Size- and Shape-Dependent Interactions of Lipid-Coated Silver Nanoparticles: An Improved Mechanistic Understanding through Model Cell Membranes and In Vivo Toxicity.

The widespread use of silver nanoparticles (AgNPs) in various applications and industries has brought to light the need for understanding the complex relationship between the physicochemical properties (shape, size, charge, and surface chemistry) of AgNPs that affect their ability to enter cells and cause toxicity. To evaluate their toxicological outcomes, this study systematically analyzed a series of homogeneous hybrid lipid-coated AgNPs spanning sizes from 5 to 100 nm with diverse shapes (spheres, triangles, and cubes). The hybrid lipid membrane comprises hydrogenated phosphatidylcholine (HPC), sodium oleate (SOA), and hexanethiol (HT), which shield the AgNP surface from surface oxidation and toxic Ag+ ion release to minimize its contribution to toxicity. To reduce any significant effects by surface chemistry, the HPC, SOA, and HT membrane composition ratio was kept constant, and the AgNPs were assessed using embryonic zebrafish (Danio rerio). While a direct comparison cannot be drawn due to the lack of complementary sizes below 40 nm for triangular plates and cubes due to synthetic challenges, significant mortality was observed for spherical AgNPs (AgNSs) of 5, 20, 40, and 60 nm at 120 h postfertilization at concentrations ≥6 mg Ag/L. In contrast, the 10, 80, and 100 nm AgNSs, 40, 70, and 100 nm triangular plate AgNPs (AgNPLs), and 55, 75, and 100 nm cubic AgNPs (AgNCs) showed no significant mortality at 5 days postfertilization following exposure to AgNPs at concentrations up to 12 mg Ag/L. With constant surface chemistry on the AgNPs, size is the dominant factor driving toxicological responses, with smaller nanoparticles (5 to 60 nm) being the most toxic. Larger AgNSs, AgNCs, and AgNPLs from 75 to 100 nm do not show any evidence of toxicity. However, when closely examining sizes between 40 and 60 nm for AgNSs, AgNCs, and AgNPLs, there is evidence that discriminates shape as a driver of toxicity since sublethal responses generally were observed to follow a pattern, suggesting toxicity is most significant for AgNSs followed by AgNPLs and then AgNCs, which is the least toxic. Sum frequency generation vibrational spectroscopy showed that irrespective of size or shape, all hybrid lipid-coated AgNPs interact with membrane surfaces and "snorkel" between phases into the lipid monolayer with minimal energetic cost. These findings decisively demonstrate that not only smaller AgNPs but also the shape of the AgNPs influences their biological compatibility.

Comparative Thermodynamic and Structural Analysis of Polyfluorinated Dodecylphosphonic Acid Adsorption to Distilled and River Water Interfaces.

As concerns rise about the health risks posed by per- and polyfluoroalkyl substances (PFAS) in the environment, there is a need to understand how these pollutants accumulate at environmental interfaces. Untangling the details of molecular adsorption, particularly when there are potential interactions with other molecules in environmental systems, can obscure the ability to focus on a particular contaminant with molecular specificity. Often adsorption studies of environmental interfaces require a reductionist approach, where laboratory experiments may not be fully tractable to environmental systems. In this work, we study polyfluorinated dodecylphosphonic acid (F21-DDPA) at the aqueous surfaces of distilled water (the most reduced "environmental" surface) and river water to explore the use of vibrational sum-frequency (VSF) spectroscopy as an experimental probe of fluorinated contaminants at natural environmental surfaces. We demonstrate how VSF spectroscopy offers advantages over nonspecific surface tension measurements when measuring PFAS adsorption isotherms at river water surfaces. VSF spectra of the C-F stretching region selectively probe the presence of F21-DDPA and can be used to extract meaningful structural insights and calculate surface concentrations, even at the complex river water surface. This study highlights the potential for VSF spectroscopy to be developed as a probe of fluorinated contaminants at natural environmental interfaces.

Formation and surface-stabilizing contributions to bare nanoemulsions created with negligible surface charge.

The stabilization of nanoemulsions, nanosized oil droplets dispersed in water, is commonly achieved through the addition of surfactants and polymers. However, nanoemulsions in the absence of emulsifiers have been observed to acquire a significant negative charge at their surface, which ultimately contributes to their stability. While the source of this negative charge is disputed to this day, its presence is taken as an inherent property of the aqueous-hydrophobic interface. This report provides a look at the molecular structure and bonding characteristics of bare aqueous-hydrophobic nanoemulsion interfaces. We report the creation of bare nanoemulsions with near zero surface charge, which are marginally stable for several days. The process of creating these low-charge nanoemulsions (LCNEs) required rigorous cleaning procedures and proper solvent storage conditions. Using vibrational sum-frequency scattering spectroscopy, we measure the structure and bonding of the interfacial aqueous and hydrophobic phases. The surfaces of these LCNE samples possess a measurable free OH vibration, not found in previous studies and indicative of a clean interface. Tuning the nanoemulsion charge through addition of anionic surfactants, modeling potential surface-active contaminants, we observe the free OH to disappear and a reorientation of the interfacial hydrophobic molecules at micromolar surfactant concentrations. Notably, the free OH vibration provides evidence for stronger dispersion interactions between water molecules and the hydrophobic phase at the LCNE surface compared with similar planar water-alkane interfaces. We propose the stronger bonding interactions, in addition to an ordered interfacial aqueous layer, contribute to the delayed droplet coalescence and subsequent phase separation.

Effects of Salt-Induced Charge Screening on AOT Adsorption to the Planar and Nanoemulsion Oil-Water Interfaces.

Nanoemulsions, nanosized droplets of oil, are easily stabilized by interfacial electric fields from the adsorption of ionic surfactants. While mean-field theories can be used to describe the impact of these interfacial fields on droplet stability, the influence of these fields on the adsorption properties of ionic surfactants is not well-understood. In this work, we study the adsorption of the surfactant sodium dioctyl sulfosuccinate (AOT) at the nanoemulsion and planar oil-water interfaces and investigate how salt-induced charge-screening affects AOT adsorption. In the absence of salt, vibrational sum-frequency scattering spectroscopy measurements reveal the ΔGads and the maximum surface density is the same for AOT at the hexadecane nanoemulsion surface as at the planar hexadecane-H2O interface. Upon the addition of NaCl, an increase in AOT surface density is detected at both interfaces, indicating that repulsive electrostatic interactions between AOT monomers are the dominant force limiting surfactant adsorption at both interfaces. The bulky alkyl chains of AOT molecules cause our observations in this study to differ from those found in previous studies investigating the adsorption of linear-chain ionic surfactants to the nanoemulsion surface. These results provide necessary information for understanding factors limiting the adsorption of ionic surfactants to nanodroplet surfaces and highlight the need for further studies into the adsorption properties of more complex macromolecules at nanoemulsion surfaces.

Probing the Molecular Structure of Coadsorbed Polyethylenimine and Charged Surfactants at the Nanoemulsion Droplet Surface.

Nanoemulsions, nanoscale oil droplets dispersed in an aqueous medium, can be stabilized by polymer-surfactant (PS) mixtures, making them ubiquitous in commercial, industrial, and pharmaceutical applications. It is well-known that the presence of PS layers coadsorbed at the droplet surface plays a significant role in droplet stability and functionality; however, little is understood about the molecular nature of this coadsorption. Such insights are especially important for application in drug delivery where physiological conditions can vary the environmental pH and significantly impact stabilization. Hence, the focus of this study examines the surface properties of ∼300 nm nanoemulsions stabilized by the coadsorption of polyethylenimine (PEI) and charged alkyl surfactants sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB). PEI is a common charge-tunable polymer used in nanocarrier templates. This study employs vibrational sum frequency scattering spectroscopy, coupled with ζ-potential and surface pressure measurements performed as a function of varying concentrations and pH. The surface specific spectroscopic results reported herein reveal that PEI adsorption and molecular ordering is influenced by both electrostatic and hydrophobic interactions. While the degree of PEI adsorption is stronger in the presence of anionic SDS than cationic DTAB, for both surfactants, PEI is molecularly disordered in acidic conditions and adopts a persistent net ordering as the solution pH becomes more basic. Both surfactants also display degrees of interfacial conformational ordering that is altered by the presence of the coadsorbed polymer. These results demonstrate the molecular-level diversity in PEI behavior at the droplet interface and provide insight into how such behavior can be controlled to yield nanocarrier technology with specific functions and enhanced efficacy.

Molecular characterization of water and surfactant AOT at nanoemulsion surfaces.

Nanoemulsions and microemulsions are environments where oil and water can be solubilized in one another to provide a unique platform for many different biological and industrial applications. Nanoemulsions, unlike microemulsions, have seen little work done to characterize molecular interactions at their surfaces. This study provides a detailed investigation of the near-surface molecular structure of regular (oil in water) and reverse (water in oil) nanoemulsions stabilized with the surfactant dioctyl sodium sulfosuccinate (AOT). Vibrational sum-frequency scattering spectroscopy (VSFSS) is used to measure the vibrational spectroscopy of these AOT stabilized regular and reverse nanoemulsions. Complementary studies of AOT adsorbed at the planar oil-water interface are conducted with vibrational sum-frequency spectroscopy (VSFS). Jointly, these give comparative insights into the orientation of interfacial water and the molecular characterization of the hydrophobic and hydrophilic regions of AOT at the different oil-water interfaces. Whereas the polar region of AOT and surrounding interfacial water molecules display nearly identical behavior at both the planar and droplet interface, there is a clear difference in hydrophobic chain ordering even when possible surface concentration differences are taken into account. This chain ordering is found to be invariant as the nanodroplets grow by Ostwald ripening and also with substitution of different counterions (Na:AOT, K:AOT, and Mg:AOT) that consequently also result in different sized nanoparticles. The results paint a compelling picture of surfactant assembly at these relatively large nanoemulsion surfaces and allow for an important comparison of AOT at smaller micellar (curved) and planar oil-water interfaces.

"Nonlinear" pursuit of understanding pollutant accumulation and chemistry at environmental and biological interfaces.

Over the past few decades, the public recognition of the prevalence of certain classes of pollutants, such as perfluoroalkyl substances and nanoplastics, within the environment, has sparked growing concerns over their potential impact on environmental and human health. Within both environmental and biological systems, the adsorption and structural organization of pollutants at aqueous interfaces can greatly impact the chemical reactivity and transformation. Experimentally probing chemical behavior at interfaces can often pose a problem due to bulk solvated molecules convoluting molecular signatures from interfacial molecules. To solve this problem, there exist interface-specific nonlinear spectroscopy techniques that can directly probe both macroscopic planar interfaces and nanoplastic interfaces in aqueous environments. These techniques can provide essential information such as chemical adsorption, structure, and reactivity at interfaces. In this perspective, these techniques are presented with obvious advantages for studying the chemical properties of pollutants adsorbed to environmental and biological interfaces.

Orientation of the Dysferlin C2A Domain is Responsive to the Composition of Lipid Membranes.

Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its ß-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its ß-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface.

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Choose your own adventure: Picosecond or broadband vibrational sum-frequency generation spectroscopy.

Vibrational sum-frequency generation (VSFG) spectroscopy is a method capable of measuring chemical structure and dynamics within the interfacial region between two bulk phases. At the core of every experimental system is a laser source that influences the experimental capabilities of the VSFG spectrometer. In this article, we discuss the differences between VSFG spectrometers built with picosecond and broadband laser sources as it will impact everything from material costs, experimental build time, experimental capabilities, and more. A focus is placed on the accessibility of the two different SFG systems to newcomers in the SFG field and provides a resource for laboratories considering incorporating VSFG spectroscopy into their research programs. This Tutorial provides a model decision tree to aid newcomers when determining whether the picosecond or femtosecond laser system is sufficient for their research program and navigates through it for a few specific scenarios.

Dynamic Duo: Vibrational Sum Frequency Scattering Investigation of pH-Switchable Carboxylic Acid/Carboxylate Surfactants on Nanodroplet Surfaces.

Surfactants containing pH-switchable, carboxylic acid moieties are utilized in a variety of environmental, industrial, and biological applications that require controlled stability of hydrophobic droplets in water. For nanoemulsions, kinetically stable oil droplets in water, surface adsorption of the anionic form of the carboxylic acid surfactant stabilizes the droplet, whereas a dominant surface presence of the neutral form leads to destabilization. Through the use of dynamic light scattering, ζ-potential, and vibrational sum frequency scattering spectroscopy (VSFSS), we investigate this mechanism and the relative surface population of the neutral and charged species as pH is adjusted. We find that the relative population of the two surfactant species at the droplet surface is distinctly different than their bulk equilibrium concentrations. The ζ-potential measurements show that the surface concentration of the charged surfactant stays nearly constant throughout the stabilizing pH range. In contrast, VSFSS shows that the neutral carboxylic acid form increasingly adsorbs to the surface with increased acidity. The spectral features of the headgroup vibrational modes confirm this behavior and go further to reveal additional molecular details of their adsorption. A significant hydrogen-bonding interaction occurs between the headgroups that, along with hydrophobic chain-chain interactions, assists in drawing more carboxylic acid surfactant to the interface. The charged surfactant provides the stabilizing force for these droplets, while the neutral surfactant introduces complexity to the interfacial structure as the pH is lowered. The results are significantly different than what has been found for the planar oil/water studies where stabilization of the interface is not a factor.

Assessing the Impact of Solvent Selection on Vibrational Sum-Frequency Scattering Spectroscopy Experiments.

The development of vibrational sum-frequency scattering (S-VSF) spectroscopy has opened the door to directly probing nanoparticle surfaces with an interfacial and chemical specificity that was previously reserved for planar interfacial systems. Despite its potential, challenges remain in the application of S-VSF spectroscopy beyond simplified chemical systems. One such challenge includes infrared absorption by an absorptive continuous phase, which will alter the spectral lineshapes within S-VSF spectra. In this study, we investigate how solvent vibrational modes manifest in S-VSF spectra of surfactant stabilized nanoemulsions and demonstrate how corrections for infrared absorption can recover the spectral features of interfacial solvent molecules. We also investigate infrared absorption for systems with the absorptive phase dispersed in a nonabsorptive continuous phase to show that infrared absorption, while reduced, will still impact the S-VSF spectra. These studies are then used to provide practical recommendations for anyone wishing to use S-VSF to study nanoparticle surfaces where absorptive solvents are present.

How Low Can You Go? Molecular Details of Low-Charge Nanoemulsion Surfaces.

Negative charge accumulation at aqueous-hydrophobic interfaces and its pH-dependent behavior are routinely ascribed to special adsorption properties of hydroxide ions. Mounting experimental and computational evidence, however, indicates that this negative charge accumulation is the result of surface-active impurities. If true, these impurities would obfuscate our fundamental understanding of the molecular structure and bonding environment at aqueous-hydrophobic interfaces. In this work, we describe the preparation and characterization of bare low-charge nanoemulsions (LCNEs), nanosized droplets of oil-absent emulsifiers. Electrophoretic mobility measurements of LCNE droplets in varying pH environments suggest that trace surface-adsorbed impurities are contributing to the lingering negative surface charge that leads to their marginal stability. We then use vibrational sum-frequency scattering spectroscopy to support this claim and to study the molecular structure and bonding environment of the interfacial aqueous and hydrophobic phases on both the LCNE surface and the surface of nanoemulsions with increasing amounts of adsorbed surfactants. For LCNE samples, our results show that interfacial water bonds more strongly to the oil phase on the droplet surface compared to similar planar interfaces. Interfacial oil molecules are found to orient mostly parallel to the bare droplet surface and reorganize upon surfactant adsorption. In summation, the results reported here provide a new look at the molecular structure and bonding of bare nanoemulsion surfaces and contribute to our evolving understanding of bare aqueous-hydrophobic interfaces.