Thermal Annealing Triggers Collapse of Biphasic Supported Lipid Bilayers into Multilayer Islands

The collapse of phase-separating single, supported lipid bilayers, consisting of mixtures of a zwitterionic phospholipid (POPC) and an anionic lipid (DPPA) upon thermal annealing in the presence of ions is examined using a combination of scanning probe, epifluorescence, and ellipsometric microscopies. We find that thermal annealing in the presence of ions in the bathing medium induces an irreversible transition from domain-textured, single supported bilayers to one comprising islands of multibilayer stacks, whose lateral area decays with lamellarity, producing pyramidal staircase “mesa” topography. The higher order lamellae are almost invariably localized above the anionic-lipid rich, gel-phase domains in the parent bilayer and depends on the ions in the bathing medium. The collapse mechanism appears to involve synergistic influences of two independent mechanisms: (1) stabilization of the incipient headgroup–headgroup interface in the emergent multibilayer configuration facilitated by ions in the bath and (2) domain-boundary templated folding. This collapse mechanism is consistent with previous theoretical predictions of topography-induced rippling instability in collapsing lipid monolayers and suggests the role of the mismatch in height and/or spontaneous curvature at domain boundaries in the collapse of phase-separated single supported bilayers

Thermal Annealing Triggers Collapse of Biphasic Supported Lipid Bilayers into Multilayer Islands

The collapse of phase-separating single, supported lipid bilayers, consisting of mixtures of a zwitterionic phospholipid (POPC) and an anionic lipid (DPPA) upon thermal annealing in the presence of ions is examined using a combination of scanning probe, epifluorescence, and ellipsometric microscopies. We find that thermal annealing in the presence of ions in the bathing medium induces an irreversible transition from domain-textured, single supported bilayers to one comprising islands of multibilayer stacks, whose lateral area decays with lamellarity, producing pyramidal staircase “mesa” topography. The higher order lamellae are almost invariably localized above the anionic-lipid rich, gel-phase domains in the parent bilayer and depends on the ions in the bathing medium. The collapse mechanism appears to involve synergistic influences of two independent mechanisms: (1) stabilization of the incipient headgroup–headgroup interface in the emergent multibilayer configuration facilitated by ions in the bath and (2) domain-boundary templated folding. This collapse mechanism is consistent with previous theoretical predictions of topography-induced rippling instability in collapsing lipid monolayers and suggests the role of the mismatch in height and/or spontaneous curvature at domain boundaries in the collapse of phase-separated single supported bilayers

Thermal Annealing Triggers Collapse of Biphasic Supported Lipid Bilayers into Multilayer Islands

The collapse of phase-separating single, supported lipid bilayers, consisting of mixtures of a zwitterionic phospholipid (POPC) and an anionic lipid (DPPA) upon thermal annealing in the presence of ions is examined using a combination of scanning probe, epifluorescence, and ellipsometric microscopies. We find that thermal annealing in the presence of ions in the bathing medium induces an irreversible transition from domain-textured, single supported bilayers to one comprising islands of multibilayer stacks, whose lateral area decays with lamellarity, producing pyramidal staircase “mesa” topography. The higher order lamellae are almost invariably localized above the anionic-lipid rich, gel-phase domains in the parent bilayer and depends on the ions in the bathing medium. The collapse mechanism appears to involve synergistic influences of two independent mechanisms: (1) stabilization of the incipient headgroup–headgroup interface in the emergent multibilayer configuration facilitated by ions in the bath and (2) domain-boundary templated folding. This collapse mechanism is consistent with previous theoretical predictions of topography-induced rippling instability in collapsing lipid monolayers and suggests the role of the mismatch in height and/or spontaneous curvature at domain boundaries in the collapse of phase-separated single supported bilayers

Lithographically Defined Macroscale Modulation of Lateral Fluidity and Phase Separation Realized via Patterned Nanoporous Silica-Supported Phospholipid Bilayers

Using lithographically defined surfaces consisting of hydrophilic patterns of nanoporous and nonporous (bulk) amorphous silica, we show that fusion of small, unilamellar lipid vesicles produces a single, contiguous, fluid bilayer phase experiencing a predetermined pattern of interfacial interactions. Although long-range lateral fluidity of the bilayer, characterized by fluorescence recovery after photobleaching, indicates a nominally single average diffusion constant, fluorescence microscopy-based measurements of temperature-dependent onset of fluidity reveals a locally enhanced fluidity for bilayer regions supported on nanoporous silica in the vicinity of the fluid–gel transition temperature. Furthermore, thermally quenching lipid bilayers composed of a binary lipid mixture below its apparent miscibility transition temperature induces qualitatively different lateral phase separation in each region of the supported bilayer: The nanoporous substrate produces large, microscopic domains (and domain-aggregates), whereas surface texture characterized by much smaller domains and devoid of any domain-aggregates appears on bulk glass-supported regions of the single-lipid bilayer. Interestingly, lateral distribution of the constituent molecules also reveals an enrichment of gel-phase lipids over nanoporous regions, presumably as a consequence of differential mobilities of constituent lipids across the topographic bulk/nanoporous boundary. Together, these results reveal that subtle local variations in constraints imposed at the bilayer interface, such as by spatial variations in roughness and substrate adhesion, can give rise to significant differences in macroscale biophysical properties of phospholipid bilayers even within a single, contiguous phase

Role of Squalene in the Organization of Monolayers Derived from Lipid Extracts of Halobacterium salinarum

We have studied interfacial compressibility and lateral organization in monolayer configurations of total (squalene containing) and polar (squalene-devoid) lipid extracts of Halobacterium salinarum NRC-1, an extremely halophilic archaeon. Pressure–area isotherms derived from Langmuir experiments reveal that packing characteristics and elastic compressibility are strongly influenced by the presence of squalene in the total lipid extract. In conjunction with control experiments using mixtures of DPhPC and squalene, our results establish that the presence of squalene significantly extends elastic area compressibility of total lipid extracts, suggesting it has a role in facilitating tighter packing of archaeal lipid mixtures. Moreover, we find that squalene also influences spatial organization in archaeal membranes. Epifluorescence and atomic force microscopy characterization of Langmuir monolayers transferred onto solid hydrophilic substrates reveal an unusual domain morphology. Individual domains of microscopic dimensions (as well as their extended networks) exhibiting a peculiar bowl-like topography are evident in atomic force microscopy images. The tall rims outlining individual domains indicate that squalene accumulates at the domain periphery in a manner similar to the accumulation of cholesterol at domain boundaries in their mixtures with phospholipids. Taken together, the results presented here support the notion that squalene plays a role in modulating molecular packing and lateral organization (i.e., domain formation) in the membranes of archaea analogous to that of cholesterol in eukaryotic membranes

Role of Squalene in the Organization of Monolayers Derived from Lipid Extracts of Halobacterium salinarum

We have studied interfacial compressibility and lateral organization in monolayer configurations of total (squalene containing) and polar (squalene-devoid) lipid extracts of Halobacterium salinarum NRC-1, an extremely halophilic archaeon. Pressure–area isotherms derived from Langmuir experiments reveal that packing characteristics and elastic compressibility are strongly influenced by the presence of squalene in the total lipid extract. In conjunction with control experiments using mixtures of DPhPC and squalene, our results establish that the presence of squalene significantly extends elastic area compressibility of total lipid extracts, suggesting it has a role in facilitating tighter packing of archaeal lipid mixtures. Moreover, we find that squalene also influences spatial organization in archaeal membranes. Epifluorescence and atomic force microscopy characterization of Langmuir monolayers transferred onto solid hydrophilic substrates reveal an unusual domain morphology. Individual domains of microscopic dimensions (as well as their extended networks) exhibiting a peculiar bowl-like topography are evident in atomic force microscopy images. The tall rims outlining individual domains indicate that squalene accumulates at the domain periphery in a manner similar to the accumulation of cholesterol at domain boundaries in their mixtures with phospholipids. Taken together, the results presented here support the notion that squalene plays a role in modulating molecular packing and lateral organization (i.e., domain formation) in the membranes of archaea analogous to that of cholesterol in eukaryotic membranes

Role of Squalene in the Organization of Monolayers Derived from Lipid Extracts of Halobacterium salinarum

We have studied interfacial compressibility and lateral organization in monolayer configurations of total (squalene containing) and polar (squalene-devoid) lipid extracts of Halobacterium salinarum NRC-1, an extremely halophilic archaeon. Pressure–area isotherms derived from Langmuir experiments reveal that packing characteristics and elastic compressibility are strongly influenced by the presence of squalene in the total lipid extract. In conjunction with control experiments using mixtures of DPhPC and squalene, our results establish that the presence of squalene significantly extends elastic area compressibility of total lipid extracts, suggesting it has a role in facilitating tighter packing of archaeal lipid mixtures. Moreover, we find that squalene also influences spatial organization in archaeal membranes. Epifluorescence and atomic force microscopy characterization of Langmuir monolayers transferred onto solid hydrophilic substrates reveal an unusual domain morphology. Individual domains of microscopic dimensions (as well as their extended networks) exhibiting a peculiar bowl-like topography are evident in atomic force microscopy images. The tall rims outlining individual domains indicate that squalene accumulates at the domain periphery in a manner similar to the accumulation of cholesterol at domain boundaries in their mixtures with phospholipids. Taken together, the results presented here support the notion that squalene plays a role in modulating molecular packing and lateral organization (i.e., domain formation) in the membranes of archaea analogous to that of cholesterol in eukaryotic membranes

Role of Squalene in the Organization of Monolayers Derived from Lipid Extracts of Halobacterium salinarum

We have studied interfacial compressibility and lateral organization in monolayer configurations of total (squalene containing) and polar (squalene-devoid) lipid extracts of Halobacterium salinarum NRC-1, an extremely halophilic archaeon. Pressure–area isotherms derived from Langmuir experiments reveal that packing characteristics and elastic compressibility are strongly influenced by the presence of squalene in the total lipid extract. In conjunction with control experiments using mixtures of DPhPC and squalene, our results establish that the presence of squalene significantly extends elastic area compressibility of total lipid extracts, suggesting it has a role in facilitating tighter packing of archaeal lipid mixtures. Moreover, we find that squalene also influences spatial organization in archaeal membranes. Epifluorescence and atomic force microscopy characterization of Langmuir monolayers transferred onto solid hydrophilic substrates reveal an unusual domain morphology. Individual domains of microscopic dimensions (as well as their extended networks) exhibiting a peculiar bowl-like topography are evident in atomic force microscopy images. The tall rims outlining individual domains indicate that squalene accumulates at the domain periphery in a manner similar to the accumulation of cholesterol at domain boundaries in their mixtures with phospholipids. Taken together, the results presented here support the notion that squalene plays a role in modulating molecular packing and lateral organization (i.e., domain formation) in the membranes of archaea analogous to that of cholesterol in eukaryotic membranes

Lipid Cross-Linking of Nanolipoprotein Particles Substantially Enhances Serum Stability and Cellular Uptake

Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100% serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ∼10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. These results collectively support the potential utility of X-NLPs for a variety of in vivo applications