Impact of Lipid Oxidization on Biophysical Properties of Model Cell Membranes

The oxidization of glycerophospholipids in cell membranes due to aging and environmental stresses may cause a variety of pathological and physiological consequences. A variety of oxidized phospholipid products (OxPl) are produced by the chemical oxidization of unsaturated hydrocarbon chains, which would significantly change the physicochemical properties of cell membranes. In this work, we constructed cell membrane models in the absence and presence of two stable oxidized lipid products and investigated their impact on physical properties of supported membranes using quartz crystal microbalance with dissipation (QCM-D) and high-energy X-ray reflectivity (XRR). Our experimental findings suggest that the lipid oxidization up to 20 mol % leads to the rupture of vesicles right after the adsorption. Our XRR analysis unravels the membrane thinning and the decrease in the lateral ordering of lipids, which can be explained by the decrease in the lateral packing of hydrocarbon chains. Further studies on mechanics of membranes incorporating oxidized lipids can be attributed to the decrease in the bending rigidity and the increase in the permeability

Impact of Lipid Oxidization on Vertical Structures and Electrostatics of Phospholipid Monolayers Revealed by Combination of Specular X‑ray Reflectivity and Grazing-Incidence X‑ray Fluorescence

The influence of phospholipid oxidization of floating monolayers on the structure perpendicular to the global plane and on the density profiles of ions near the lipid monolayer has been investigated by a combination of grazing incidence X-ray fluorescence (GIXF) and specular X-ray reflectivity (XRR). Systematic variation of the composition of the floating monolayers unravels changes in the thickness, roughness and electron density of the lipid monolayers as a function of molar fraction of oxidized phospholipids. Simultaneous GIXF measurements enable one to qualitatively determine the element-specific density profiles of monovalent (K⁺ or Cs⁺) and divalent ions (Ca²⁺) in the vicinity of the interface in the presence and absence of two types of oxidized phospholipids (PazePC and PoxnoPC) with high spatial accuracy (±5 Å). We found the condensation of Ca²⁺ near carboxylated PazePC was more pronounced compared to PoxnoPC with an aldehyde group. In contrast, the condensation of monovalent ions could hardly be detected even for pure oxidized phospholipid monolayers. Moreover, pure phospholipid monolayers exhibited almost no ion specific condensation near the interface. The quantitative studies with well-defined floating monolayers revealed how the elevation of lipid oxidization level alters the structures and functions of cell membranes

Morphology and Adhesion Strength of Myoblast Cells on Photocurable Gelatin under Native and Non-native Micromechanical Environments

We have quantitatively determined how the morphology and adhesion strength of myoblast cells can be regulated by photocurable gelatin gels, whose mechanical properties can be fine-tuned by a factor of 10³ (0.1 kPa ≤ <i>E</i> ≤ 140 kPa). The use of such gels allows for the investigation of mechanosensing of cells not only near the natural mechanical microenvironments (<i>E</i> ∼ 10 kPa) but also far below and beyond of the natural condition. Optical microscopy and statistical image analysis revealed that myoblast cells sensitively adopt their morphology in response to the substrate elasticity at <i>E</i> ∼ 1–20 kPa, which can be characterized by the significant changes in the contact area and order parameters of actin cytoskeletons. In contrast, the cells in contact with the gels with lower elastic moduli remained almost round, and the increase in the elasticity beyond <i>E</i> ∼ 20 kPa caused no distinct change in morphology. In addition to the morphological analysis, the adhesion strength was quantitatively evaluated by measuring the critical detachment pressure with an aid of intensive pressure waves induced by picosecond laser pulses. This noninvasive technique utilizing extremely short pressure waves (pulse time width ∼100 ns) enables one to determine the critical pressure for cell detachment with reliable statistics while minimizing the artifacts arising from the inelastic deformation of cells. The adhesion strength also exhibited a transition from weak adhesion to strong adhesion within the same elasticity range (<i>E</i> ∼ 1–20 kPa). A clear correlation between the cell morphology and adhesion strength suggests the coupling of the strain of the substrate and the mechanosensors near focal adhesion sites

Grazing-Incidence Neutron-Induced Fluorescence Probes Density Profiles of Labeled Molecules at Solid/Liquid Interfaces

We report on the use of characteristic prompt γ-fluorescence after neutron capture induced by an evanescent neutron wave to probe densities and depth profiles of labeled molecules at solid/liquid interfaces. In contrast to classical scattering techniques and X-ray fluorescence, this method of “grazing-incidence neutron-induced fluorescence” combines direct chemical specificity, provided by the label, with sensitivity to the interface, inherent to the evanescent wave. We demonstrate that the formation of a supported lipid membrane can be quantitatively monitored from the characteristic fluorescence of ¹⁵⁷Gd³⁺ ions bound to the headgroup of chelator lipids. Moreover, we were able to localize the ¹⁵⁷Gd³⁺ ions along the surface normal with nanometer precision. This first proof of principle with a well-defined model system suggests that the method has a great potential for biology and soft matter studies where spatial resolution and chemical sensitivity are required

Generic Role of Polymer Supports in the Fine Adjustment of Interfacial Interactions between Solid Substrates and Model Cell Membranes

To understand the generic role of soft, hydrated biopolymers in adjusting interfacial interactions at biological interfaces, we designed a defined model of the cell–extracellular matrix contacts based on planar lipid membranes deposited on polymer supports (polymer-supported membranes). Highly uniform polymer supports made out of regenerated cellulose allow for the control of film thickness without changing the surface roughness and without osmotic dehydration. The complementary combination of specular neutron reflectivity and high-energy specular X-ray reflectivity yields the equilibrium membrane–substrate distances, which can quantitatively be modeled by computing the interplay of van der Waals interaction, hydration repulsion, and repulsion caused by the thermal undulation of membranes. The obtained results help to understand the role of a biopolymer in the interfacial interactions of cell membranes from a physical point of view and also open a large potential to generally bridge soft, biological matter and hard inorganic materials

Combination of MD Simulations with Two-State Kinetic Rate Modeling Elucidates the Chain Melting Transition of Phospholipid Bilayers for Different Hydration Levels

The phase behavior of membrane lipids plays an important role in the formation of functional domains in biological membranes and crucially affects molecular transport through lipid layers, for instance, in the skin. We investigate the thermotropic chain melting transition from the ordered <i>L</i>_β phase to the disordered <i>L</i>_α phase in membranes composed of dipalmitoylphosphatidylcholine (DPPC) by atomistic molecular dynamics simulations in which the membranes are subject to variable heating rates. We find that the transition is initiated by a localized nucleus and followed by the propagation of the phase boundary. A two-state kinetic rate model allows characterizing the transition state in terms of thermodynamic quantities such as transition state enthalpy and entropy. The extrapolated equilibrium melting temperature increases with reduced membrane hydration and thus in tendency reproduces the experimentally observed dependence on dehydrating osmotic stress

Fine Adjustment of Interfacial Potential between pH-Responsive Hydrogels and Cell-Sized Particles

We quantitatively determined interfacial potentials between cell-sized particles and stimulus-responsive hydrogels using a microinterferometer. The hydrogel is based on physically interconnected ABA triblock copolymer micelles comprising an inner biocompatible PMPC block and two outer pH-responsive PDPA blocks. The out-of-plane temporal fluctuation in the position of the cell-sized particles was calculated from changes in the interference pattern measured by Reflection Interference Contrast Microscopy (RICM), thus yielding the particle-substrate interaction potential <i>V</i> (Δ<i>h</i>). Measurements in pH buffers ranging from 7.0 to 7.8 resulted in a systematic reduction in height of the potential minima ⟨Δ<i>h</i>⟩ and a concomitant increase in the potential curvature <i>V</i>″ (Δ<i>h</i>). The experimental data were analyzed by applying the modified Ross and Pincus model for polyelectrolytes, while accounting for gravitation, lubrication and van der Waals interactions. Elastic moduli calculated from <i>V</i>″ (Δ<i>h</i>) were in good agreement with those measured by Atomic Force Microscopy. The ability to fine-tune both the gel elasticity and the interfacial potential at around physiological pH makes such triblock copolymer hydrogels a promising biocompatible substrate for dynamic switching of cell–material interactions

DSC thermograms of myelin (20 Mm) under different aqueous media.

<p>In the cases where homogenization takes place–bi-distilled water (black line) and near physiological medium (red line)–a maximum is reached after which a stabilization of the C_p is observed near physiological temperature. In the case of high [Ca²⁺] (blue line), a continuous variation of C_p according to a phase redistribution of components is observed.</p

Number (<i>n</i>) of correlated membranes for the different phases as a function of temperature for whole myelin (empty) and DIGs (filled).

<p>In black is marked the <i>n</i> for the native period, in red for the expanded period and in blue for the compact period. In the presence of physiological (diamonds) and high [Ca²⁺] (circles) media.</p

X-ray diffraction pattern of isolated myelin as a function of temperature in Ringer’s solution.

<p>At high temperature (37–46°C) two single rings Peaks 2 and 4 are observed. At 30°C the faint smallest peak (1) is observed near the beamstop. At 25°C beam splitting is more easily observed in the peak 4 and IV which is also evident in peaks 2 and II at 10°C.</p