This work presents molecular-level investigations of how wellcharacterized silica-supported phospholipid bilayers formed from either pure DOPC or a 9:1 mixture of DOPC:DOTAP interact with positively and negatively charged 4 nm gold metal nanoparticles at pH 7.4 and NaCl concentrations ranging from 0.001 to 0.1 M. Second harmonic generation (SHG) charge screening measurements indicate the supported bilayers carry a negative interfacial potential. Resonantly enhanced SHG measurements probing electronic transitions within the gold core of the nanoparticles show the particles interact irreversibly with the supported bilayers at a range of concentrations. At 0.1 M NaCl, surface coverages for the particles functionalized with the negatively charged ligand mercaptopropionic acid (MPA) or wrapped in the cationic polyelectrolyte poly(allylamine) hydrochloride (PAH) are estimated from a joint analysis of QCM-D, XPS, AFM, and ToF-SIMS to be roughly 1 × 10 7 and 1 × 10 11 particles cm −2 , respectively. Results from complementary SHG charge screening experiments point to the possibility that the surface coverage of the MPA-coated particles is more limited by interparticle Coulomb repulsion due to the charges within their hydrodynamic volumes than with the PAH-wrapped particles. Yet, SHG adsorption isotherms indicate that the interaction strength per particle is independent of ionic strength and particle coating, highlighting the importance of multivalent interactions. 1 H NMR spectra of the lipids within vesicles suspended in solution show little change upon interaction with either particle type but indicate loosening of the gold-bound PAH polymer wrapping upon attachment to the vesicles. The thermodynamic, spectroscopic, and electrostatic data presented here may serve to benchmark experimental and computational studies of nanoparticle attachment processes at the nano−bio interface.

Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.

Mechanistic insight into how polycations disrupt and cross cell membranes is needed for understanding and controlling polycation-membrane interactions, yet such information is surprisingly difficult to obtain at the molecular level. We use second harmonic and vibrational sum frequency generation spectroscopies along with quartz crystal microbalance with dissipation monitoring and computer simulations to quantify the interaction of poly(allylamine) hydrochloride (PAH) and its monomeric precursor allylamine hydrochloride (AH) with lipid bilayers. We find PAH adsorption to be reversible and nondisruptive to the bilayer under the conditions of our experiments. With an observed free adsorption energy of -52.7 ± 0.6 kJ/mol, PAH adsorption was found to be surprisingly less favorable relative to AH (-14.6 ± 0.4 kJ/mol) when considering a simple additive model. By experimentally quantifying the number of adsorbates and the average amount of charge carried by each adsorbate, we find that the PAH is associated with only 70% of the positive charges it could hold while the AH remains mostly charged while attached to the membrane. Simulations indicate that PAH pulls in condensed counterions from solution to avoid charge-repulsion along its backbone and with other PAH molecules to attach to, and completely cover, the bilayer surface. In addition, computations indicate that the amine groups shift their pK values due to the confined environment upon adsorption to the surface. Our results provide experimental constraints for theoretical calculations, which yield atomistic views of the structures that are formed when polycations interact with lipid membranes that will be important for predicting polycation-membrane interactions.

Mechanisms of corona formation around nanomaterials remain enigmatic. Here, we provide evidence for spontaneous lipid corona formation that engenders new particle properties without the need for active mixing upon attachment to stationary and suspended lipid bilayer membranes. The mechanism of lipid corona formation can be used to improve control over nano-bio interactions and to help understand why some nanomaterial-ligand combinations are detrimental to organisms but others are not. SUMMARYAlthough mixing nanoparticles with certain biological molecules can result in coronas that afford some control over how engineered nanomaterials interact with living systems, corona formation mechanisms remain enigmatic. Here, we report results from experiments and computer simulations that provide concrete lines of evidence for spontaneous lipid corona formation without active mixing upon attachment to stationary and suspended lipid bilayer membranes. Experiments show that polycation-wrapped particles disrupt the tails of zwitterionic lipids, increase bilayer fluidity, and leave the membrane with reduced z potentials. Computer simulations suggest that the contact ion pairing between the lipid head groups and the polycations' ammonium groups leads to the formation of stable, albeit fragmented, lipid bilayer coronas. The mechanistic insight regarding lipid corona formation can be used to improve control over nanobio interactions and to help understand why some nanomaterial-ligand combinations are detrimental to organisms but others are not.

Second harmonic generation (SHG) is useful for studying the properties of interfaces, including the surfaces of nanoparticles and the interaction of nanoparticles with biologically relevant surfaces. Gold nanoparticles at the biological membrane represent a particularly interesting system to be probed by SHG spectroscopy given the rich electronic structure of gold nanoparticles and the charged nature of the nano-bio interface. Here we describe the interplay between the resonant and nonresonant components of the second harmonic response as 4 and 14 nm spherical gold nanoparticles (AuNPs) wrapped in the cationic polyelectrolyte poly­(allylamine hydrochloride) (PAH) adsorb to negatively charged supported lipid bilayers. In contrast to the SHG response of 4 nm PAH-AuNPs, that we have shown previously to be dominated by resonance enhancement, the SHG response from the adsorption of the 14 nm PAH-AuNPs, with similar hydrodynamic diameters, to a 9:1 DOPC:DOTAP bilayer is dominated by the nonresonant, interfacial, potential-dependent component of the signal. We hypothesize that the difference in the SHG response is attributable to the differences in the number of PAH molecules associated with the particles and, therefore, differences in the number of positively charged ammonium groups associated with the 4 vs the 14 nm particles. For 14 nm PAH-AuNPs with larger hydrodynamic diameters, we determined two regimes in the adsorption behavior, one where the resonance enhancement from the gold core of the nanoparticle dominates the signal and a second where the nonresonant, interfacial, potential-dependent term dominates the signal. The results presented in this study provide insight into the interplay between resonant and nonresonant components of the second harmonic signal from the adsorption of charged AuNPs and are valuable for future studies with other functionalized particles and lipid systems by SHG.