in HEK293 cells while maintaining high viability. Polarized ATR-FTIR provides structural context, indicating predominantly membrane-associated -helical conformations across lipid compositions. Together, these results establish lipid asymmetry and composition as actionable biointerface parameters that tune CPP function between translocation and leakage and demonstrate an experimentally benchmarked framework for predicting membrane outcomes across complex lipid environments.

Proteins in solution adsorb to the corona of nanoparticles such as single-walled carbon nanotubes (SWCNTs), but these interactions are difficult to predict and analyze due to ambiguities in the structure of the latter. In this work, we employ ss(GT)15-DNA wrapped SWCNTs, a commonly used fluorescent sensor construct, to examine protein adsorption by quantifying binding dissociation constants and characterizing the corresponding photophysical effects. A library of 20 proteins are used to evaluate adsorption-induced changes in photoluminescence (PL) intensity ({Delta}I/I0) and emission wavelength upon solution phase binding. We find that 15 proteins produce monotonic dose-response behavior well described using a single-site Langmuir model. Alternatively, five proteins exhibited more complex, non-monotonic behavior consistent with a two-step binding model representing protein-protein interactions coupled to adsorption. The study reveals that metalloproteins, which comprised 12 of the 20 proteins in the library, induced greater PL quenching compared with metal-free proteins for this system, with maximum binding-associated quenching ({Delta}I/I0) of 94% for metalloproteins versus 20% for metal-free proteins. For metalloproteins, we introduce a proximity-based quenching framework in which protein size provides a coarse proxy for cofactor-SWCNT separation, offering a mechanistic interpretation of the observed quenching variation across proteins. Together, these results establish the use of metal coordination sites, such as those in metalloproteins, to assist the transduction of certain nanoparticle fluorescent sensors, helping with sensor probe design and interpretation in biological environments.

Recent genetic and bioinformatic studies have led to the discovery that many bacterial species encode the genes required to produce sphingolipids. Shotgun lipidomic studies have identified numerous sphingolipid species with novel structures that do not exist in eukaryotic organisms. The impacts of these lipids on the biophysical properties of bacterial membranes have not yet been determined. In this study, we purify a novel anionic bacterial sphingolipid, ceramide phosphoglycerate (CPG), and investigate its effect on membrane zeta potential and bending stiffness. CPG and its precursor, ceramide 1-phosphate (C1P), are shown to increase the magnitude of the membrane zeta potential. These sphingolipids also increase the stiffness of these membranes, with CPG increasing rigidity more than C1P or ceramide. This work provides experimental and computational methods of lipid isolation and characterization that may be broadly applicable to a variety of uncharacterized bacterial sphingolipids. SIGNIFICANCEThe diversity of bacterial sphingolipids far exceeds those found in eukaryotes. However, the function and biophysical properties of these lipids are unknown. Characterization of these lipids is a challenge as they are not commercially available. In this study, we developed experimental methods to purify the anionic sphingolipid ceramide phosphoglycerate and incorporate it into liposomes for analysis. Furthermore, we built computational tools to determine the bending stiffness of sphingolipid-containing vesicles from thermal fluctuation data.

Hyperbranched poly(beta-amino ester) (hPBAE) nanoparticles represent a promising platform for nucleic acid delivery, particularly to the lungs. In this study, we evaluate the potential of hPBAE nanoparticles to deliver defective interfering RNA (diRNA) antivirals targeting betacoronaviruses under a range of formulations and storage conditions. hPBAE-diRNA nanoparticles demonstrated efficient cellular uptake of functional diRNA across diverse cell types, conferred protection against nuclease-mediated degradation, and exhibited low in vitro cytotoxicity. In vivo, these nanoparticles enabled effective delivery of functional diRNA to the lungs of golden hamsters without inducing adverse physiological effects. Collectively, these findings support hPBAE nanoparticles as a safe and effective platform for diRNA delivery for the treatment of respiratory viral infections. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC="FIGDIR/small/721911v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@2fc386org.highwire.dtl.DTLVardef@1cda4f9org.highwire.dtl.DTLVardef@9f61borg.highwire.dtl.DTLVardef@1fc8461_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO Defective interfering RNA was mixed with hyperbranched poly(beta-amino ester) nanoparticles and delivered to cells in vitro and to golden hamsters in vivo, to measure toxicity and the replication potential of the RNA. C_FIG

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.