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

Observation of Stripe Superstructure in the β-Two-Phase Coexistence Region of Cholesterol–Phospholipid Mixtures in Supported Membranes

Visualization of phase coexistence in the β region of cholesterol–phospholipid mixtures consisting of high cholesterol concentrations has proved elusive in lipid bilayers. Here, using the solvent-assisted lipid bilayer approach to prepare supported membranes with high cholesterol fractions close to the cholesterol solubility limit, we report the observation of coexisting liquid phases using fluorescence microscopy. At ∼63 mol % cholesterol, supported membranes consisting of mixtures of DOPC and cholesterol exhibit large-area striping reminiscent of the stripe superstructures that characterize the proximity of the second critical point in the miscibility phase diagram. The properties of the two phases are consistent with condensed complex-rich and cholesterol-rich liquids. Both phases exhibit long-range lateral mobility, and diffusion through a given phase is favored over hopping across the phase boundary, producing an “archipelago effect” and a complex percolation path.NMRC (Natl Medical Research Council, S’pore

Fluorescence blinking, exciton dynamics, and energy transfer domains in single conjugated polymer chains.

In order to understand exciton migration and fluorescence intensity fluctuation mechanisms in conjugated polymer single molecules, we studied fluorescence decay dynamics at "on" and "off" fluorescence intensity levels with 20 ps time resolution using MEH-PPV [poly(2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene] dispersed in PMMA. Two types of intensity fluctuations were distinguished for single chains of conjugated polymers. Abrupt intensity fluctuations (blinking) were found to be always accompanied by corresponding changes in fluorescence lifetime. On the contrary, during "smooth" intensity fluctuations no lifetime change was observed. Time-resolved data in combination with data on fluorescence emission and excitation anisotropy lead to a picture where a single polymer molecule is seen as consisting of several energy transfer domains. Exciton migration is efficient within a domain and not efficient between domains. Each domain can have several emitting low-energy sites over which the exciton continuously migrates until it decays. Emission of individual domains is often highly polarized. Fluorescence from a domain can be strongly quenched by Forster energy transfer to a quencher (hole polaron) if the domain overlaps with the quenching sphere

Membrane attack complex formation on a supported lipid bilayer: initial steps towards a CARPA predictor nanodevice

The rapid advance of nanomedicines and biologicals in pharmacotherapy gives increasing importance to a common adverse effect of these modern therapeutics: complement (C) activation-related pseudoallergy (CARPA). CARPA is a relatively frequent and potentially lethal acute immune toxicity of many intravenous drugs that contain nanoparticles or proteins, whose prediction by laboratory or in vivo testing has not yet been solved. Preliminary studies suggest that proneness of the drug to cause C activation in the blood of patients may predict the individual risk of CARPA, thus, a sensitive and rapid bedside assay for individualized assessment of a drug’s C activating potential could alleviate the CARPA problem. The goal of the present study was to lay down the foundations of a novel approach for real-time sensing of C activation on a supported lipid bilayer platform. We utilized the quartz crystal microbalance with dissipation (QCM-D) monitoring technique to measure the self-assembly of C terminal complex (or membrane attack complex [MAC]) on supported lipid bilayers rapidly assembled by the solvent-assisted lipid bilayer (SALB) formation method, as an immediate measure of C activation. By measuring the changes in frequency and energy dissipation of deposited protein, the technique allows extremely sensitive real-time quantification of the sequential assembly of MAC from its molecular components (C5b-6, C7, C8 and C9) and hence, measure C activation in the ambient medium. The present paper delineates the technique and our initial evidence with purified C proteins that the approach enables sensitive and rapid (real-time) quantification of MAC formation on a silicon-supported planar (phospho) lipid bilayer, which can be used as an endpoint in a clinically useful bedside C activation assay.NRF (Natl Research Foundation, S’pore)Published versio

A Numerical Study on the Effect of Particle Surface Coverage on the Quartz Crystal Microbalance Response

The quartz crystal microbalance (QCM) is a surface-sensitive measurement technique to characterize adsorption processes at solid–fluid interfaces. While QCM measurements are routinely applied to study homogeneous thin films, characterizing heterogeneous films of adsorbed particles remains challenging because QCM is sensitive to not only the mass of adsorbed particles but also to that of hydrodynamically coupled fluid. To extract information about adsorbed particles, it is necessary to model these hydrodynamic effects, however, current QCM models are restricted to the limit of either a very low surface coverage or to the extrapolated limit of saturation coverage. Herein, we investigated QCM measurement responses in the intermediate surface coverage regime, by conducting lattice Boltzmann simulations of monodisperse, spherical particles that are attached to an oscillating surface. From the simulations, we relate the overtone-dependent QCM frequency and bandwidth shifts to particle size, interparticle distance, and the relevant hydrodynamic length scale. The corresponding results are in qualitative agreement with experimental QCM data for sub-100 nm, gel-phase liposomes. Furthermore, the data provide a theoretical basis for extracting particle sizes from QCM data in the high surface coverage limit

Effect of Glucose on the Mobility of Membrane-Adhering Liposomes

Enclosed lipid bilayer structures, referred to as liposomes or lipid vesicles, have a wide range of biological functions, such as cellular signaling and membrane trafficking. The efficiency of cellular uptake of liposomes, a key step in many of these functions, is strongly dependent on the contact area between a liposome and a cell membrane, which is governed by the adhesion force <i>w</i>, the membrane bending energy κ, and the osmotic pressure Δ<i>p</i>. Herein, we investigate the relationship between these forces and the physicochemical properties of the solvent, namely, the presence of glucose (a nonionic osmolyte). Using fluorescence microscopy, we measure the diffusivity <i>D</i> of small (∼50 nm radius), fluorescently labeled liposomes adhering to a supported lipid bilayer or to the freestanding membrane of a giant (∼10 μm radius) liposome. It is observed that glucose in solution reduces <i>D</i> on the supported membrane, while having negligible effect on <i>D</i> on the freestanding membrane. Using well-known hydrodynamic theory for the diffusivity of membrane inclusions, these observations suggest that glucose enhances the contact area between the small liposomes and the underlying membrane, while not affecting the viscosity of the underlying membrane. In addition, quartz crystal microbalance experiments showed no significant change in the hydrodynamic height of the adsorbed liposomes, upon adding glucose. This observation suggests that instead of osmotic deflation, glucose enhances the contact area via adhesion forces, presumably due to the depletion of the glucose molecules from the intermembrane hydration layer