[Show abstract][Hide abstract] ABSTRACT: We describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laser-activated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine.
Published by Elsevier Ltd.
[Show abstract][Hide abstract] ABSTRACT: 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, < ~ 38 mN/m; DMPE has an LE phase for < ~ 7 mN/m. In their respective LE phases, DLPE and DMPE show no measurable change in surface viscosity with , consistent with a surface viscosity < 10-9 Ns/m, 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 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 measureable 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 measureable elasticity is also seen in DLPE, even though no indication of a LC-S transition is visible in the isotherms.
[Show abstract][Hide abstract] ABSTRACT: Building additional functionality into both the membrane and the internal compartments of biocompatible liposomes can provide ways of enhancing colloidal stability and spatial and temporal control of contents release. An interdigitation-fusion process is used to encapsulate near infra-red 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 02/2015; 112(8). DOI:10.1073/pnas.1419033112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 poly-histidine 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.
[Show abstract][Hide abstract] ABSTRACT: Liposome drug delivery systems are faced by the quandary of minimizing non-specific drug release and initiating fast release at the site of interest. A novel strategy is to separate the mechanism of drug release from drug retention by using an external agent to trigger drug release. Near infra-red (NIR) light is an appropriate choice as it is physiologically friendly with minimal thermal injury to normal tissues and light penetration of several cm. We can synthesize Hollow Gold Nanoparticles (HGN) to absorb NIR light over the range from 700-900 nm by manipulating the diameter to shell thickness ratio. NIR picosecond pulsed laser light absorbed by liposome encapsulated or tethered HGN is rapidly converted into thermal energy; the large temperature gradients lead to the formation of transient vapor nanobubbles in aqueous solution. The collapse of the nanobubbles ruptures cell, endosome, or liposome membranes; thereby releasing their contents with minimal damage to the contents or surroundings. We find smaller (10 – 15 nm) HGN show higher specific absorbance of NIR light than larger (30-50 nm) HGN, which leads to more efficient membrane rupture and delivery of liposome contents. We have systematically synthesized monodisperse HGN of sizes from 10 – 40 nm and determined the relative efficacy of laser power and duration on the rate of contents release from 200 nm liposomes. A fluorescent dye was encapsulated within a liposome as a model agent to study release kinetics triggered by a picosecond pulsed NIR laser irradiation of the HGN coupled to the liposome. Subsequently, an anticancer drug was encapsulated within the liposomes to deliver the drug to prostate cancer cell in vitro; near-complete cell killing was observed. The study provides design parameters for engineering drug delivery systems that ultimately will be apply to tumor targeting therapeutics.
[Show abstract][Hide abstract] ABSTRACT: A novel drug carrier is presented consisting of plasmonic hollow gold nanoshells (HGN) chemically tethered to liposomes made temperature sensitive with lysolipids (TSL). Continuous-wave irradiation by physiologically friendly near-infra-red light at 800 nm for 2.5 min at laser intensities an order of magnitude below that known to damage skin generates heating localized to the liposome membrane. The heating increases the liposome permeability in an irradiation dose dependent, but reversible manner, resulting in rapid release of small molecules such as the self-quenching dye carboxyfluorescein or the chemotherapeutic doxorubicin, without raising the bulk temperature. The local rise in nanoshell temperature under laser irradiation is inferred by comparing dye release rates from the TSL via bulk heating to that induced by irradiation. Laser-irradiation of TSL enables precise control of contents release with low temperature gradients confined to areas irradiated by the laser focus. The combined effects of rapid local release and localized hyperthermia provide a synergistic effect as shown by a near doubling of androgen resistant PPC-1 prostate cancer cell toxicity compared to the same concentration of free doxorubicin.
Particle and Particle Systems Characterization 11/2014; 31(11). DOI:10.1002/ppsc.201400035 · 3.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chemoradiation-resistant cancer cells and unresectable micro-tumors limit treatment efficacy and lead to high nonspecific toxicity or recurrence in head and neck cancers. We show the cancer cell-specific, on-demand enhancement of the chemo- and chemoradiation therapy with mechanical intracellular impact of plasmonic nanobubbles, a laser pulseinduced explosive nano-event, not a particle. We report cellular mechanisms of cancer cell-specific detection and enhancement of the entry drug and X-ray dose and validate these mechanisms in vitro and in vivo for head and neck squamous cell carcinoma. Plasmonic nanobubble technology showed more than 10-fold enhancement of the therapeutic efficacy compared to standard chemoradiation in murine models of primary, microscopic residual and recurrent diseases. At the same time our technology efficiently spared adjacent normal tissues due to the reduction of the effective therapeutic doses of drug by 30-40 fold, X-rays by 15-fold and the treatment time to a single procedure. The developed plasmonic nanobubble technology transforms a standard macro-therapy into a cell-level on-demand theranostic treatment for primary, adjuvant and adjunct applications.
Proceedings of SPIE - The International Society for Optical Engineering 02/2014; 8926. DOI:10.1117/12.2038277 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The surface viscosity of dipalmitoylphosphatidylcholine (DPPC) monolayers decreases by two orders of magnitude on addition of 3.7 mol% cholesterol, followed by a sharp increase in the monolayer elasticity above 3.7 mol%. We correlate these intriguing rheological properties with changes in the molecular organization as revealed by Grazing Incidence X-ray Diffraction (GIXD). Adding cholesterol at constant surface pressure decreases the tilt of the DPPC lattice in the same way as increasing the surface pressure at constant cholesterol. The correlation length of the DPPC lattice decreases with cholesterol fraction, suggesting that the increase in defect density leads to the decrease in surface viscosity. Above 4 mol%, the DPPC lattice parameter and correlation length saturates, showing that the observed increase in monolayer elasticity comes about from a percolated, emulsion-like cholesterol network.
Cholesterol is hypothesized to alter the fluidity, permeability and phase transitions of monolayers, bilayers, and cell membranes, but the mechanisms by which this happens are still obscure. At different concentrations, cholesterol organizes phospholipid molecules into “liquid-ordered” phases; while at other concentrations, cholesterol inhibits phospholipid crystallization into solid or liquid condensed phases. Despite their ubiquity in mammalian cell membranes, there is little quantitative information on the surface viscosity and elasticity of lipid/cholesterol monolayers, and how the viscosity and elasticity relate to interactions between cholesterol and phospholipids. For example, nanometer scale variations in membrane fluidity are hypothesized to be essential to the dynamics and function of membrane-associated proteins in self-organizing “rafts.” Monolayer fluidity also plays an important, but as yet unappreciated role in the spreading and surface tension lowering properties of lung surfactants. Lung surfactant must reliably and reproducibly reduce the surface tension at the air-water interface of the alveoli to near zero to minimize the work of breathing. This requires fast spreading of the surfactant over the alveolar air-liquid interfaces during inspiration as the lung area rapidly increases. During exhalation, surfactant must resist Marangoni flow due to the surface tension differences between the deep lung and the bronchi. A lack of surfactant in premature infants causes potentially fatal neonatal Respiratory Distress Syndrome, which is currently treated with animal-derived replacement surfactants. However, the optimal concentration of many of the lipid and protein species, especially cholesterol is unknown. Even the presence of cholesterol in replacement lung surfactants is controversial as blood and cell debris extracted with surfactant from animal lungs complicate the cholesterol analysis. FDA-approved clinical surfactants Survanta and Curosurf have all cholesterol removed, while FDA-approved Infasurf has 4-5 wt% cholesterol. One common factor between all replacement surfactants is that DPPC is the dominant (50 – 80 mol%) phospholipid.
Here we show that the rheological properties of mixed DPPC/Chol monolayers at low cholesterol (≤ 3.7 mol%) arise from changes in the DPPC lattice, but the increased elasticity at higher cholesterol mole fractions is the result of the mesoscopic evolution of the nanodomain morphology. GIXD shows that increasing the Chol fraction at constant surface pressure, or increasing the surface pressure at constant Chol fraction decreases the tilt in the same way. DPPC tilts to accommodate the mismatch between the larger projected area of the phosphocholine headgroup relative to the alkane tailgroup at low surface pressure. Palmitic acid (PA, used in Survanta) and hexadecanol (HD, used in the replacement surfactant, Exosurf) also decrease the molecular tilt at a given surface pressure. Cholesterol, like PA and HD, has a relatively larger tailgroup and a smaller headgroup, which mitigates the area mismatch, leading to the reduction in tilt. However, unlike PA or HD, the correlation length of the DPPC lattice decreases from ~ 10 nm to ~ 1 nm as Chol is increased from 0 to 7 mol%. This decreased correlation length is consistent with the cholesterol sterol ring disrupting the packing of the tailgroup lattice of DPPC. The correlation length can be thought of as the distance between lattice defects; hence the reduction in lattice correlations with increasing cholesterol fraction suggest an increased defect density, which corresponds to an increase in the “free area” available to the molecules to enhance diffusion and hence, reduce the surface viscosity. The reduction in lattice correlation and tilt saturates at ~ 4 mol% Chol, at the same concentration we see the change from a viscous to elastic monolayer (Fig. 4). However, the reduction in correlations are not consistent with an increase in the elasticity of the monolayer, but rather suggests the opposite. However, at mole fractions > 3.7%, the nanodomains percolate to form a bicontinuous morphology and the DPPC domain size decreases. In analogy to 3-D surfactant-stabilized emulsions, even small stresses deform the emulsion network structure, leading to an elastic modulus, G’ ~ λR, in which λ, the line tension, is the two-dimension equivalent of the surface tension in 3-D emulsions. R is a characteristic length scale of the DPPC domains, which decreases with increasing Chol content as the nanodomains form a percolating network.
[Show abstract][Hide abstract] ABSTRACT: Various methods have been employed to measure the interfacial rheological properties of surfactant monolayers, but it is common to find disagreement in the literature in the values obtained by these techniques. Many of these measurements cannot fully account for effects such as history-dependence, yield stress, and viscoelastic recovery which are known to exist in some monolayers. We use electromagnets to drive microfabricated, micron-scale magnetic probes of prescribed shape, embedded within insoluble surfactant monolayers at the air-water interface. Small amplitude oscillatory shear experiments provide a linear response, recovering purely shear moduli from the angular displacement of circular probes under applies magnetic torques. We describe a series of experiments to investigate the different rheological responses to shear, dilation, and extension that arise under different forcings with different probes. During each of these experiments, the response of the condensed domains to the magnetic probe is simultaneously visualized with fluorescence microscopy. Thus we correlate the measured response of the monolayer under different driving forces/torques with the observed microstructural deformations, rearrangements, and behavior more generally.
[Show abstract][Hide abstract] ABSTRACT: Liposomes, lipid bilayer capsules, have been widely evaluated as drug nanocarriers, however, they are often limited by slow and non-specific release. A novel strategy is to initiate drug release by an external agent such as near infra-red pulsed laser light adsorbed by encapsulated or tethered nanoparticles. The light energy is rapidly converted into thermal energy that generates nanobubble formation that can mechanically disrupt the liposome membranes, thereby releasing the drug with temporal and spatial control. This study presents a comparison of the photoactivated liposomal drug release induced by two different types of nanoparticles (NPs): hollow gold nanoshells (HGN) and cupper sulfide nanoparticles (CSN). Both NPs exhibit strong absorption of near infra-red (NIR) light and offer the opportunity to convert optical energy into thermal energy. The absorbance of NIR light is desirable because it causes minimal thermal injury to normal tissues with optimal light penetration. A picosecond pulsed NIR laser is used to trigger and target the release of the drug. The photothermal heating of the nanoparticles generated by short laser pulses results in large temperature gradients that leads to the formation of transient vapor nanobubbles in aqueous solution. The collapse of the nanobubbles induces the rupture of the liposome membrane and the subsequent drug release with minimal damage to the surroundings. We are particularly interested in the relative light intensity necessary to initiate nanobubble formation for the ~ 10 nm diameter CSN and the ~ 50 nm diameter HGN. We are also interested in the ease of encapsulating the smaller CSN compared to the larger HGN within 100 nm liposomes most used for drug delivery. The study will lead to a better understanding of the capabilities of multi-functional nanomaterials for NIR laser-controlled drug release in order to facilitate the development of more complex therapeutic studies.
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 07/2013; 104(2). DOI:10.1073/pnas.1303304110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nanoparticles have opened new exciting avenues for both diagnostic and therapeutic applications in human disease, and targeted nanoparticles are increasingly used as specific drug delivery vehicles. The precise quantification of nanoparticle internalization is of importance to measure the impact of physical and chemical properties on the uptake of nanoparticles into target cells or into cells responsible for rapid clearance. Internalization of nanoparticles has been measured by various techniques, but comparability of data between different labs is impeded by lack of a generally accepted standardized assay. Furthermore, the distinction between associated and internalized particles has been a challenge for many years, although this distinction is critical for most research questions. Previously used methods to verify intracellular location are typically not quantitative and do not lend themselves to high throughput analysis. Here we developed a mathematical model which integrates the data from high throughput flow cytometry measurements with data from quantitative confocal microscopy. The generic method described here will be a useful tool in biomedical nanotechnology studies. The method was then applied to measure the impact of surface coatings of vesosomes on their internalization by cells of the reticuloendothelial system (RES). RES cells are responsible for rapid clearance of nanoparticles, and the resulting fast blood clearance is one of the major challenges in biomedical applications of nanoparticles. Coating of vesosomes with long chain polyethylene glycol showed a trend for lower internalization by RES cells.
[Show abstract][Hide abstract] ABSTRACT: We present the asymmetric responses of condensed phase dipalmitoylphosphatidylcholine (DPPC) monolayers due to clockwise and counterclockwise deformations. The phospholipid, especially DPPC, forms condensed grainy liquid crystal monolayer, whose individual grains are chiral: due to tilt of tail group at the interface and a chiral point within the molecular structure, bio-derived DPPC, R enantiomer, grains curl in counterclockwise direction while S-DPPC , made by only synthetically, curls in clockwise direction. By applying constant stress to the interlocked condensed domains of R DPPC at condensed phase, we show that the strain responses are asymmetric: deformations are easier in clockwise direction than counterclockwise direction. In addition, we manipulate the degree of chirality of DPPC grains by adding racemic DPPC which contains no chiral properties, and we show that this asymmetry of strains decreases as adding more racemic DPPC to R DPPC. We conclude that this chirality of DPPC domains leads to a chiral viscoelastic responses.
[Show abstract][Hide abstract] ABSTRACT: Plasmonic nanobubbles, new multifunctional cellular nano-agents, are selectively generated in target cancer cells with a short near-infrared laser pulse. The enhanced cellular specificity and tunable transient nature of plasmonic nanobubbles provide fast, efficient and selective dual therapeutic effects: intracellular delivery of a drug and direct mechanical destruction of cells while sparing normal cells, and reduce drug dose and overcome drug-resistance of cancer cells.
[Show abstract][Hide abstract] ABSTRACT: We investigated a model of acute respiratory distress syndrome in which the serum protein albumin adsorbs to an air-liquid interface and prevents the thermodynamically preferable adsorption of the clinical lung surfactant Survanta by inducing steric and electrostatic energy barriers analogous to those that prevent colloidal aggregation. Chitosan and polyethylene glycol (PEG), two polymers that traditionally have been used to aggregate colloids, both allow Survanta to quantitatively displace albumin from the interface, but through two distinct mechanisms. Direct visualization with confocal microscopy shows that the polycation chitosan coadsorbs to interfacial layers of both Survanta and albumin, and also colocalizes with the anionic domains of Survanta at the air-liquid interface, consistent with it eliminating the electrostatic repulsion by neutralizing the surface charges on albumin and Survanta. In contrast, the PEG distribution does not change during the displacement of albumin by Survanta, consistent with PEG inducing a depletion attraction sufficient to overcome the repulsive energy barrier toward adsorption.
[Show abstract][Hide abstract] ABSTRACT: The size distribution of domains in phase-separated lung surfactant monolayers influences monolayer viscoelasticity and compressibility which, in turn, influence monolayer collapse and set the compression at which the minimum surface tension is reached. The surfactant-specific protein SP-B decreases the mean domain size and polydispersity as shown by fluorescence microscopy. From the images, the line tension and dipole density difference are determined by comparing the measured size distributions with a theory derived by minimizing the free energy associated with the domain energy and mixing entropy. We find that SP-B increases the line tension, dipole density difference, and the compressibility modulus at surface pressures up to the squeeze-out pressure. The increase in line tension due to SP-B indicates the protein avoids domain boundaries due to its solubility in the more fluid regions of the film.
[Show abstract][Hide abstract] ABSTRACT: The temporal and spatial control over the delivery of materials such as siRNA or drugs into cells remains a significant technical challenge. We demonstrate the pulsed near-infrared (NIR) laser dependent release of siRNA from coated 40 nm gold nanoshells. Tat-lipid coating mediates the cellular uptake of the nanomaterial at pM concentration, while spatiotemporal silencing of a reporter gene (green fluorescence protein) was studied using photomasking. The NIR laser induced release of siRNA from the nanoshells is found to be power and time dependent, through surface-linker bond cleavage, while the escape of the siRNA from endosomes occurs above a critical pulse energy attributed to local heating and cavitation. NIR laser controlled drug release from functional nanomaterials should facilitate more sophisticated developmental biology and therapeutic studies.
[Show abstract][Hide abstract] ABSTRACT: We present systematic measurements of the surface rheology of monolayers of liquid-condensed(LC) dipalmitoylphosphatidylcholine (DPPC)/cholesterol mixtures at the air/water interface. Using microfabricated, ferromagnetic ‘microbuttons’ as new microrheological probes, we measure the linear viscoelastic moduli of DPPC/cholesterol monolayers as both surface pressure and frequency are varied as a function of cholesterol concentration. Visualization of this interface using fluorescence microscopy reveals that the interlocked, spiral liquid crystalline domains that comprise an LC-DPPC monolayer expand and elongate on addition of cholesterol, and are separated by cholesterol-rich fluid domains, suggesting that cholesterol is line-active. In addition, the monolayer domains give rise to a visco-elastic solid response, analogous to a two-dimensional emulsion, and the measured viscoelastic moduli qualitatively agree with predictions based on a 2D concentrated emulsion model. Surprisingly, 1 - 2 wt% cholesterol decreases the surface viscosity of DPPC monolayers by orders of magnitude; we have correlated this decrease with a log-additive mixing rule, similar to polymer blends, with the cholesterol-rich phase having a very low viscosity and the LC DPPC phases being very viscous. Atomic Force Microscopy imaging was used to verify the structure of the mixed monolayer at higher surface pressures to show that the spiral domain structure persisted. These rheological observations have important implications for replacement human lung surfactants in which the role and even presence of cholesterol is hotly debated.