Langmuir

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.

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.

Cholesterol is a key component of cellular membranes, regulating membrane organization, fluidity, and signaling. However, cholesterol analysis remains technically challenging, as no single method currently allows both accurate quantification and spatially resolved visualization. Biochemical assays provide accurate quantification but lack spatial resolution, whereas imaging strategies can perturb membrane organization or cholesterol accessibility. Here, we describe optimized protocols using fluorescent D4 probes derived from the cholesterol-binding domain of perfringolysin O (D4-mCherry and D4-GFP) to detect, visualize, and quantify cholesterol in biological samples. We detail procedures for probe production, purification, and application, and establish conditions that ensure robust and reproducible labeling of membrane-accessible cholesterol. By combining fluorescence-based imaging with quantitative analysis, this approach enables the assessment of cholesterol distribution while preserving its native membrane environment. The proposed methodology provides a versatile and reliable framework for studying cholesterol in a wide range of experimental systems.

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.

The apparent pKa of ionizable lipids in lipid nanoparticles (LNPs) is a key determinant of RNA encapsulation during formulation and endosomal release after cellular uptake. However, it is difficult to predict the effective pKa of a given ionizable lipid solely from its solution pKa, because it is sensitive to the membranes composition, as well as solution conditions such as the salt concentration. We developed a simple continuum electrostatics model, based on Gouy-Chapman theory, to predict the shift in effective pKa for ionizable lipids in lipid bilayers as a function of salt concentration and membrane composition. We derive equations for the surface potential and fraction of lipids charged, which are solved self-consistently as a function of solution pH to extract the titration curve and effective pKa. The model shows that the shift in effective pKa is largest when the concentration of titratable lipid is high, and the effect is diminished by increasing salt concentration. We provide a python implementation of the model and an interactive notebook that will allow users to further easily explore the predicted pKa shifts as a function of formulation variables.

Saturated fatty acids such as stearic acid (SA) can exhibit both antimicrobial and growth-promoting effects on bacteria, depending on their concentration and chemical structure. However, the physical properties of the bacterial cell envelope in response to such molecules remain under-explored compared to their biochemical pathways. In this study, a comprehensive investigation is presented on the interaction of SA with the Gram-positive bacterium, Staphylococcus epider-midis (S. epi). SA alters bacterial growth, reflected in a higher maximum specific growth rate, a shorter lag phase, and an extended exponential phase, consistent with a prebiotic effect. Using fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy, we show that SA incorporation leads to significant fluidization of the lipid membrane, characterized by enhanced lateral diffusion and reduced membrane viscosity. Coarse-grained molecular dynamics (CG-MD) simulations demonstrate spontaneous insertion of SA into the membrane and a significant increase in mean-square displacement after insertion, supporting our experimental observations. Importantly, atomic force microscopy measurements show an increase in cell-envelope stiffness, reflected by a higher Youngs modulus which can be attributed to modulations in the glycan-peptide linkage density based on earlier studies that correlate stiffness changes to peptidoglycan (PG) crosslinking in Gram-positive strains [1]. These results provide direct evidence linking membrane fluidization induced by SA and increased cell wall stiffness due to transport modifications in the membrane mediated PG synthesis pathways to enhance bacterial cell viability.

This study investigates the interaction between the cationic antimicrobial peptide protamine and bacterial porin OmpF (E. coli) at the single-molecule level. Using high-resolution conductance measurements in planar lipid bilayers, strong voltage- and concentration-dependent ion current blockages with OmpF, indicating significant protamine binding were observed. Further analysis revealed that peptide length influences binding kinetics, with longer peptides showing reduced affinity and slower exchange rates. These findings demonstrate that OmpF is a tractable model for studying cationic peptide-channel interactions and translocation mechanisms relevant to antimicrobial action.

Efficient integration of proteins into amphiphilic polymer membranes offers new opportunities in synthetic biology and nanotechnology. Long-term protein reconstitution into artificial membranes remains challenging due to a lack of stabilising protein-membrane interactions found in native lipid bilayers. Here, we redesigned the transmembrane region of a CytK-4D {beta}-barrel nanopore for stable insertion into 3.5-6.6 nm thick PBD-PEO (poly(1,2-butadiene)-b-poly(ethylene oxide)) bilayers. PBD-PEO membranes offer high mechanical and chemical stability and low electrical noise, but the thick membrane hinders anchoring of biological nanopores. By systematically investigating the elongation of the {beta}-barrel, we engineered nanopore constructs suitable for PBD11PEO8 and PBD22PEO14 membranes. Efficient insertions were observed by adding amino acids that stabilised the transmembrane {beta}-barrel structure and enhanced anchoring of the nanopore into the membrane. Molecular dynamics simulations and single-molecule assays revealed that nanopores folded naturally into PBD-PEO bilayers, enabling successful detection of cyclodextrins and translocation of polypeptides and full-length proteins. Our study offers important lessons for the reconstitution of membrane proteins into artificial membranes. Moreover, these highly robust nanopore-membrane interfaces can be readily integrated into biosensing devices, enabling peptide and protein analysis directly from complex solutions.

Extracellular vesicles (EVs) are cell-secreted biological nanoparticles that play a crucial role in intercellular communication and are gaining increasing attention as diagnostic biomarkers, therapeutic agents, and drug delivery vehicles. Consequently, the development of robust and sensitive methods for their characterization is essential. Herein we present the use of a microscope-mounted nanofluidic device for direct size determination and multi-parametric (3-color) fluorescence-based phenotyping of single biological nanoparticles that are in the size range of 20-200 nm in a method we denote Nano-SMF (SMF; size and multiplexed fluorescence). We demonstrate that it is possible to accurately determine the size of nanoparticles by analyzing their one-dimensional Brownian motion during directional flow through nanochannels, achieving size distributions for monodisperse nanoparticle solutions that are on par with TEM analysis, and size discrimination of nanoparticle mixtures that is significantly improved compared to conventional nanoparticle tracking analysis (NTA). Furter, we demonstrate that the method can be applied to analyze EVs directly in minute volumes of cell supernatant, avoiding pre-isolation or concentration steps. The method was applied to phenotype CD63- and CD81-positive EVs from a human embryonic kidney cell model, demonstrating that vesicle sub-populations defined by these two tetraspanin biomarkers differ significantly in size.

To achieve a therapeutic effect, nanoparticles delivering nucleic acids must facilitate endosomal escape (EE) of their cargo. Despite extensive research, the mechanisms that lead to an effective EE are not sufficiently understood. Herein, we utilized Molecular Dynamics (MD) simulations in All Atom (AA) and Coarse Grained (CG) resolutions to differentiate the interaction of four polymeric formulations (polyplexes) and one lipid nanoparticle (LNP) with endosomal membranes. On the one hand, the results emphasize the benefit of hydrophobic residues in the nanoparticles. On the other hand, the role of anionic lipids in the biological membranes is demonstrated. Furthermore, the identified interaction patterns were successfully correlated to the in vitro performance of the formulations. For the first time, different EE mechanisms of polyplex formulations are visualized in simulation and therefore distinguishable from one another. Hence, this work highlights the power of MD simulations for taking a big step towards better understanding EE efficiency. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/711661v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@abba74org.highwire.dtl.DTLVardef@5e2b8eorg.highwire.dtl.DTLVardef@7db144org.highwire.dtl.DTLVardef@1034e_HPS_FORMAT_FIGEXP M_FIG C_FIG

The conformational dynamics of a model cationic protein in water and in the presence of anionic sodium dodecyl sulphate (SDS) and cationic cetyltrimethylamonium bromide (CTAB) surfactants at different concentrations were investigated using all-atom molecular dynamics simulations. Free-energy landscapes constructed along principal components reveal a compact, well-defined native basin at 25 {degrees}C in water, whereas elevated temperature (100 {degrees}C) induces a broadening of the conformational space and the emergence of multiple metastable states. The presence of surfactants further modulates this behavior in a concentration-dependent manner. Cluster population analysis shows that SDS promotes a highly heterogeneous ensemble characterized by reduced dominance of the native-like cluster, while CTAB partially protects the protein from thermal denaturation at higher concentrations. Radial distribution functions demonstrate strong accumulation of SDS headgroups around the protein and pronounced insertion of SDS alkyl tails into hydrophobic protein regions, indicating direct hydrophobic destabilization and micelle-assisted unfolding. In contrast, CTAB exhibits weaker headgroup association owing to electrostatic repulsion and reduced tail-hydrophobic contacts, suggesting a less disruptive interaction mechanism. At high concentration, CTAB aggregates provide a structured hydrophobic environment that stabilizes the folded state and suppresses denaturation. Together, these results provide a molecular-level picture of how surfactant chemistry and concentration govern the conformational stability of a cationic protein, highlighting the dominant role of hydrophobic interactions in surfactant-induced denaturation at high temperature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/717321v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@dcf96aorg.highwire.dtl.DTLVardef@17acdc7org.highwire.dtl.DTLVardef@15bdc2borg.highwire.dtl.DTLVardef@1d39f3c_HPS_FORMAT_FIGEXP M_FIG C_FIG

The optimization of mRNA-lipid nanoparticles (mRNA-LNPs) for therapeutic applications is limited in part by the inadequate characterization of mRNA payload heterogeneity. One current challenge is accurately measuring the number of mRNA copies within individual LNPs, where the standard method of intensity-based mRNA number determination is sensitive to fluorescent dye-dye interactions and heterogeneity of mRNA labeling. Here we present a single-particle microscopy method that combines direct counting of the mRNA copies per LNP with LNP size measurements. While confined in microwells, individual mRNA-LNPs are lysed to release their cargo and stained with a dye such that the number of mRNA molecules in each well can be directly counted using fluorescence microscopy. Since the method stains the mRNA cargo in situ, it enables characterization of LNPs formulated with therapeutic grade (e.g., unlabeled) mRNA. We applied this approach to two Onpattro(R)-based LNP formulations prepared using different formulation buffers, where the two formulations had different average mRNA copy number, particle size, and fraction of LNPs lacking mRNA. The ability to directly count the number of mRNA molecules in LNPs establishes a complimentary method to intensity-based mRNA number determination and supports the characterization and screening of clinically relevant LNP formulations.

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.

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

In mammalian cells, lipid monolayers support the integrity of lipid droplets (LDs), organelles that function as storage for neutral lipids. Liver-targeting illnesses such as liver cancer interrupt normal LD metabolism and prompt changes in the chemical content of these organelles, which can have effects on structural and organizational behavior of the lipids. In LDs, liver cancer induces concentric crystalline phases of cholesteryl esters (CEs) and triglycerides near the NL-monolayer interface, which become more pronounced as CE concentration increases. Yet, there is little known about how this phenomenon may link to persistence of undigested LDs in liver cancer patients. To shed light on this, all-atom molecular dynamics simulations were used to model LD micropipette aspiration experiments and gain insight into the effect of CE concentration on partitioning, structural, and mechanical properties of LDs. We successfully model micropipette aspiration by application of constant surface tension laterally, which stretches lipid bilayers and monolayers as the magnitude increased. The results show increased phospholipid packing due to insertion of CE fatty tails into the monolayer. Increasing CE concentration induces a non-linear change in surface packing defects on the LDs, notable rigidification, and stiffness. Taken together, these insights improve our understanding of the physical properties at the LD monolayer-core interface during liver cancer progression.

Predicting high-concentration viscosity of monoclonal antibodies such as IgG1 is crucial for their development as therapeutics for subcutaneous delivery. Unfortunately, traditional experimental rheometry methods for assessing viscosity are low-throughput. This study evaluates Self-Interaction Nanoparticle Spectroscopy (SINS) assays--specifically charge-stabilized SINS (CS-SINS) and PEG-stabilized SINS (PS-SINS)--for high-throughput viscosity prediction. We characterized 96 IgG1 antibodies, assessing SINS against in silico descriptors and dynamic light scattering (DLS) data. CS-SINS showed strong correlation with charge, offering limited additional utility. In contrast, PS-SINS provided orthogonal information; integrating it with in silico data and DLS significantly improved random forest model accuracy for binary viscosity classification. PS-SINS measurements in multiple buffers captured complementary information, achieving comparable accuracy without DLS. Importantly, PS-SINS scores exhibited a strong logarithmic relationship (r=0.98) with high-concentration viscosity in Fc variants of clinical antibodies, suggesting a direct mechanistic link. Furthermore, PS-SINS performed reliably with one column purified (protein A) samples, supporting its early-stage application. These findings establish PS-SINS as a high-throughput tool to accelerate the developability assessment of antibody candidates.

Efficient intracellular delivery of nucleic acids, proteins, and other biomolecules is critical to advancing therapeutic strategies and genome-editing technologies. Lipid nanoparticles (LNPs) have emerged as highly promising delivery vehicles owing to their self-assembly properties, biocompatibility, and capacity to encapsulate large molecular cargos. Their biological performance is determined largely by lipid composition, which influences particle stability, cellular uptake, membrane fusion, and intracellular trafficking. In this study, we designed and optimized LNP formulations inspired by the lipid architecture of enveloped viruses. Four distinct formulations were generated and systematically evaluated in mammalian cell culture, leading to the identification of two lead candidates with superior delivery characteristics. The biodistribution and translocation properties of these formulations were subsequently assessed using an in vitro brain endothelial barrier model to mimic brain environment. Furthermore, we demonstrated that the selected LNPs enable efficient and functional delivery of CRISPR-Cas ribonucleoprotein complexes to mammalian cells. Together, these findings underscore the potential of rationally engineered LNPs as versatile, safe, and effective non-viral delivery platforms for advanced genome-editing applications.

mRNA-lipid nanoparticles (LNP) have proven their potential as a rapidly adaptable vaccine platform and promise to revolutionize numerous therapeutic areas. A major hurdle towards the widespread adoption of mRNA-LNP vaccines and therapeutics is their limited liquid shelf-life compared to more established modalities currently necessitating an ultralow temperature cold-chain to enable their distribution and storage. While ongoing efforts aim to improve liquid stability through chemical modification of mRNA and lipid components, complementary strategies that are broadly applicable across chemistries may further accelerate translation. Here, we present an approach to improve the liquid shelf-life of mRNA-LNPs that does not rely on modifications to the mRNA or LNP chemistry. In particular, we show that bleb formation induced by high ionic strength acidic citrate buffers during LNP formation reduces mRNA degradation and retains in vitro activity during extended liquid storage. We observed an increase in the in vitro activity storage half-life from 2.8 to 18.9 days at 25{degrees}C when prepared using high ionic strength buffers translating into a [~]7-fold improvement in the liquid shelf-life of MC3-LNPs. This enhanced stability of LNPs with large amount of bleb formation was mainly attributed to reduced rates of lipid-mRNA adduct formation and mRNA fragmentation. Furthermore, the acidic buffer dependent stabilization was observed across different ionizable lipids with the extent dependent on the ionizable lipid head group. We envision that the induction of bleb formation via selection of appropriate acidic mixing buffers may represent a universal approach to enhance mRNA-LNPs stability and enable extended long-term refrigerated storage.

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.

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.