Understanding the Retention of Vaping Additives in the Lungs: Model Lung Surfactant Membrane Perturbation by Vitamin E and Vitamin E Acetate.

Deviations from the normal physicochemical and functional properties of pulmonary surfactants are associated with the incidence of lung injury and other respiratory disorders. This study aims to evaluate the alteration of the 2D molecular organization and morphology of pulmonary surfactant model membranes by the electronic cigarette additives α-tocopherol (vitamin E) and α-tocopherol acetate (vitamin E acetate), which have been associated with lung injury, termed e-cigarette or vaping-use-associated lung injury (EVALI). The model membranes used contained a 7:3 molar ratio of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to which α-tocopherol and α-tocopherol acetate were added to form mixtures of up to 20 mol % additive. The properties of the neat tocopherol additives and DPPC/POPG (7:3) mixtures with increasing molar proportions of additive were evaluated by surface pressure-area isotherms, excess area calculations, Brewster angle microscopy, grazing incidence X-ray diffraction, X-ray reflectivity, and atomic force microscopy. The addition of either additive alters the essential phase balance of the model pulmonary surfactant membrane by generating a greater proportion of the fluid phase. Despite this net fluidization, both tocopherol additives have space-filling effects on the liquid-expanded and condensed phases, yielding negative excess areas in the liquid-expanded phase and reduced tilt angles in the condensed phase. Both tocopherol additives alter the stability of the fluid phase, pushing the eventual collapse of this phase to higher surface pressures than the model membrane in the absence of an additive.

Impact of Pollutant Ozone on the Biophysical Properties of Tear Film Lipid Layer Model Membranes.

Ozone exposure from environmental smog has been implicated as a risk factor for developing dry eye disease (DED). The tear film lipid layer (TFLL), which is the outermost layer of the tear film and responsible for surface tension reduction while blinking, is in direct contact with the environment and serves as the first line of defense against external aggressors such as environmental pollution. The impact of exposure to ozone on the biophysical properties of three TFLL model membranes was investigated. These model membranes include a binary mixture of cholesteryl oleate (CO) and L-α-phosphatidylcholine (egg PC), a ternary mixture of CO, glyceryl trioleate (GT) and PC, as well as a quaternary mixture of CO, GT, a mixture of free fatty acids palmitic acid and stearic acid (FFAs) and PC. Biophysical impacts were evaluated as changes to the surface activity, respreadability, morphology and viscoelastic properties of the films. Expansion to higher molecular areas was observed in all the TFLL model membrane films which is attributable to the accommodation of the cleaved chains in the film. Significant morphological changes were observed, namely fluidization and the disruption of the phase transition behaviour of GT, and multilayer formation of CO. This fluidization reduces the hysteresis loops for the model membranes. On the other hand, the viscoelastic properties of the films exhibited differential impacts from ozone exposure as a function of composition. These findings are correlated to chemical changes to the lipids determined using ESI-MS.

A biophysical study of tear film lipid layer model membranes.

The tear film lipid layer (TFLL), the final layer of the human tear film is responsible for surface tension reduction while blinking, water evaporation retardation and maintaining the stability of the tear film. The study of the composition-structure-function relationship of TFLL is paramount, as a compromised structure of TFLL leads to the emergence of dry eye disease (DED) which is one the most prevalent ophthalmic surface diseases of the modern world, associated with chronic pain and reduced visual capability. In this model membrane study, a systematic approach is used to study the biophysical properties of TFLL model membranes as a function of composition. Three mixed-lipid model membranes are studied along with their individual components comprising cholesteryl oleate (CO), glyceryl trioleate (GT), L-α-phosphatidylcholine (egg PC) and a free fatty acid mixture. The models become progressively more complex from binary to quaternary mixtures, allowing the role of each individual lipid to be derived. Langmuir balance, Brewster Angle Microscopy (BAM) and Profile Analysis Tensiometer (PAT) are used to study the surface activity and compression-expansion cycles, morphology, and rheological behaviour of the model membranes, respectively. Evidence of multilayering is observed with inclusion of CO and a reversible collapse is associated with the GT phase transition. An initially more coherent film is observed due to the addition of polar PC. Notably, these individual behaviours are retained in the mixed films and suggest a possible role for each physiological component of TFLL.

Opposites Attract: Electrostatically Driven Loading of Antimicrobial Peptides into Phytoglycogen Nanocarriers.

Antimicrobial peptides, such as GL13K, have a high binding selectivity toward bacterial membranes, while not affecting healthy mammalian cells at therapeutic concentrations. However, delivery of these peptides is challenging since they are susceptible to proteolytic hydrolysis and exhibit poor cellular uptake. A protective nanocarrier is thus proposed to overcome these obstacles. We investigate the potential to employ biodegradable phytoglycogen nanoparticles as carriers for GL13K using a simple loading protocol based on electrostatic association rather than chemical conjugation, eliminating the need for control of chemical cleavage for release of the peptide in situ. Both the native (quasi-neutral) and carboxymethylated (anionic) phytoglycogen were evaluated for their colloidal stability, loading capacity, and release characteristics. We show that the anionic nanophytoglycogen carries a greater cationic GL13K load and exhibits slower release kinetics than native nanophytoglycogen. Isotope exchange measurements demonstrate that the antimicrobial peptide is entrapped in the pores of the dendritic-like macromolecule, which should provide the necessary protection for delivery. Importantly, the nanoformulations are active against a Pseudomonas aeruginosa clinical isolate at concentrations comparable to those of the free peptide and representative, small molecule antibiotics. The colloidal nanocarrier preserves peptide stability and antimicrobial activity, even after long periods of storage (at least 8 months).

Mechanisms of hypericin incorporation to explain the photooxidation outcomes in phospholipid biomembrane models.

Cell membranes are the first barriers for drug binding and key for the action of photosensitizers (PS). Herein, we report on the incorporation of the PS hypericin into Langmuir monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) to represent eukaryotic cell membranes, and 1,2-dioleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DOPG) to mimic bacterial membranes. Surface pressure (π) vs mean molecular area (Å) isotherms showed a high degree of interaction (binding, penetration and relative solubilization) of hypericin into DPPC and DOPC monolayers. On the other hand, electrostatic repulsions govern the interactions with DOPG and DOPS, favoring hypericin self-aggregation, as visualized by Brewster angle microscopy (BAM). Indeed, the larger domains in BAM were consistent with the greater expansion of DOPG monolayers with incorporated hypericin, owing to stronger electrostatic repulsions. In contrast to DPPC, light-irradiation of DOPC monolayers containing hypericin induced loss of material due to hydrocarbon chain cleavage triggered by contact-dependent reactions between triplet excited state of hypericin and chain unsaturations. The mild effects noted for both irradiated DOPS and DOPG monolayers are attributed to hypericin self-aggregation, which may have decreased the singlet oxygen quantum yield (Φ1O2) via self-quenching, despite the increased instability induced in the monolayers.

Are Plant-Based Carbohydrate Nanoparticles Safe for Inhalation? Investigating Their Interactions with the Pulmonary Surfactant Using Langmuir Monolayers.

Nanoparticle carriers show promise for drug delivery, including by inhalation, where the first barrier for uptake in the lungs is the monolayer pulmonary surfactant membrane that coats the air/alveoli interface and is critical to breathing. It is imperative to establish the fate of potential nanocarriers and their effects on the biophysical properties of the pulmonary surfactant. To this end, the impact of the nanoparticle surface charge on the lateral organization, thickness, and recompressibility of Langmuir monolayers of model phospholipid-only and phospholipid-protein mixtures was investigated using native and modified forms of nanophytoglycogen, a carbohydrate-based dendritic polymer extracted from corn as monodisperse nanoparticles. We show that the native (quasi-neutral) and anionic nanophytoglycogens have little impact on the phase behavior and film properties. By contrast, cationic nanophytoglycogen alters the film morphology and increases the hysteresis associated with the work of breathing due to its electrostatic interaction with the anionic phospholipids in the model systems. These findings specifically highlight the importance of surface charge as a selection criterion for inhaled nanoformulations.

Interactions between the Cell Membrane Repair Protein S100A10 and Phospholipid Monolayers and Bilayers.

Protein S100A10 participates in different cellular mechanisms and has different functions, especially at the membrane. Among those, it forms a ternary complex with annexin A2 and the C-terminal of AHNAK and then joins the dysferlin membrane repair complex. Together, they act as a platform enabling membrane repair. Both AHNAK and annexin A2 have been shown to have membrane binding properties. However, the membrane binding abilities of S100A10 are not clear. In this paper, we aimed to study the membrane binding of S100A10 in order to better understand its role in the cell membrane repair process. S100A10 was overexpressed by E. coli and purified by affinity chromatography. Using a Langmuir monolayer as a model membrane, the binding parameters and ellipsometric angles of the purified S100A10 were measured using surface tensiometry and ellipsometry, respectively. Phosphorus-31 solid-state nuclear magnetic resonance spectroscopy was also used to study the interaction of S100A10 with lipid bilayers. In the presence of a lipid monolayer, S100A10 preferentially interacts with unsaturated phospholipids. In addition, its behavior in the presence of a bilayer model suggests that S100A10 interacts more with the negatively charged polar head groups than the zwitterionic ones. This work offers new insights on the binding of S100A10 to different phospholipids and advances our understanding of the parameters influencing its membrane behavior.

Thermal properties of lipid bilayers derived from the transient heating regime of upconverting nanoparticles.

Heat transfer and thermal properties at the nanoscale can be challenging to obtain experimentally. These are potentially relevant for understanding thermoregulation in cells. Experimental data from the transient heating regime in conjunction with a model based on the energy conservation enable the determination of the specific heat capacities for all components of a nanoconstruct, namely an upconverting nanoparticle and its conformal lipid bilayer coating. This approach benefits from a very simple, cost-effective and non-invasive optical setup to measure the thermal parameters at the nanoscale. The time-dependent model developed herein lays the foundation to describe the dynamics of heat transfer at the nanoscale and were used to understand the heat dissipation by lipid bilayers.

The antibacterial activity of p-tert-butylcalix[6]arene and its effect on a membrane model: molecular dynamics and Langmuir film studies.

The antibacterial activity of a calixarene derivative, p-tert-butylcalix[6]arene (Calix6), was assessed and was shown not to inhibit the growth of E. coli, S. aureus and B. subtilis bacteria. With the aim of gaining more insights into the absence of antibacterial activity of Calix6, the interaction of this derivative with DPPG, a bacterial cell membrane lipid, was studied. Langmuir monolayers were used as the model membrane. Pure DPPG and pure Calix6 monolayers, as well as binary DPPG:Calix6 mixtures were studied using surface pressure measurements, compressional modulus, Brewster angle and fluorescence microscopies, ellipsometry, polarization-modulation infrared reflection absorption spectroscopy and molecular dynamics simulations. Thermodynamic properties of the mixed monolayers were additionally calculated using thermodynamic parameters. The analysis of isotherms showed that Calix6 significantly affects the DPPG monolayers, modifying the isotherm profile and increasing the molecular area, in agreement with the molecular dynamics simulations. The presence of Calix6 in the mixed monolayers decreased the interfacial elasticity, indicating that calixarene disrupts the strong intermolecular interactions of DPPG hindering its organization into a compact arrangement. At low molar ratios of Calix6, the DPPG:Calix6 interactions are preferentially attractive, due to the interactions between the hydrophobic tails of DPPG and the tert-butyl groups of Calix6. Increasing the proportion of calixarene generates repulsive interactions. Calix6 significantly affects the hydrophobic tail organization, which was confirmed by PM-IRRAS measurements. Calix6 appears to be expelled from the mixed films at a biologically relevant surface pressure, π = 30 mN m-1, indicating a low interaction with the cell membrane model related to the absence of antibacterial activity.

ω-Thiolation of Phenolic Surfactants Enables Controlled Conversion between Extended, Bolaform, and Multilayer Conformations.

The self-assembly of ω-thiolated surfactants onto gold is a well-studied phenomenon; however, control over the final organization within the thin films is either limited or requires extensive pre- and post-deposition chemical modifications. On the other hand, Langmuir-Blodgett deposition from the air-water interfaces affords a high degree of control over lateral organization within the film, yet it is generally employed to create physisorbed, soft matter films. Despite this, relatively little is known about the impact of the ω-thiolation on either the air-water of deposited film organization. Here, we show that the introduction of a terminal hydrophilic thiol on a phenolic surfactant does not necessarily disrupt a highly organized film nor does it necessarily induce a bolaform conformation at the interface. We show that the relative proportions of different conformations can be controlled using pH, relaxation time, surface pressure, and combinations thereof. Moreover, at high pH, the system undergoes a monolayer-to-multilayer transition wherein well-defined multilayer structures and morphologies are generated. These multilayers appear to comprise a single bolaform conformation atop an extended-chain condensed phase. We demonstrate that these structures can be transferred using Langmuir-Blodgett deposition demonstrating that combining these two approaches has the potential to achieve greater control over the functional properties of robust, chemisorbed films.

AHNAK C-Terminal Peptide Membrane Binding-Interactions between the Residues 5654-5673 of AHNAK and Phospholipid Monolayers and Bilayers.

The dysferlin membrane repair complex contains a small complex, S100A10-annexin A2, which initiates membrane repair by recruiting the protein AHNAK to the membrane, where it interacts via binding sites in the C-terminal region. However, no molecular data are available for the membrane binding of the various proteins involved in this complex. Therefore, the present study investigated the membrane binding of AHNAK to elucidate its role in the cell membrane repair process. A chemically synthesized peptide (pAHNAK), comprising the 20 amino acids in the C-terminal domain of AHNAK, was applied to Langmuir monolayer models, and the binding parameters and insertion angles were measured with surface tensiometry and ellipsometry. The interaction of pAHNAK with lipid bilayers was studied using 31P solid-state nuclear magnetic resonance. pAHNAK preferentially and strongly interacted with phospholipids that comprised negatively charged polar head groups with unsaturated lipids. This finding provides a better understanding of AHNAK membrane behavior and the parameters that influence its function in membrane repair.

Interfacial Self-Assembly of Antimicrobial Peptide GL13K into Non-Fibril Crystalline ß-Sheets.

The need for new and potent antibiotics in an era of increasing multidrug resistance in bacteria has driven the search for new antimicrobial agents, including the design of synthetic antimicrobial peptides (AMPs). While a number of ß-sheet forming AMPs have been proposed, their similarity to ß-amyloids raises a number of concerns associated with neurodegenerative states. GL13K is an effective, synthetic AMP that selectively folds into ß-sheets at anionic interfaces. Moreover, it is one of relatively few AMPs that preferentially fold into ß-sheets without bridging disulfides. The interfacial activity of GL13K and its propensity to form amyloid fibrils have not been investigated. Using structural studies at the air/water interface and in the absence of anionic lipids, we demonstrate that while GL13K does form crystalline ß-sheets, it does not self-assemble into fibrils. This work emphasizes the requirement for a single charged amino acid in the hydrophobic face to prevent fibril formation in synthetic peptides.

Strong Headgroup Interactions Drive Highly Directional Growth and Unusual Phase Co-Existence in Self-Assembled Phenolic Films.

Self-assembled materials as surface coatings are used to confer functional properties to substrates, but such properties are highly dependent on molecular organization that can be controlled through tailoring the noncovalent interactions. For monomolecular films, it is well-known that strong, dipolar interactions can oppose line tension generating noncircular domain growth. While many surfactant films exhibit liquid crystalline arrangement of the alkyl chains, there are relatively few reports of crystalline headgroups. Here, we report the self-assembly of phenolic surfactants where the combination of hydrogen bonding and π-stacking leads to a herringbone arrangement of the headgroups, generating a molecular super-lattice that can be observed using grazing incidence X-ray diffraction; such an arrangement has been previously proposed for related phenolic systems but never experimentally observed. We also investigated using pH to modulate the intermolecular interactions and the response of the system in terms of molecular organization. The first hydroxyl deprotonation does not appear to impact the structure but has significant impact on the domain size and morphology. Higher pH generates both strong directional domain growth and a loss of the molecular lattice structure, attributed to a second deprotonation. In contrast, a shorter chain surfactant, lauryl gallate, forms a liquid expanded phase that can contract upon deprotonation. In the condensed phase, the deprotonation kinetics are unusually slow for which an internal charge re-organization is proposed. The slow kinetics leads to the co-existence of three distinct phases for a single component system over relatively long timescales and provides evidence of a liquid-mediated polymorphic transformation process in two-dimensional, soft-matter films. This work has implications for understanding the long-range ordering in aromatic self-assembled structures and the mechanisms underlying Langmuir monolayer polymorphism.

Structural organization and phase behaviour of meta-substituted dioctadecylaminobenzoquinones at the air/water interface.

The structural organization and phase behaviour of an amphiphilic zwitterionic quinonemonoimine at the air/water interface are presented. Brewster angle microscopy reveals multiple co-existing phases are observed over the entire isotherm while grazing incidence X-ray diffraction (GIXD) shows that these comprise both tilted, untilted and multilayer structures with crystalline headgroups. Despite the heterogeneity, the phase transitions are highly reversible over multiple cycles.

Cellular Uptake, Cytotoxicity and Trafficking of Supported Lipid-Bilayer-Coated Lanthanide Upconverting Nanoparticles in Alveolar Lung Cancer Cells.

An understanding of the cellular uptake and trafficking of nanoparticles is important for the design of efficient nanoparticle-based nanomedicines. Herein we compare the uptake and cytotoxicity of diamond-shape, lanthanide upconverting nanoparticles (LiYF4:Tm3+/Yb3+ UCNPs) with different surface properties. Coating the UCNP with a supported lipid bilayer yielded negligible cytotoxicity on A549 human lung cancer cells, albeit with a lower, but still significant, UCNP uptake compared to oleate-capped and oleate-free UCNPs. Using inhibition studies and cellular imaging we demonstrate that the UCNPs are internalized by endocytosis and energy independent pathways and trafficked to the endoplasmic reticulum, Golgi apparatus, and lamellar and lipid bodies. Upon incorporation of a photostimulus within the bilayer coating, release of a Nile red as a hydrophobic drug model was demonstrated.

Model Lung Surfactant Films: Why Composition Matters.

Lung surfactant replacement therapies, Survanta and Infasurf, and two lipid-only systems both containing saturated and unsaturated phospholipids and one containing additional palmitic acid were used to study the impact of buffered saline on the surface activity, morphology, rheology, and structure of Langmuir monolayer model membranes. Isotherms and Brewster angle microscopy show that buffered saline subphases induce a film expansion, except when the cationic protein, SP-B, is present in sufficient quantities to already screen electrostatic repulsion, thus limiting the effect of changing pH and adding counterions. Grazing incidence X-ray diffraction results indicate an expansion not only of the liquid expanded phase but also an expansion of the lattice of the condensed phase. The film expansion corresponded in all cases with a significant reduction in the viscosity and elasticity of the films. The viscoelastic parameters are dominated by liquid expanded phase properties and do not appear to be dependent on the structure of the condensed phase domains in a phase separated film. The results highlight that the choice of subphase and film composition is important for meaningful interpretations of measurements using model systems.

Surface Behavior of Boronic Acid-Terminated Silicones.

Silicone polymers, with their high flexibility, lie in a monolayer at the air-water interface as they are compressed until a critical pressure is reached, at which point multilayers are formed. Surface pressure measurements demonstrate that, in contrast, silicones that are end-modified with polar groups take up lower surface areas under compression because the polar groups submerge into the water phase. Boronic acids have the ability to undergo coordination with Lewis bases. As part of a program to examine the surface properties of boronic acids, we have prepared boronic acid-modified silicones (SiBAs) and examined them at the air-water interface to better understand if they behave like other end-functional silicones. Monolayers of silicones, aminopropylsilicones, and SiBAs were characterized at the air-water interface as a function of end functionalization and silicone chain length. Brewster angle and atomic force microscopies confirm domain formation and similar film morphologies for both functionalized and non-functionalized silicone chains. There is a critical surface pressure (10 mN m(-1)) independent of chain length that corresponds to a first-order phase transition. Below this transition, the film appears to be a homogeneous monolayer, whose thickness is independent of the chain length. Ellipsometry at the air-water interface indicates that the boronic acid functionality leads to a significant increase of film thickness at low molecular areas that is not seen for non-functionalized silicone chains. What differentiates the boronic acids from simple silicones or other end-functionalized silicones, in particular, is the larger area occupied by the headgroup when under compression compared to other or non-end-functionalized silicones, which suggests an in-plane rather than submerged orientation that may be driven by boronic acid self-complexation.

Membrane selectivity and biophysical studies of the antimicrobial peptide GL13K.

GL13K is a short (13 amino acid) antimicrobial peptide derived from the parotid secretory protein. GL13K has been found to exhibit anti-inflammatory and antibacterial activities in physiological salt conditions. We investigated the mechanism of interaction of GL13K, with model membranes comprising 1, 2-dioleoylphosphatidylcholine (DOPC) and 1, 2-dioleoylphosphatidylglycerol (DOPG) using various biophysical and imaging techniques. Circular dichroism studies showed that GL13K adopts a ß-sheet structure in the presence of negatively charged DOPG liposomes while it retains its random coil structure with zwitterionic DOPC liposomes. GL13K did not cause any fusion of these liposomes but was able to selectively disrupt the negatively charged membranes of DOPG leading to vesicular leakage. There was no or minimal evidence of GL13K interaction with DOPC liposomes, however an analysis of supported lipid bilayers (SLBs) using atomic force microscopic (AFM) imaging and dual polarization interferometry (DPI) suggested that GL13K can interact with the surface of a DOPC planar bilayer. In the case of DOPG bilayers, AFM and DPI clearly showed membrane thinned regions where a portion of lipid molecules has been removed. These results suggest that the mechanism of GL13K action on bacterial membranes involves localized removal of lipid from the membrane via peptide-induced micellization.

A molecular chalice with hydrophobic walls and a hydrophilic rim: self-assembly and complexation properties.

We describe the synthesis of a diphenylglycoluril/dibenzo-crown-6 molecular chalice, the self-assembly at the air/water interface and its complexation properties in solution and at the water/chloroform interface.

Space-filling trialkoxysilane: synthesis and self-assembly into low-density monolayers.

A novel space-filling trialkoxysilane derivative was synthesized using a two-step strategy from commercially available starting materials to produce the precursor for the formation of low-density self-assembled monolayers. Self-assembled monolayers of the synthesized compound were prepared on three different substrates (Si/SiO(2), glass and ITO) and were characterized using contact angle, ellipsometry and sum-frequency generation spectroscopy. Removal of the space-filling protecting group, (2-chlorophenyl)diphenyl methanol, yields a carboxy-terminated surface. Correspondingly, the contact angle and film thickness decrease and the SFG spectra clearly indicate an increase in gauche defect concentration characteristic of a low-density disordered monolayer.