Recapitulating the Lateral Organization of Membrane Receptors at the Nanoscale.

Many cell membrane functions emerge from the lateral presentation of membrane receptors. The link between the nanoscale organization of the receptors and ligand binding remains, however, mostly unclear. In this work, we applied surface molecular imprinting and utilized the phase behavior of lipid bilayers to create platforms that recapitulate the lateral organization of membrane receptors at the nanoscale. We used liposomes decorated with amphiphilic boronic acids that commonly serve as synthetic saccharide receptors and generated three lateral modes of receptor presentation─random distribution, nanoclustering, and receptor crowding─and studied their interaction with saccharides. In comparison to liposomes with randomly dispersed receptors, surface-imprinted liposomes resulted in more than a 5-fold increase in avidity. Quantifying the binding affinity and cooperativity proved that the boost was mediated by the formation of the nanoclusters rather than a local increase in the receptor concentration. In contrast, receptor crowding, despite the presence of increased local receptor concentrations, prevented multivalent oligosaccharide binding due to steric effects. The findings demonstrate the significance of nanometric aspects of receptor presentation and generation of multivalent ligands including artificial lectins for the sensitive and specific detection of glycans.

Förster Resonance Energy Transfer Nanoplatform Based on Recognition-Induced Fusion/Fission of DNA Mixed Micelles for Nucleic Acid Sensing.

The dynamic nature of micellar nanostructures is employed to form a self-assembled Förster resonance energy transfer (FRET) nanoplatform for enhanced sensing of DNA. The platform consists of lipid oligonucleotide FRET probes incorporated into micellar scaffolds, where single recognition events result in fusion and fission of DNA mixed micelles, triggering the fluorescence response of multiple rather than a single FRET pair. In comparison to conventional FRET substrates where a single donor interacts with a single acceptor, the micellar multiplex FRET system showed ∼20- and ∼3-fold enhancements in the limit of detection and FRET efficiency, respectively. This supramolecular signal amplification approach could potentially be used to improve FRET-based diagnostic assays of nucleic acid and non-DNA based targets.

Hybrid Biomimetic Interfaces Integrating Supported Lipid Bilayers with Decellularized Extracellular Matrix Components.

This paper describes the functionalization of solid supported phospholipid bilayer with decellularized extracellular matrix (dECM) components, toward the development of biomimetic platforms that more closely mimic the cell surface environment. The dECM was obtained through a combination of chemical and enzymatic treatments of mouse adipose tissue that contains collagen, fibronectin, and glycosaminoglycans (GAGs). Using amine coupling chemistry, the dECM components were attached covalently to the surface of a supported lipid bilayer containing phospholipids with reactive carboxylic acid headgroups. The bilayer formation and the kinetics of subsequent dECM conjugation were monitored by quartz crystal microbalance with dissipation (QCM-D). Fluorescence recovery after photobleaching (FRAP) confirmed the fluidity of the membrane after functionalization with dECM. The resulting hybrid biomimetic interface supports the attachment and survival of the human hepatocyte Huh 7.5 and maintains the representative hepatocellular function. Importantly, the platform is suitable for monitoring the lateral organization and clustering of cell-binding ligands and growth factor receptors in the presence of the rich biochemical profile of tissue-derived ECM components.

Dynamic Cellular Interactions with Extracellular Matrix Triggered by Biomechanical Tuning of Low-Rigidity, Supported Lipid Membranes.

The behavior of cells in a tissue is regulated by chemical as well as physical signals arising from their microenvironment. While gel-based substrates have been widely used for mimicking a range of substrate rigidities, there is a need for the development of low rigidity substrates for mimicking the physical properties of soft tissues. In this study, the authors report the development of a supported lipid bilayer (SLB)-based low rigidity substrate for cell adhesion studies. SLBs are functionalized with either collagen I or fibronectin via covalent, amine coupling to a carboxyl group-modified lipid molecule. While the lipid molecules in the bilayer show long-range lateral mobility, the covalently functionalized extracellular matrix (ECM) proteins are immobile on the bilayer surface. Specific adhesion of cells results in an enrichment of the protein on the bilayer and the appearance of a zone of depletion around the cells. Further, the lateral reorganization of the ECM proteins is controlled by altering the fluidity of lipid molecules in the substrate. Thus, the experimental platform developed in this study can be utilized for addressing basic questions related to cell adhesion on low rigidity substrates as well as biomedical applications requiring adhesion of cells to low rigidity substrates.

Optimizing the Performance of Supported Lipid Bilayers as Cell Culture Platforms Based on Extracellular Matrix Functionalization.

Strategies to fabricate biofunctionalized surfaces are essential for many biotechnological applications. Zwitterionic lipid bilayer coatings doped with lipids with chemically selective headgroups provide a robust platform for immobilization of biomolecules in an antifouling, protein resistant background. Herein, we assess the biological activity of two important components of the extracellular matrix (ECM), collagen type I (Col I) and fibronectin (FN), which are covalently attached to a supported lipid bilayer (SLB), and compare their activity with the same proteins, nonspecifically adsorbed onto a SiO2 surface. The characterization of protein coatings by quartz crystal microbalance with dissipation revealed that Col I and FN attached to SLB are less dense and have higher structural flexibility than when adsorbed onto SiO2. Cell adhesion, proliferation, and function, as well as Col I-FN interactions, were more efficient on the ECM-functionalized SLB, making it a promising platform for cell-based diagnostics, tissue engineering, medical implants, and biosensor development.

Size-dependent, stochastic nature of lipid exchange between nano-vesicles and model membranes.

The interaction of nanoscale lipid vesicles with cell membranes is of fundamental importance for the design and development of vesicular drug delivery systems. Here, we introduce a novel approach to study vesicle-membrane interactions whereby we are able to probe the influence of nanoscale membrane properties on the dynamic adsorption, exchange, and detachment of vesicles. Using total internal reflection fluorescence (TIRF) microscopy, we monitor these processes in real-time upon the electrostatically tuned attachment of individual, sub-100 nm vesicles to a supported lipid bilayer. The observed exponential vesicle detachment rate depends strongly on the vesicle size, but not on the vesicle charge, which suggests that lipid exchange occurs during a single stochastic event, which is consistent with membrane stalk formation. The fluorescence microscopy assay developed in this work may enable measuring of the probability of stalk formation in a controlled manner, which is of fundamental importance in membrane biology, offering a new tool to understand nanoscale phenomena in the context of biological sciences.

Multistep Compositional Remodeling of Supported Lipid Membranes by Interfacially Active Phosphatidylinositol Kinases.

The multienzyme catalytic phosphorylation of phosphatidylinositol (PI) in a supported lipid membrane platform is demonstrated for the first time. One-step treatment with PI 4-kinase IIIß (PI4Kß) yielded PI 4-phosphate (PI4P), while a multistep enzymatic cascade of PI4Kß followed by PIP 5-kinase produced PI-4,5-bisphosphate (PI(4,5)P2 or PIP2). By employing quartz crystal microbalance with dissipation monitoring, we were able to track membrane association of kinase enzymes for the first time as well as detect PI4P and PI(4,5)P2 generation based on subsequent antibody binding to the supported lipid bilayers. Pharmacologic inhibition of PI4Kß by a small molecule inhibitor was also quantitatively assessed, yielding an EC50 value that agrees well with conventional biochemical readout. Taken together, the development of a PI-containing supported membrane platform coupled with surface-sensitive measurement techniques for kinase studies opens the door to exploring the rich biochemistry and pharmacological targeting of membrane-associated phosphoinositides.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Fabrication of charged membranes by the solvent-assisted lipid bilayer (SALB) formation method on SiO2 and Al2O3.

In this study, we employed the solvent-assisted lipid bilayer (SALB) formation method to fabricate charged membranes on solid supports. The SALB formation method exploits a ternary mixture of lipid-alcohol-aqueous buffer to deposit lamellar phase structures on solid supports upon gradual increase of the buffer fraction. Using the quartz crystal microbalance with dissipation (QCM-D) technique, we investigated the formation of negatively and positively charged membranes via the SALB formation method and directly compared with the vesicle fusion method on two different oxide films. Bilayers containing an increasing fraction of negatively charged DOPS lipid molecules were successfully formed on both SiO2 and Al2O3 substrates using the SALB formation method at physiological pH (7.5). In contrast, the vesicle fusion method did not support bilayer formation on Al2O3 and those containing more than 10% DOPS ruptured on SiO2 only under acidic conditions (pH 5). Characterization of the fraction of negatively charge DOPS by in situ annexin 5A binding assay revealed that the fraction of DOPS lipid molecules in the bilayers formed on Al2O3 is significantly higher than that formed on SiO2. This suggests that the SALB self-assembly of charged membranes is predominantly governed by the electrostatic interaction. Furthermore, our findings indicate that when multicomponent lipid mixtures are used, the relative fraction of lipids in the bilayer may differ from the fraction of lipids in the precursor mixture.