Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein.

The rapid screening of protein binding affinity for poly- and perfluoroalkyl substances (PFAS) benefits risk assessment and fate and transport modelling. PFAS are known to bioaccumulate in livestock through contaminated food and water. One excretion pathway is through milk, which may be facilitated by binding to milk proteins such as bovine serum albumin (BSA). We report a label-free differential scanning fluorimetry approach to determine PFAS-BSA binding over a broad temperature range. This method utilizes the tryptophan residue within the protein binding pocket as an intrinsic fluorophore, eliminating the need for fluorophore labels that may influence binding. BSA association constants were determined by (a) an equilibrium-based model at the melting temperature of BSA and (b) the Hill adsorption model to account for temperature dependent binding and binding cooperativity. Differences in binding between PFAS and fatty acid analogs revealed that a combination of size and hydrophobicity drives PFAS binding.

Electric Potential Induced Prevention and Removal of an Algal Biofoulant from Planar SERS Substrates.

Ulva zoospores are widespread marine macroalgae and a common organism found in biofouling communities due to their strong adhesive properties and quick settlement times. Using Ulva as a model organism, a strategy is presented where direct-current (DC) electric potentials are applied in conjunction with surface-enhanced Raman spectroscopy (SERS) to characterize, remove, and prevent Ulva from forming a biofilm on gold-capped nanopillar SERS substrates. Experiments were conducted within a poly(tetrafluoroethylene) (PTFE) flow channel device where the SERS substrates were used as an electrode. Ulva density, determined in situ by SERS and ex situ by electron and fluorescence microscopy, decreased under successively increasing low negative potentials up to -1.0 V. The presence of damaged Ulva suggests that the applied potential led to spore rupture. At the highest negative applied potential (-1.0 V), microparticles containing copper, which is known for its antimicrobial properties, were associated with Ulva on the SERS substrate and the lowest Ulva density was observed. These findings indicate that (1) SERS can be employed to study biofilm formation on nanostructured metal surfaces and (2) applying low-voltage electric potentials may be used to control Ulva biofouling on SERS marine sensors.

Organic Anion Detection with Functionalized SERS Substrates via Coupled Electrokinetic Preconcentration, Analyte Capture, and Charge Transfer.

Detecting ultralow concentrations of anionic analytes in solution by surface-enhanced Raman spectroscopy (SERS) remains challenging due to their low affinity for SERS substrates. Two strategies were examined to enable in situ, liquid phase detection using 5(6)-carboxyfluorescein (5(6)-FAM) as a model analyte: functionalization of a gold nanopillar substrate with cationic cysteamine self-assembled monolayer (CA-SAM) and electrokinetic preconcentration (EP-SERS) with potentials ranging from 0 to +500 mV. The CA-SAM did not enable detection without an applied field, likely due to insufficient accumulation of 5(6)-FAM on the substrate surface limited by passive diffusion. 5(6)-FAM could only be reliably detected with an applied electric field with the charged molecules driven by electroconvection to the substrate surface and the SERS intensity following the Langmuir adsorption model. The obtained limits of detection (LODs) with an applied field were 97.5 and 6.4 nM on bare and CA-SAM substrates, respectively. For the CA-SAM substrates, both the ligand and analyte displayed an ∼15-fold signal enhancement with an applied field, revealing an additional enhancement due to charge-transfer resonance taking place between the metal and 5(6)-FAM that improved the LOD by an order of magnitude.

PFAS fluidize synthetic and bacterial lipid monolayers based on hydrophobicity and lipid charge.

Poly- and Perfluoroalkyl substances (PFASs) are pollutants of emerging concern that persist in nature and pose environmental health and safety risks. PFAS disrupt biological membranes resulting in cellular inhibition, but the mechanism of disruption and the role of lipid composition remain unclear. We examine the role of phospholipid saturation and headgroup charge on the interactions between PFASs and phospholipid monolayers comprised of synthetic phosphocholine (PC) and phosphoglycerol (PG) lipids and prepared from bacteria membrane extracts rich in PG lipids from an environmentally relevant marine bacterium Alcanivorax borkumensis. When deposited on a buffered subphase containing PFAS, PFAS mixed within and fluidized zwitterionic and net-anionic monolayers leading to increases in monolayer compressibility that were driven by a combination of PFAS hydrophobicity and monolayer charge density. Differences in the monolayer response using saturated or unsaturated lipids are attributed to the ability of the unsaturated lipids to accommodate PFAS within 'void space' arising from the bent lipid tails. Similar fluidization and compressibility behavior were also observed in A. borkumensis lipid monolayers. This work provides new insight into PFAS partitioning into bacterial membranes and the effect PFAS have on the physicomechanical properties of zwitterionic and charged lipid monolayers.

Replacement per- and polyfluoroalkyl substances (PFAS) are potent modulators of lipogenic and drug metabolizing gene expression signatures in primary human hepatocytes.

Per- and polyfluoroalkyl substances (PFAS) are a class of environmental toxicants, and some, such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), have been associated with hepatic steatosis in rodents and monkeys. It was hypothesized that perfluorosulfonic acids (C4, 6, 8), perfluorocarboxylic acids (C4-14), perfluoro(2-methyl-3-oxahexanoic) acid (HFPO-DA), 1H, 1H, 2H, 2H-perfluorooctanesulfonic acid (6:2 FTS) along with 3 PFOS precursors could induce expression of lipid metabolism genes and lipid deposition in human hepatocytes. Five-donor pooled cryopreserved human hepatocytes were cultured and treated with 0.1% DMSO vehicle or various PFAS (0.25 to 25 µM) in media. After a 48-h treatment, mRNA transcripts related to lipid transport, metabolism, and synthesis were measured using a Quantigene Plex assay. After 72-h treatments, hepatocytes were stained with Nile Red dye to quantify intracellular lipids. Overall, PFAS were transcriptionally active at 25 µM. In this model, lipid accumulation was not observed with C8-C12 treatments. Shorter chain PFAS (C4-C5), 6:2 FTS, and PFOS precursor, metFOSA, induced significant liver lipid accumulation, and gene activation at lower concentrations than legacy PFAS. In summary short chain PFAS and other alternative PFAS were more potent gene inducers, and potential health effects of replacement PFAS should be critically evaluated in humans.

Transformation of Lipid Vesicles into Micelles by Adding Nonionic Surfactants: Elucidating the Structural Pathway and the Intermediate Structures.

The phospholipid lecithin (L) and the nonionic surfactant Tween 80 (T) are used together in various contexts, including in drug delivery and oil spill remediation. There is hence a need to elucidate the nanostructures in LT mixtures, which is the focus of this paper. We study these mixtures using cryogenic transmission electron microscopy (cryo-TEM), coupled with dynamic light scattering and small-angle neutron scattering. As the concentration of Tween 80 is increased, the vesicles formed by lecithin are transformed into spherical micelles. We identify bicelles (i.e., disc-like micelles) as well as cylindrical micelles as the key stable nanostructures formed at intermediate L/T ratios. The bicelles have diameters ∼13-26 nm, and the bicelle size decreases as the Tween 80 content increases. We propose that the lecithin lipids form the body of the discs, while the Tween 80 surfactants occupy the rims. This hypothesis is consistent with geometric arguments because lecithin is double-tailed and favors minimal curvature, whereas the single-tailed Tween 80 molecules prefer curved interfaces. In the case of cylindrical micelles, cryo-TEM reveals that the micelles are short (length < 22 nm) and flexible. We are able to directly visualize the microstructure of the aggregates formed by lecithin-Tween 80 mixtures, thereby enhancing the understanding of morphological changes in the lecithin-Tween 80 system.

Critical new insights into the binding of poly- and perfluoroalkyl substances (PFAS) to albumin protein.

With an increasing number of health-related impacts of per- and polyfluoroalkyl substances (PFAS) being reported, there is a pressing need to understand PFAS transport within both the human body and the environment. As proteins can serve as a primary transport mechanism for PFAS, understanding PFAS binding to proteins is essential for predictive physiological models where accurate values of protein binding constants are vital. In this work we present a critical analysis of three common models for analyzing PFAS binding to bovine serum albumin (BSA) based on fluorescence quenching: the Stern-Volmer model, the modified Stern-Volmer model, and the Hill equation. The PFAS examined include perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorobutanesulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanesulfonic acid (PFOS), and the replacement compound 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate (HFPO-DA or GenX). While all three models capture the general effects of hydrophobicity and steric limitations to PFAS binding, the Hill equation highlighted a unique relationship between binding cooperativity and the number of fluorinated carbons, with PFOA exhibiting the greatest binding cooperativity. The significance of steric limitations was confirmed by comparing results obtained by fluorescence quenching, which is an indirect method based on specific binding, to those obtained by equilibrium dialysis where PFAS binding directly correlated with traditional measures of hydrophobicity. Finally, the binding constants were correlated with PFAS physicochemical properties where van der Waals volume best described the steric limitations observed by fluorescence quenching.

Using Microemulsion Phase Behavior as a Predictive Model for Lecithin-Tween 80 Marine Oil Dispersant Effectiveness.

Marine oil dispersants typically contain blends of surfactants dissolved in solvents. When introduced to the crude oil-seawater interface, dispersants facilitate the breakup of crude oil into droplets that can disperse in the water column. Recently, questions about the environmental persistence and toxicity of commercial dispersants have led to the development of "greener" dispersants consisting solely of food-grade surfactants such as l-α-phosphatidylcholine (lecithin, L) and polyoxyethylenated sorbitan monooleate (Tween 80, T). Individually, neither L nor T is effective at dispersing crude oil, but mixtures of the two (LT blends) work synergistically to ensure effective dispersion. The reasons for this synergy remain unexplained. More broadly, an unresolved challenge is to be able to predict whether a given surfactant (or a blend) can serve as an effective dispersant. Herein, we investigate whether the LT dispersant effectiveness can be correlated with thermodynamic phase behavior in model systems. Specifically, we study ternary "DOW" systems comprising LT dispersant (D) + a model oil (hexadecane, O) + synthetic seawater (W), with the D formulation being systematically varied (across 0:100, 20:80, 40:60, 60:40, 80:20, and 100:0 L:T weight ratios). We find that the most effective LT dispersants (60:40 and 80:20 L:T) induce broad Winsor III microemulsion regions in the DOW phase diagrams (Winsor III implies that the microemulsion coexists with aqueous and oil phases). This correlation is generally consistent with expectations from hydrophilic-lipophilic deviation (HLD) calculations, but specific exceptions are seen. This study then outlines a protocol that allows the phase behavior to be observed on short time scales (ca. hours) and provides a set of guidelines to interpret the results. The complementary use of HLD calculations and the outlined fast protocol are expected to be used as a predictive model for effective dispersant blends, providing a tool to guide the efficient formulation of future marine oil dispersants.

In situ SERS detection of dissolved nitrate on hydrated gold substrates.

The accurate and fast measurement of nitrate in seawater is important for monitoring and controlling water quality to prevent ecologic and economic disasters. In this work we show that the in situ detection of nitrate in aqueous solution is feasible at nanomolar concentrations through surface enhanced Raman spectroscopy (SERS) using native nanostructured gold substrates without surface functionalization. Spectra were analyzed as collected or after standard normal variate (SNV) normalization, which was shown through Principal Component Analysis (PCA) to reduce spectral variations between sample sets and improve Langmuir adsorption model fits. An additional normalization approach based on the substrate silicon template showed that silicon provided an internal standard that accounted for the spectral variance without the need for SNV normalization. Nitrate adsorption was well-described by the Langmuir adsorption model, consistent with an adsorbed monolayer, and a limit of detection of 64 nM nitrate was obtained in ultrapure water, representing environmentally relevant concentrations. Free energy calculations based on the Langmuir adsorption constants, approximating equilibrium adsorption constants, and calculated self-energy arising from image charge, accounting for electrostatic interactions with a polarizable nanostructured substrate, suggest that nitrate adsorption was partially driven by an entropy gain presumably due to dehydration of the gold substrate and/or nitrate ion. This work is being extended to determine if similar statistical and normalization methods can be applied to nitrate detection in complex natural waters where non-target ions and molecules are expected to interfere.

Dominant entropic binding of perfluoroalkyl substances (PFASs) to albumin protein revealed by 19F NMR.

Mechanistic insight into protein binding by poly- and perfluoroalkyl substances (PFASs) is critical to understanding how PFASs distribute and accumulate within the body and to developing predictive models within and across classes of PFASs. Fluorine nuclear magnetic resonance spectroscopy (19F NMR) has proven to be a powerful, yet underutilized tool to study PFAS binding; chemical shifts of each fluorine group reflect the local environment along the length of the PFAS molecule. Using bovine serum albumin (BSA), we report dissociation constants, Kd, for four common PFASs well below reported critical micelle concentrations (CMCs) - perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexanesulfonic acid (PFHxS), and perfluorooctanesulfonic acid (PFOS) - as a function of temperature in phosphate buffered saline. Kd values were determined based on the difluoroethyl group adjacent to the anionic headgroups and the terminal trifluoromethyl groups. Our results indicate that the hydrophobic tails exhibit greater binding affinity relative to the headgroup, and that the binding affinities are generally consistent with previous results showing that greater PFAS hydrophobicity leads to greater protein binding. However, the binding mechanism was dominated by entropic hydrophobic interactions attributed to desolvation of the PFAS tails within the hydrophobic cavities of the protein and on the surface of the protein. In addition, PFNA appears to form hemimicelles on the protein surfaces below reported CMC values. This work provides a renewed approach to utilizing 19F NMR for PFAS-protein binding studies and a new perspective on the role of solvent entropy.

Radiofrequency and Near-Infrared Responsive Core-Shell Nanostructures Using Layersome Templates for Cancer Treatment.

We report a multifunctional nanotherapeutic platform based on liposomes loaded with drug and iron oxide nanoparticles (IONs) coated with a gold nanoshell synthesized using a polyelectrolyte (layersome) soft templating technique. IONs and gold nanoshells were used to provide combined hyperthermia and triggered drug release via radio frequency (RF) or near-infrared (NIR) stimulation. IONs and the anticancer drug doxorubicin (DOX) were coencapsulated inside liposomes composed of zwitterionic phosphatidylcholine, anionic phosphatidylglycerol, and cholesterol lipids. Coating the magneto-liposomes with positively charged poly-l-lysine enriched the interface with gold anions to form a dense gold nanoshell and protected the structure against deformation and DOX cargo release during shell formation. After modification with thiol-terminated polyethylene glycol, intracellular delivery and release of DOX from the nanostructures was examined in A549 human lung cancer cells. The nanostructures retained their DOX cargo and remained in the cytosol after cellular uptake. Only when triggered by RF or NIR stimuli did the nanostructures release DOX, which then entered the cell nucleus. Compared to the single photothermal therapy or radio frequency treatment, the carriers with combined DOX and RF or NIR stimulation displayed higher therapeutic effect on A549 cells.

Does the Solvent in a Dispersant Impact the Efficiency of Crude-Oil Dispersion?

Dispersants, used in the mitigation of oil spills, are mixtures of amphiphilic molecules (surfactants) dissolved in a solvent. The recent large-scale use of dispersants has raised environmental concerns regarding the safety of these materials. In response to these concerns, our lab has developed a class of eco-friendly dispersants based on blends of the food-grade surfactants, soy lecithin (L) and Tween 80 (T), in a solvent. We have shown that these "L/T dispersants" are very efficient at dispersing crude oil into seawater. The solvent for dispersants is usually selected based on factors like toxicity, volatility, or viscosity of the overall mixture. However, with regard to the dispersion efficiency of crude oil, the solvent is considered to play a negligible role. In this paper, we re-examine the role of solvent in the L/T system and show that it can actually have a significant impact on the dispersion efficiency. That is, the dispersion efficiency can be altered from poor to excellent simply by varying the solvent while keeping the same blend of surfactants. We devise a systematic procedure for selecting the optimal solvents by utilizing Hansen solubility parameters. The optimal solvents are shown to have a high affinity for crude oil and limited hydrophilicity. Our analysis further enables us to identify solvents that combine high dispersion efficiency, good solubility of the L/T surfactants, a low toxicity profile, and a high flash point.

Phospholipid Bilayer Softening Due to Hydrophobic Gold Nanoparticle Inclusions.

Liposome-nanoparticle assemblies (LNAs) are vital in the context of novel targeted drug-delivery systems, in addition to investigating nanoparticle-lipid bilayer interactions. Quantifying membrane structural properties and dynamics in presence of nanoparticle inclusions provides a simple model to elucidate nanoparticle effects on membrane biophysical properties. We present experimental evidences of bilayer softening due to small hydrophobic gold nanoparticle inclusions. LNA structure has been investigated by a combination of cryo-transmission electron microscopy, dynamic light scattering, and small-angle neutron scattering. Neutron spin echo spectroscopy demonstrated a remarkable ∼15% bending modulus decrease for LNAs relative to pure liposomes. Clear dependence of bending modulus on gold nanoparticle diameter and concentration was observed from our observations. Our findings point toward local bilayer fluidization by nanoparticle inclusions leading to an overall bilayer softening. These findings add valuable information to liposomal drug-delivery vehicle design and membrane biophysics research.

Attachment of Alcanivorax borkumensis to Hexadecane-In-Artificial Sea Water Emulsion Droplets.

Alcanivorax borkumensis (AB) is a marine bacterium that dominates bacterial communities around many oil spills because it enzymatically degrades the oil while using it as a nutrient source. Several dispersants have been used to produce oil-in-water emulsions following a spill. Compared to surface slicks, the additional oil-water surface area produced by emulsification provides greater access to the oil and accelerates its degradation. We deliberately cultured AB cells using hexadecane as the only nutrient source. We then examined the first critical step of the biodegradation process, the attachment of these AB cells to hexadecane-water interfaces, using fluorescence microscopy and cryogenic scanning electron microscopy. The hexadecane-in-artificial sea water (ASW) emulsions were produced by gentle shaking and were stabilized either by AB alone, by Corexit 9500, by Tween 20, or by carbon black particles. When no dispersants were used, AB stabilizes the emulsion, and bacterial cells attach to the hexadecane droplets within the first 3 days. When Corexit 9500 was used as the dispersant, AB did not attach to the hexadecane droplets over 3 days, and many AB cells in the aqueous phase appeared dead. Only limited attachment was observed after 7 days. No AB attachment was observed over 3 days when Tween 20 was used as the dispersant. However, the bacteria used Tween 20 in the ASW as a nutrient. Large amounts of AB attached to carbon black stabilized hexadecane droplets within 3 days. An analysis that accounts for van der Waals and electrostatic interactions is unable to predict all of these observations, indicating that the attachment of AB to the hexadecane is a complex phenomenon that goes beyond simple physiochemical effects. While these experiments do not mimic conditions in the open ocean where the large amount of water dilutes any emulsion stabilizer, they provide important insights on bacteria adhesion to oil, a critical step in the oil degradation process following a marine spill.

Surface Activity of Poly(ethylene glycol)-Coated Silver Nanoparticles in the Presence of a Lipid Monolayer.

We have investigated the surface activity of poly(ethylene glycol) (PEG)-coated silver nanoparticles (Ag-PEG) in the presence or absence of lipid monolayers comprised of monounsaturated dioleoylphosphocholine and dioleoylphosphoglycerol (DOPC/DOPG; 1:1 mol ratio). Dynamic measurements of surface pressure demonstrated that Ag-PEG were surface-active at the air/water interface. Surface excess concentrations suggested that at high Ag-PEG subphase concentrations, Ag-PEG assembled as densely packed monolayers in the presence and absence of a lipid monolayer. The presence of a lipid monolayer led to only a slight decrease in the excess surface concentration of Ag-PEG. Surface pressure-area isotherms showed that in the absence of lipids Ag-PEG increased the surface pressure up to 45 mN m-1 upon compression before the Ag-PEG surface layer collapsed. Our results suggest that surface activity of Ag-PEG was due to hydrophobic interactions imparted by a combination of the amphiphilic polymer coating and the hydrophobic dodecanethiol ligands bound to the Ag-PEG surface. With lipid present, Ag-PEG + lipid surface pressure-area (π-A) isotherms reflected Ag-PEG incorporation within the lipid monolayers. At high Ag-PEG concentrations, the π-A isotherms of the Ag-PEG + lipid films closely resembled that of Ag-PEG alone with a minimal contribution from the lipids present. Analysis of the subphase silver (Ag) and phosphorus (P) concentrations revealed that most of the adsorbed material remained at the air/lipid/water interface and was not forced into the aqueous subphase upon compression, confirming the presence of a composite Ag-PEG + lipid film. While interactions between "water-soluble" nanoparticles and lipids are often considered to be dominated by electrostatic interactions, these results provide further evidence that the amphiphilic character of a nanoparticle coating can also play a significant role.

Tuning the Multifunctionality of Iron Oxide Nanoparticles Using Self-Assembled Mixed Lipid Layers.

We present an approach to tuning the multifunctionality of iron oxide nanoparticles (IONs) using mixed self-assembled monolayers of cationic lipid and anionic polyethylene glycol (PEG) lipid. By forming stable, monodispersed lipid-coated IONs (L-IONs) through a solvent-exchange technique, we were able to demonstrate the relationship between surface charge, the magnetic transverse relaxivity (r2 from T2-weighted images), and the binding capacity of small interfering ribonucleic acids (siRNAs) as a function of the cationic-to-anionic (PEG) lipid ratio. These properties were controlled by the cationic charge and the PEG conformation; relaxivity and siRNA binding could be varied in the mushroom and brush regimes but not at high brush densities. In vitro results combining cell viability, uptake, and transfection efficiency using HeLa cells suggest that the functional physicochemical and biological properties of L-IONs may be best achieved using catanionic lipid coatings near equimolar ratios of cationic to anionic PEG-lipids.

Effects of Membrane Defects and Polymer Hydrophobicity on Networking Kinetics of Vesicles.

The kinetics of clustering unilamellar vesicles induced by inverse Pluronics [poly(propylene oxide)m-poly(ethylene oxide)n-poly(propylene oxide)m, POm-EOn-POm] was investigated via experiments and molecular dynamic simulations. Two important factors for controlling the networking kinetics are the membrane defects, presumably located at the interfacial region between two lipid domains induced by acyl chain mismatch, and the polymer hydrophobicity. As expected, the clustering rate increases significantly with increasing bilayer defects on the membrane where the insertion of PPO is likely to take place because of the reduced energy barrier for the insertion of PO. The hydrophobic interaction between the PO blocks and membranes with the defects region dictates the "anchoring" kinetics, which is controlled by the association-dissociation of PO with the lipid membrane. As a result, the dependence of clustering rate on polymer concentration is strongly influenced by the hydrophobicity of the PO blocks. Nevertheless, longer PO blocks show stronger association with the membrane, resulting in faster consumption of the "active" sites made of these defect regions (causing mostly "invalid" insertions) with increasing polymer concentration, hence inhibiting the formation of large networking clusters, while shorter PO blocks undergo more frequent association with/dissociation from the defects, allowing continuous formation of larger clusters with increasing polymer concentration. This study provides important insights into how the organization and dynamics of a biomembrane influence its interaction with foreign amphiphilic molecules.

Near-Infrared Responsive Gold-Layersome Nanoshells.

Anionic liposomes coated with cationic polyelectrolyte poly-l-lysine (PLL), or layersomes, were used as soft, self-assembled templates for synthesizing gold nanoshells that absorb near-infrared radiation. The gold nanoshells were formed using two techniques: (a) direct reduction of tetrachloroauric acid on the layersomes and (b) the reduction of a tetrachloroauric acid/potassium carbonate "growth" solution on nanosized gold seeds bound to the surface of layersomes. The resulting structures were characterized by transmission and scanning electron microscopy and visible-near-infrared spectroscopy. Direct reduction produced discrete gold nanoparticles on the layersomes. The slower reduction from the growth solution on the gold seeds resulted in more complete shells. The absorption spectra of these suspensions were sensitive to the synthesis method. The morphology of the gold shells was tuned for absorption at biologically safe and tissue-penetrating NIR wavelengths, and laser irradiation at 810 nm produced significant heat. These gold-layersome nanoshells have the potential to be used for photothermal therapy, photothermally mediated drug delivery, and biomedical imaging.

Hydrophobic Nanoparticles Modify the Thermal Release Behavior of Liposomes.

Understanding the effect of embedded nanoparticles on the characteristics and behavior of lipid bilayers is critical to the development of lipid-nanoparticle assemblies (LNAs) for biomedical applications. In this work we investigate the effect of hydrophobic nanoparticle size and concentration on liposomal thermal release behavior. Decorated LNAs (D-LNAs) were formed by embedding 2 nm (GNP2) and 4 nm (GNP4) dodecanethiol-capped gold nanoparticles into DPPC liposomes at lipid to nanoparticle ratios (L:N) of 25,000:1, 10,000:1, and 5,000:1. D-LNA structure was investigated by cryogenic transmission electron microscopy, and lipid bilayer permeability and phase behavior were investigated based on the leakage of a model drug, carboxyfluorescein, and by differential scanning calorimetry, respectively. The presence of bilayer nanoparticles caused changes in the lipid bilayer release and phase behavior compared to pure lipid controls at very low nanoparticle to bilayer volume fractions (0.3%-4.6%). Arrhenius plots of the thermal leakage show that GNP2 led to greater increases in the leakage energy barrier compared to GNP4, consistent with GNP4 causing greater bilayer disruption due to their size relative to the bilayer thickness. Embedding hydrophobic nanoparticles as permeability modifiers is a unique approach to controlling liposomal leakage based on nanoparticle size and concentration.

Patchy Layersomes Formed by Layer-by-Layer Coating of Liposomes with Strong Biopolyelectrolytes.

Layer-by-layer deposition of polyelectrolytes (PEs) onto self-assembled liposomes represents an alternative to PE deposition on solid particles for the formation of hollow nanoscale capsules. This work examines how competition between PE-liposome and inter-PE interactions drives the structure and colloidal stability of layersomes. Unlike solid particles, liposomes respond to adsorbed material through lipid reorganization and changes in size and shape. This responsive nature could yield new types of layered PE structures. We show that sequential deposition of strong biopolyelectrolytes, dextran sulfate-sodium salt (DxS-) and poly-l-arginine (PA+), onto cationic liposomes in water yields the expected charge inversion behavior commonly observed for dispersed particles. However, cryogenic transmission electron microscopy results show that the layersomes formed and their PE coatings were heterogeneous. The PE coatings contained PE complexes (PECs) that were formed when an even number of layers (2 or 4) was deposited. PECs remained attached as patches that were spatially distinguishable. This behavior was confirmed through fluorescence anisotropy measurements of liposome bilayer fluidity, where PA+ counteracted the ordering effects of DxS- on the lipid bilayer through charge neutralization and local PEC desorption. With increased charge screening, DxS- desorbed from the layersomes, whereas the patchy layersomes terminating in PA+ retained their PE coatings and colloidal stability at higher salt concentrations. To our knowledge, this is the first time such patchy layersome structures have been observed.