Evolution of interfacial mechanics of lung surfactant mimics progression of acute respiratory distress syndrome.

How acute respiratory distress syndrome progresses from underlying disease or trauma is poorly understood, and there are no generally accepted treatments resulting in a 40% mortality rate. However, during the inflammation that accompanies this disease, the phospholipase A2 concentration increases in the alveolar fluids leading to the hydrolysis of bacterial, viral, and lung surfactant phospholipids into soluble lysolipids. We show that if the lysolipid concentration in the subphase reaches or exceeds its critical micelle concentration, the surface tension, γ, of dipalmitoyl phosphatidylcholine (DPPC) or Curosurf monolayers increases and the dilatational modulus, [Formula: see text], decreases to that of a pure lysolipid interface. This is consistent with DPPC being solubilized in lysolipid micelles and being replaced by lysolipid at the interface. These changes lead to [Formula: see text] which is the criterion for the Laplace instability that can lead to mechanical instabilities during lung inflation, potentially causing alveolar collapse. These findings provide a mechanism behind the alveolar collapse and uneven lung inflation during ARDS.

Reply to the 'Comment on "Bilayer aggregate microstructure determines viscoelasticity of lung surfactant suspensions"' by J.-F. Berret, DOI: 10.1039/d2sm00653g.

In their comment, Berret suggests that Curosurf, one of three clinical lung surfactant aqueous suspensions examined in the Soft Matter, 2021, 17, 5170-51820 is a Newtonian liquid rather than a shear-thinning soft solid with a small, but measurable yield stress. We postulate that these discrepancies may be due to the size of the magnetic wire measurement probe used in their paper (Thai et al., Colloids Surf., B, 2019, 178, 337-345) the diameter of which is similar in size to the Curosurf bilayer agregates (1-10 µm). The cone and plate rheometer used by Ciutara and Zasadzinski measures averaged effects over the entire macroscopic sample. Our combined results point out that the local viscoelastic properties of a moderately dense suspension may be different than its bulk properties.

Probing Neuropeptide Volume Transmission In Vivo by Simultaneous Near-Infrared Light-Triggered Release and Optical Sensing.

Neuropeptides are abundant signaling molecules in the central nervous system. Yet remarkably little is known about their spatiotemporal spread and biological activity. Here, we developed an integrated optical approach using Plasmonic nAnovesicles and cell-based neurotransmitter fluorescent engineered reporter (CNiFER), or PACE, to probe neuropeptide signaling in the mouse neocortex. Small volumes (fL to pL) of exogenously supplied somatostatin-14 (SST) can be rapidly released under near-infrared light stimulation from nanovesicles implanted in the brain and detected by SST2 CNiFERs with nM sensitivity. Our measurements reveal reduced but synchronized SST transmission within 130 µm, and markedly smaller and delayed transmission at longer distances. These measurements enabled a quantitative estimation of the SST loss rate due to peptide degradation and binding. PACE offers a new tool for determining the spatiotemporal scales of neuropeptide volume transmission and signaling in the brain.

Bilayer aggregate microstructure determines viscoelasticity of lung surfactant suspensions.

Neonatal respiratory distress syndrome (NRDS) is treated by intratracheal delivery of suspensions of animal-derived lung surfactant in saline. Lung surfactants are extracted via organic solvents from animal lung lavage, followed by solvent removal and surfactant re-hydration to form multi-bilayer particles suspended in saline. Following intra-tracheal administration, the surfactant suspension spreads throughout the lungs by surface tension gradient induced flow; the spreading rate is limited by suspension viscoelasticity. Here we examine the rheology of three clinical lung surfactant suspensions: Survanta (bovine lung), Curosurf (porcine lung), and Infasurf (calf lung). These surfactants have widely different rheological properties that depend on the lipid composition and bilayer organization. The steady shear viscosity is related to the bilayer particle volume fraction as for a suspension of hard spheres, but the lipid volume fraction is not simply related to the mass loading. Optical and electron microscopy and small angle X-ray scattering show that the viscosity variation is due to the temperature and composition dependent bilayer aggregate shapes and internal particle organization. Survanta forms crystalline bilayers at 37 °C, resulting in high aspect ratio asymmetric particles. Infasurf forms aggregates of unilamellar vesicles containing water pockets, while Curosurf forms onion-like multi-layered liposomes. While the mass loading of the three clinical surfactants is different, the different bilayer organization causes the particle volume fractions to be similar. Adding polyethylene glycol dehydrates and partially flocculates the bilayer aggregates in all suspensions, leading to smaller particle volume fractions and a reduced suspension viscosity even though the solvent viscosity increases almost six-fold.

Dilatational rheology of water-in-diesel fuel interfaces: effect of surfactant concentration and bulk-to-interface exchange.

Micrometer-sized water droplets dispersed in diesel fuel are stabilized by the fuel's surface-active additives, such as mono-olein and poly(isobutylene)succinimide (PIBSI), making the droplets challenging for coalescing filters to separate. Dynamic material properties found from interfacial rheology are known to influence the behavior of microscale droplets in coalescing filters. In this work, we study the interfacial dilatational properties of water-in-fuel interfaces laden with mono-olein and PIBSI, with a fuel phase of clay-treated ultra-low sulphur diesel (CT ULSD). First, the dynamic interfacial tension (IFT) is measured using pendant drop tensiometry, and a curvature-dependent form of the Ward and Tordai diffusion equation is applied for extracting the diffusivity of the surfactants. Additionally, Langmuir kinetics are applied to the dynamic IFT results to obtain the maximum surface concentration (Γ∞) and ratio of adsorption to desorption rate constants (κ). We then use a capillary pressure microtensiometer to measure the interfacial dilatational modulus, and further extract the characteristic frequency of surfactant exchange (ω0) by fitting a model assuming diffusive exchange between the interface and bulk. In this measurement, 50-100 µm diameter water droplets are pinned at the tip of a glass capillary in contact with the surfactant-containing fuel phase, and small amplitude capillary pressure oscillations over a range of frequencies from 0.45-20 rad s-1 are applied to the interface, inducing changes in interfacial tension and area to yield the dilatational modulus, E*(ω). Over the range of concentrations studied, the dilatational modulus of CT ULSD with either mono-olein or PIBSI increases with a decrease in bulk concentration and plateaus at the lowest concentrations of mono-olein. Characteristic frequency (ω0) values extracted from the fit are compared with those calculated using equilibrium surfactant parameters (κ and Γ∞) derived from pendant drop tensiometry, and good agreement is found between these values. Importantly, the results imply that diffusive exchange models based on the equilibrium relationships between surfactant concentration and interfacial tension can be used to infer the dynamic dilatational behavior of complex surfactant systems, such as the water-in-diesel fuel interfaces in this study.

Interfacial curvature effects on the monolayer morphology and dynamics of a clinical lung surfactant.

The morphology of surfactant monolayers is typically studied on the planar surface of a Langmuir trough, even though most physiological interfaces are curved at the micrometer scale. Here, we show that, as the radius of a clinical lung surfactant monolayer-covered bubble decreases to ∼100 µm, the monolayer morphology changes from dispersed circular liquid-condensed (LC) domains in a continuous liquid-expanded (LE) matrix to a continuous LC linear mesh separating discontinuous LE domains. The curvature-associated morphological transition cannot be readily explained by current liquid crystal theories based on isotropic domains. It is likely due to the anisotropic bending energy of the LC phase of the saturated phospholipids that are common to all natural and clinical lung surfactants. This continuous LC linear mesh morphology is also present on bilayer vesicles in solution. Surfactant adsorption and the dilatational modulus are also strongly influenced by the changes in morphology induced by interfacial curvature. The changes in morphology and dynamics may have physiological consequences for lung stability and function as the morphological transition occurs at alveolar dimensions.

Inflammation product effects on dilatational mechanics can trigger the Laplace instability and acute respiratory distress syndrome.

In the lungs, the Laplace pressure, ΔP = 2γ/R, would be higher in smaller alveoli than larger alveoli unless the surface tension, γ decreases with alveolar interfacial area, A, such that 2ε > γ in which ε = A(dγ/dA) is the dilatational modulus. In Acute Respiratory Distress Syndrome (ARDS), lipase activity due to the immune response to an underlying trauma or disease causes single chain lysolipid concentrations to increase in the alveolar fluids via hydrolysis of double-chain phospholpids in bacterial, viral, and normal cell membranes. Increasing lysolipid concentrations decrease the dilatational modulus dramatically at breathing frequencies if the soluble lysolipid has sufficient time to diffuse off the interface, causing 2ε < γ, thereby potentially inducing the "Laplace Instability", in which larger alveoli have a lower internal pressure than smaller alveoli. This can lead to uneven lung inflation, alveolar flooding, and poor gas exchange, typical symptoms of ARDS. While the ARDS lung contains a number of lipid and protein species in the alveolar fluid in addition to lysolipids, the surface activity and frequency dependent dilatational modulus of lysolipid suggest how inflammation may lead to the lung instabilities associated with ARDS. At high frequencies, even at high lysolipid concentrations, 2ε - γ > 0, which may explain the benefits ARDS patients receive from high frequency oscillatory ventilation.

Near-Infrared Light Triggered-Release in Deep Brain Regions Using Ultra-photosensitive Nanovesicles.

Remote and minimally-invasive modulation of biological systems with light has transformed modern biology and neuroscience. However, light absorption and scattering significantly prevents penetration to deep brain regions. Herein, we describe the use of gold-coated mechanoresponsive nanovesicles, which consist of liposomes made from the artificial phospholipid Rad-PC-Rad as a tool for the delivery of bioactive molecules into brain tissue. Near-infrared picosecond laser pulses activated the gold-coating on the surface of nanovesicles, creating nanomechanical stress and leading to near-complete vesicle cargo release in sub-seconds. Compared to natural phospholipid liposomes, the photo-release was possible at 40 times lower laser energy. This high photosensitivity enables photorelease of molecules down to a depth of 4 mm in mouse brain. This promising tool provides a versatile platform to optically release functional molecules to modulate brain circuits.

Perfluoroheptane-Loaded Hollow Gold Nanoshells Reduce Nanobubble Threshold Flux.

The threshold flux for nanobubble formation and liposome rupture is reduced by 50-60% by adding a liquid mixture of tetradecanol and perfluoroheptane to the interior cavity of 40 nm diameter hollow gold nanoshells (HGN), and allowing the tetradecanol to solidify to hold the perfluoroheptane in place. On absorption of picosecond pulses of near-infrared light, the perfluoroheptane vaporizes to initiate cavitation-like nanobubbles as the HGN temperature increases. The lower spinodal temperature and heat capacity of perfluoroheptane relative to water causes the threshold flux for nanobubble formation to decrease. The perfluoroheptane-containing HGN can be linked via thiol-PEG-lipid tethers to carboxyfluorescein-containing liposomes and shows a similar decreased flux necessary for liposome contents release.

Comparison of Line Tension Measurement Methods for Lipid Monolayers at Liquid-Liquid Coexistence.

Several methods of measuring the line tension between phase-separated liquid-ordered-liquid -disordered domains in phospholipid-cholesterol systems have been proposed. These experimental techniques are typically internally self-consistent, but the measured line tension values vary widely among these techniques. To date, no measurement of line tension has utilized multiple experimental techniques to look at the same monolayer system. Here we compare two nonperturbative methods, Fourier analysis of boundary fluctuations (BA) and one proposed by Israelachvili involving the analysis of domain size distributions (SD), to extract the line tension in a 70 mol % DMPC/30 mol % dihydrocholesterol (DChol) mixture as a function of surface pressure. We show that BA predicts the expected variation in line tension measurements consistent with the theoretical critical exponent whereas SD does not. From this comparison, we conclude that the size distribution of monolayer domains is metastable and primarily determined by the kinetics of domain nucleation and subsequent aging.

Interfacial rheology and direct imaging reveal domain-templated network formation in phospholipid monolayers penetrated by fibrinogen.

Phospholipids are found throughout the natural world, including the lung surfactant (LS) layer that reduces pulmonary surface tension and enables breathing. Fibrinogen, a protein involved in the blood clotting process, is implicated in LS inactivation and the progression of disorders such as acute respiratory distress syndrome. However, the interaction between fibrinogen and LS at the air-water interface is poorly understood. Through a combined microrheological, confocal and epifluorescence microscopy approach we quantify the interfacial shear response and directly image the morphological evolution when a model LS monolayer is penetrated by fibrinogen. When injected into the subphase beneath a monolayer of the phospholipid dipalmitoylphosphatidylcholine (DPPC, the majority component of LS), fibrinogen preferentially penetrates disordered liquid expanded (LE) regions and accumulates on the boundaries between LE DPPC and liquid condensed (LC) DPPC domains. Thus, fibrinogen is line active. Aggregates grow from the LC domain boundaries, ultimately forming a percolating network. This network stiffens the interface compared to pure DPPC and imparts the penetrated monolayer with a viscoelastic character reminiscent of a weak gel. When the DPPC monolayer is initially compressed beyond LE-LC coexistence, stiffening is significantly more modest and the penetrated monolayer retains a viscous-dominated, DPPC-like character.

Optimizing the NIR Fluence Threshold for Nanobubble Generation by Controlled Synthesis of 10 - 40 nm Hollow Gold Nanoshells.

The laser fluence to trigger nanobubbles around hollow gold nanoshells (HGN) with near infrared light was examined through systematic modification of HGN size, localized surface plasmon resonance (LSPR), HGN concentration, and surface coverage. Improved temperature control during silver template synthesis provided monodisperse, silver templates as small as 9 nm. 10 nm HGN with < 2 nm shell thickness were prepared from these templates with a range of surface plasmon resonances from 600 - 900 nm. The fluence of picosecond near infrared (NIR) pulses to induce transient vapor nanobubbles decreased with HGN size at a fixed LSPR wavelength, unlike solid gold nanoparticles of similar dimensions that require an increased fluence with decreasing size. Nanobubble generation causes the HGN to melt with a blue shift of the LSPR. The nanobubble threshold fluence increases as the irradiation wavelength moves off the nanoshell LSPR. Surface treatment did not influence the threshold fluence. The threshold fluence increased with decreasing HGN concentration, suggesting that light localization through multiple scattering plays a role. The nanobubble threshold to rupture liposomes is 4 times smaller for 10 nm than for 40 nm HGN at a given LSPR, allowing us to use HGN size, LSPR, laser wavelength and fluence to control nanobubble generation.

Light-Triggered Genome Editing: Cre Recombinase Mediated Gene Editing with Near-Infrared Light.

A light-activated genome editing platform based on the release of enzymes from a plasmonic nanoparticle carrier when exposed to biocompatible near-infrared light pulses is described. The platform relies on the robust affinity of polyhistidine tags to nitrilotriacetic acid in the presence of copper which is attached to double-stranded nucleic acids self-assembled on the gold nanoparticle surface. A protein fusion of the Cre recombinase containing a TAT internalization peptide sequence to achieve endosomal localization is also employed. High-resolution gene knock-in of a red fluorescent reporter is observed using a commercial two-photon microscope. High-throughput irradiation is described to generate useful quantities of edited cells.

Nonlinear chiral rheology of phospholipid monolayers.

Microbutton rheometry reveals that the chiral morphology of dipalmitoylphosphatidylcholine (DPPC) monolayers imparts a chiral nonlinear rheological response. The nonlinear elastic modulus and yield stress of DPPC monolayers are greater when sheared clockwise (C), against the natural winding direction of DPPC domains, than counter-clockwise (CC). Under strong CC shear strains, domains deform plastically; by contrast, domains appear to fracture under strong C shearing. After CC shearing, extended LC domains develop regular patterns of new invaginations as they recoil, which we hypothesize reflect the nucleation and growth of new defect lines across which the tilt direction undergoes a step change in orientation. The regular spacing of these twist-gradient defects is likely set by a competition between the molecular chirality and the correlation length of the DPPC lattice. The macroscopic mechanical consequences of DPPC's underlying molecular chirality are remarkable, given the single-component, non-cross-linked nature of the monolayers they form.

Visualizing monolayers with a water-soluble fluorophore to quantify adsorption, desorption, and the double layer.

Contrast in confocal microscopy of phase-separated monolayers at the air-water interface can be generated by the selective adsorption of water-soluble fluorescent dyes to disordered monolayer phases. Optical sectioning minimizes the fluorescence signal from the subphase, whereas convolution of the measured point spread function with a simple box model of the interface provides quantitative assessment of the excess dye concentration associated with the monolayer. Coexisting liquid-expanded, liquid-condensed, and gas phases could be visualized due to differential dye adsorption in the liquid-expanded and gas phases. Dye preferentially adsorbed to the liquid-disordered phase during immiscible liquid-liquid phase coexistence, and the contrast persisted through the critical point as shown by characteristic circle-to-stripe shape transitions. The measured dye concentration in the disordered phase depended on the phase composition and surface pressure, and the dye was expelled from the film at the end of coexistence. The excess concentration of a cationic dye within the double layer adjacent to an anionic phospholipid monolayer was quantified as a function of subphase ionic strength, and the changes in measured excess agreed with those predicted by the mean-field Gouy-Chapman equations. This provided a rapid and noninvasive optical method of measuring the fractional dissociation of lipid headgroups and the monolayer surface potential.

Effect of cholesterol nanodomains on monolayer morphology and dynamics.

At low mole fractions, cholesterol segregates into 10- to 100-nm-diameter nanodomains dispersed throughout primarily dipalmitoylphosphatidylcholine (DPPC) domains in mixed DPPC:cholesterol monolayers. The nanodomains consist of 6:1 DPPC:cholesterol "complexes" that decorate and lengthen DPPC domain boundaries, consistent with a reduced line tension, λ. The surface viscosity of the monolayer, ηs, decreases exponentially with the area fraction of the nanodomains at fixed surface pressure over the 0.1- to 10-Hz range of frequencies common to respiration. At fixed cholesterol fraction, the surface viscosity increases exponentially with surface pressure in similar ways for all cholesterol fractions. This increase can be explained with a free-area model that relates ηs to the pure DPPC monolayer compressibility and collapse pressure. The elastic modulus, G', initially decreases with cholesterol fraction, consistent with the decrease in λ expected from the line-active nanodomains, in analogy to 3D emulsions. However, increasing cholesterol further causes a sharp increase in G' between 4 and 5 mol% cholesterol owing to an evolution in the domain morphology, so that the monolayer is elastic rather than viscous over 0.1-10 Hz. Understanding the effects of small mole fractions of cholesterol should help resolve the controversial role cholesterol plays in human lung surfactants and may give clues as to how cholesterol influences raft formation in cell membranes.

Targeted intracellular delivery of proteins with spatial and temporal control.

While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

Monitoring phases and phase transitions in phosphatidylethanolamine monolayers using active interfacial microrheology.

Active interfacial microrheology is a sensitive tool to detect phase transitions and headgroup order in phospholipid monolayers. The re-orientation of a magnetic nickel nanorod is used to explore changes in the surface rheology of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), which differ by two CH2 groups in their alkyl chains. Phosphatidylethanolamines such as DLPE and DMPE are a major component of cell membranes in bacteria and in the nervous system. At room temperature, DLPE has a liquid expanded (LE) phase for surface pressure, Π < ∼38 mN m(-1); DMPE has an LE phase for Π < ∼7 mN m(-1). In their respective LE phases, DLPE and DMPE show no measurable change in surface viscosity with Π, consistent with a surface viscosity <10(-9) N s m(-1), the resolution of our technique. However, there is a measurable, discontinuous change in the surface viscosity at the LE to liquid condensed (LC) transition for both DLPE and DMPE. This discontinuous change is correlated with a significant increase in the surface compressibility modulus (or isothermal two-dimensional bulk modulus). In the LC phase of DMPE there is an exponential increase in surface viscosity with Π consistent with a two-dimensional free area model. The second-order LC to solid (S) transition in DMPE is marked by an abrupt onset of surface elasticity; there is no measurable elasticity in the LC phase. A measurable surface elasticity in the S phase suggests a change in the molecular ordering or interactions of the DMPE headgroups that is not reflected in isotherms or in grazing incidence X-ray diffraction. This onset of measurable elasticity is also seen in DLPE, even though no indication of a LC-S transition is visible in the isotherms.

Inside-outside self-assembly of light-activated fast-release liposomes.

Building additional functionality into both the membrane and the internal compartments of biocompatible liposomes by self-assembly can provide ways of enhancing colloidal stability and spatial and temporal control of contents release. An interdigitation-fusion process is used to encapsulate near infrared light absorbing copper sulfide nanoparticles in the interior compartments of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol liposomes. Once formed, the liposome membrane is modified to include lysolipids and polyethylene glycol lipids by partitioning from lysolipid and PEG-lipid micelles in solution. This results in sterically stable, thermosensitive liposomes with a permeability transition near physiological temperature that can be triggered by NIR light irradiation. Rapid changes in local concentration can be induced with spatial and temporal control using NIR laser light.

Cryogenic electron microscopy study of nanoemulsion formation from microemulsions.

We examine a process of preparing oil-in-water nanoemulsions by quenching (diluting and cooling) precursor microemulsions made with nonionic surfactants and a cosurfactant. The precursor microemulsion structure is varied by changing the concentration of the cosurfactant. Water-continuous microemulsions produce initial nanoemulsion structures that are small and simple, mostly unilamellar vesicles, but microemulsions that are not water-continuous produce initial nanoemulsion structures that are larger and multilamellar. Examination of these structures by cryo-electron microscopy supports the hypothesis that they are initially vesicular structures formed via lamellar intermediate structures, and that if the lamellar structures are too well ordered they fail to produce small simple structures.