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The plasma membrane of eukaryotic cells is asymmetric with respect to its phospholipid composition. Analysis of the lipid composition of the outer leaflet is important for understanding cell membrane biology in health and disease. Here, a method based on cyclodextrin-mediated lipid exchange to characterize the phospholipids in the outer leaflet of red blood cells (RBCs) is reported. Methyl-α-cyclodextrin, loaded with exogenous lipids, was used to extract phospholipids from the membrane outer leaflet, while delivering lipids to the cell to maintain cell membrane integrity. Thin layer chromatography and lipidomics demonstrated that the extracted lipids were from the membrane outer leaflet. Phosphatidylcholines (PC) and sphingomyelins (SM) were the most abundant phospholipids in the RBCs outer leaflet with PC 34:1 and SM 34:1 being the most abundant species. Fluorescence quenching confirmed the delivery of exogenous lipids to the cell outer leaflet. The developed lipid exchange method was then used to remove phosphatidylserine, a phagocyte recognition marker, from the outer leaflet of senescent RBCs. Senescent RBCs with reconstituted membranes were phagocytosed in significantly lower amounts compared to control cells, demonstrating the efficiency of the lipid exchange process and its application in modifying cell–cell interactions.

Fluorescence-based techniques have been an integral factor in the study of cellular and model membranes. Fluorescence studies carried out on model membranes have provided valuable structural information and have helped reveal mechanistic detail regarding the formation and properties of ordered lipid domains, commonly known as lipid rafts. This chapter focuses on four techniques, based on fluorescence spectroscopy or microscopy, which are commonly used to analyze lipid rafts. The techniques described in this chapter may be used in a variety of ways and in combination with other techniques to provide valuable information regarding lipid order and domain formation, especially in model membranes.

The plasma membrane of eukaryotic cells is commonly believed to contain ordered lipid domains. The interest in understanding the origin of such domains has led to extensive studies on the phase behavior of mixed lipid systems. Three-component phase diagrams, composed of a high melting temperature (Tm) lipid, cholesterol, and a low Tm lipid have been valuable in studying lipid phase behavior. However, developing phase diagrams over the entire composition space and with precise tie-lines requires significant experimental effort. In this study, a machine learning approach was used to predict the Tm of lipids and generate phase diagrams from lipid mixtures. First, artificial neural network (ANN) was used for the prediction of Tm. The network was trained using available Tm data and was able to generate Tm values that closely matched literature results for its testing dataset. This model was then used to predict the Tm for lipids that have not yet been experimentally tested. Then, random forests (RF) and support vector machines (SVM) were trained and tested for their ability to predict a test three-component phase diagram. The model from the RF algorithm was able to generate a diagram that closely matched published results. This model was then used to generate phase diagrams for lipid mixtures at various temperatures and various degrees of unsaturation. This machine learning approach to the generation of lipid phase diagrams has the potential to save significant time and resources in studies of lipid phase behavior.

With the increasing industrial and biomedical applications of engineered nanomaterials (ENMs), concerns have been raised regarding the increased risk of exposure. Exposure to ENMs can potentially lead to adverse health effects including cell toxicity. The plasma membrane, a lipid bilayer surrounding all cells, is the first cellular entity that comes into contact with foreign particles, and membrane damage by ENMs is one of the potential mechanisms through which ENMs induce cytotoxicity. In recent years, significant effort has been focused on elucidating the complex interactions at the particle–plasma membrane interface. Such studies have primarily relied on membrane models to tease out what particle physicochemical properties might perturb the structure and function of the cell plasma membrane. However, the diversity of membrane models has made it difficult to translate the results obtained from studies with one model to another and ultimately to live cells. This review summarizes the current knowledge on ENM–plasma membrane interactions based on studies in membrane models including lipid monolayers, supported lipid bilayers, and lipid vesicles. The mechanisms of membrane disruption by the ENMs in each model have been discussed in detail and the role of the membrane model itself in modulating the results has been described. In addition, results in membrane models have been compared with the current knowledge about cells. Finally, some of the challenges toward improving the current membrane models and enhancing the environmental relevance of studies are discussed.

Plasma membrane damage is one of the primary mechanisms through which engineered nanoparticles induce cell toxicity. Over the past decade, a number of mechanistic studies have used membrane models to examine how nanoparticles alter membrane structure and integrity. However, the role of membrane lipid asymmetry, considering that the plasma membrane has a different lipid composition in the exofacial leaflet compared to the cytofacial leaflet, in regulating nanoparticle–membrane interactions has remained obscure. In the current study, the role of individual membrane leaflets in regulating the interactions of membrane models and erythrocytes with engineered silica nanoparticles (50 and 100 nm) was examined. It was found that silica nanoparticles bind to and disrupt synthetic vesicles mimicking the exofacial leaflet but not those mimicking the cytofacial leaflet of the erythrocyte plasma membrane. Forster resonance energy transfer (FRET) studies revealed that nanoparticles disrupted vesicle integrity by inducing pore formation in vesicles. Imaging giant unilamellar vesicles and further FRET experiments revealed that nanoparticles that disrupt membrane integrity show significant localization at the vesicle surface. Nanoparticles that disrupted vesicles mimicking the exofacial leaflet also induced hemolysis in erythrocytes, suggesting that the exofacial leaflet is the primary regulator of nanoparticle-induced membrane damage. This was confirmed by demonstrating that nanoparticles caused a similar disruptive behavior in symmetric and asymmetric vesicles, which had similar outer leaflet, but different inner leaflet lipid compositions. Together, these studies reveal that membrane lipid asymmetry plays a minor role in nanoparticle-induced membrane disruption with the exofacial leaflet being the primary regulator of interactions.

Understanding the mechanisms by which engineered nanomaterials disrupt the cell plasma membrane is crucial in advancing the industrial and biomedical applications of nanotechnology. While the role of nanoparticle properties in inducing membrane damage has received significant attention, the role of the lipid chemical structure in regulating such interactions is less explored. Here, we investigated the role of the lipid chemical structure in the disruption of lipid vesicles by unmodified silica, carboxyl-modified silica, and unmodified polystyrene nanoparticles (50 nm). The role of the lipid headgroup was examined by comparing nanoparticle effects on vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vs an inverse phosphocholine (PC) with the same acyl chain structure. The role of acyl chain saturation was examined by comparing nanoparticle effects on saturated vs unsaturated PCs and sphingomyelins. Nanoparticle effects on PCs (glycerol backbone) vs sphingomyelins (sphingosine backbone) were also examined. Results showed that the lipid headgroup, backbone, and acyl chain saturation affect nanoparticle binding to and disruption of the membranes. A low headgroup tilt angle and the presence of a trimethylammonium moiety at the vesicle surface are required for unmodified nanoparticles to induce membrane disruption. Lipid backbone structure significantly affects nanoparticle-membrane interactions, with carboxyl-modified particles only disrupting lipids containing cis unsaturation and a sphingosine backbone. Acyl chain saturation makes vesicles more resistant to particles by increasing lipid packing in vesicles, impeding molecular interactions. Finally, nanoparticles were capable of changing the lipid packing, resulting in pore formation in the process. These observations are important in interpreting nanoparticle toxicity to biological membranes.

Despite a number of fundamental studies, the interactions of engineered nanoparticles with the cell plasma membrane are still not well understood. Membrane models, such as lipid monolayers and bilayers, have been used to provide a mechanistic understanding of nanoparticle effects on the structure and integrity of the cell membrane. However, the role of the membrane model itself in regulating nanoparticle–lipid interactions has generally been overlooked. In an effort to elucidate how changing the membrane model affects the resulting interactions with nanoparticles, the current study investigates the interaction of silica nanoparticles (104 ± 5 nm) with different surface-functional groups: plain (silanol), amine, and poly(ethylene glycol) (PEG), with various molecular weights of 2k, 5k, and 20k Daltons, with lipid monolayers and bilayers of the same composition. Studies with lipid monolayers demonstrated that PEGylated particles, regardless of the PEG molecular weight, and unlike plain and amine-modified particles, were able to penetrate the monolayer and disrupt the structure of lipids as evidenced by tensiometry and atomic force microscopy. In contrast, when interacting with lipid bilayers in suspension (i.e. vesicles), plain, amine-modified, and PEG 20k-modified particles disrupted vesicle structure, causing significant leakage, while PEG 2k- and 5k-modified particles did not affect the integrity of the vesicles. In summary, while the interactions of silica nanoparticles with membrane models is dependent on particle surface properties, a clear difference is observed in particle interactions with lipid monolayers compared to bilayers.

Fungal glucosylceramide (GlcCer) is a plasma membrane sphingolipid in which the sphingosine backbone is unsaturated in carbon position 8 (C8) and methylated in carbon position 9 (C9). Studies in the fungal pathogen, Cryptococcus neoformans, have shown that loss of GlcCer synthase activity results in complete loss of virulence in the mouse model. However, whether the loss of virulence is due to the lack of the enzyme or to the loss of the sphingolipid is not known. In this study, we used genetic engineering to alter the chemical structure of fungal GlcCer and studied its effect on fungal growth and pathogenicity. Here we show that unsaturation in C8 and methylation in C9 is required for virulence in the mouse model without affecting fungal growth in vitro or common virulence factors. However, changes in GlcCer structure led to a dramatic susceptibility to membrane stressors resulting in increased cell membrane permeability and rendering the fungal mutant unable to grow within host macrophages. Biophysical studies using synthetic vesicles containing GlcCer revealed that the saturated and unmethylated sphingolipid formed vesicles with higher lipid order that were more likely to phase separate into ordered domains. Taken together, these studies show for the first time that a specific structure of GlcCer is a major regulator of membrane permeability required for fungal pathogenicity.

Antarctic notothenioids are noted for extreme stenothermy, yet underpinnings of their thermal limits are not fully understood. We hypothesized that properties of ventricular membranes could explain previously observed differences among notothenioids in temperature onset of cardiac arrhythmias and persistent asystole. Microsomes were prepared using ventricles from six species of notothenioids, including four species from the hemoglobin-less (Hb−) family Channichthyidae (icefishes), which also differentially express cardiac myoglobin (Mb), and two species from the (Hb+) Nototheniidae. We determined membrane fluidity and structural integrity by quantifying fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and leakage of 5(6)-carboxyfluorescein, respectively, over a temperature range from ambient (0 °C) to 20 °C. Compositions of membrane phospholipids and cholesterol contents were also quantified. Membranes from all four species of icefishes exhibited greater fluidity than membranes from the red-blooded species N. coriiceps. Thermal sensitivity of fluidity did not vary among species. The greatest thermal sensitivity to leakage occurred between 0 and 5 °C for all species, while membranes from the icefish, Chaenocephalus aceratus (Hb−/Mb−) displayed leakage that was nearly 1.5-fold greater than leakage in N. coriiceps (Hb+/Mb+). Contents of phosphatidylethanolamine (PE) were approximately 1.5-fold greater in icefishes than in red-blooded fishes, and phospholipids had a higher degree of unsaturation in icefishes than in Hb + notothenioids. Cholesterol contents were lowest in Champsocephalus gunnari (Hb−/Mb−) and highest in the two Hb+/Mb + species, G. gibberifrons and N. coriiceps. Our results reveal marked differences in membrane properties and indicate a breach in membrane fluidity and structural integrity at a lower temperature in icefishes than in red-blooded notothenioids.

Disruption of plasma membrane integrity is a primary mechanism of nanoparticle toxicity in cells. Mechanistic studies on nanoparticle-induced membrane damage have been commonly performed using model membranes with a focus on symmetric bilayers, overlooking the fact that the membrane has an asymmetric phospholipid composition. In this study, erythrocytes with normal and scrambled membrane asymmetry were utilized to examine how the loss of membrane asymmetry and the resulting alterations in the outer leaflet lipid composition affect nanoparticle-membrane interactions. Unmodified, amine-modified, and carboxyl-modified silica (30 nm) were used as nanoparticle models. Loss of membrane asymmetry was achieved by induction of eryptosis, using a calcium ionophore. Erythrocyte membrane disruption (hemolysis) by unmodified silica nanoparticles was significantly reduced in eryptotic compared to healthy cells. Amine- and carboxyl-modified particles did not cause hemolysis in either cell. In agreement, a significant reduction in the binding of unmodified silica nanoparticles to the membrane was observed upon loss of membrane asymmetry. Unmodified silica particles also caused significant cell deformation, changing healthy erythrocytes into a spheroid shape. In agreement with findings in the cells, unmodified particles disrupted vesicles mimicking the erythrocyte outer leaflet lipid composition. The degree of disruption and nanoparticle binding to the membrane was reduced in vesicles mimicking the composition of scrambled membranes. Cryo-electron microscopy revealed the presence of lipid layers on particle surfaces, pointing to lipid adsorption as the mechanism for vesicle damage. Together, findings indicate an important role for the lipid composition of the membrane outer leaflet in nanoparticle-induced membrane damage in both vesicles and erythrocytes.

Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.

The emergent applications of nanomaterials in food, cosmetics, bio-sensing, electronics, and medical products necessitates evaluation of their toxicity upon human exposure. Once inside the body, nanomaterials can interact with human cells. The cell membrane, a lipid bilayer that separates the cell from the outer environment, is the first cellular entity that “meets” the nanoparticles. Thus, understanding the interactions of nanoparticles with the cell membrane is expected to provide an understanding of the potential toxicity of such materials. However, despite a number of studies, a clear understanding of the mechanisms of nanoparticle-cell membrane interactions is still lacking and the role of nanoparticle physicochemical properties in such interactions remains ill-defined. In this talk, I will give an overview of my studies on the interactions of engineered nanoparticles with lipid interfaces, including lipid monolayers and bilayers representing simple models of biological membranes. Mechanisms of interactions between engineered polystyrene and silica nanoparticles and lipid interfaces will be discussed and an overview on how nanoparticle physicochemical properties might affect their interactions with lipid interfaces will be provided.