Hillary L. Smith

Early stages of oxidative stress-induced membrane permeabilization: a neutron reflectometry study.

Neutron reflectometry was used to probe in situ the structure of supported lipid bilayers at the solid-liquid interface during the early stages of UV-induced oxidative degradation. Single-component supported lipid bilayers composed of gel phase, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and fluid phase, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), phospholipids were exposed to low-dose oxidative stress generated by UV light and their structures were examined by neutron reflectometry. An interrupted illumination mode, involving exposures in 15 min increments with 2 h intervals between subsequent exposures, and a continuous mode involving a single 60 (or 90) min exposure period were employed. In both cases, pronounced differences in the structure of the lipid bilayer after exposure were observed. Interrupted exposure led to a substantial decrease in membrane coverage but preserved its total thickness at reduced scattering length densities. These results indicate that the initial phase during UV-induced membrane degradation involves the formation of hydrophilic channels within the membrane. This is consistent with the loss of some lipid molecules we observe and attendant reorganization of residual lipids forming hemimicellar edges of the hydrophilic channels. In contrast, continuous illumination produced a graded interface of continuously varied scattering length density (and hence hydrocarbon density) extending 100-150 A into the liquid phase. Exposure of a DPPC bilayer to UV light in the presence of a reservoir of unfused vesicles showed low net membrane disintegration during oxidative stress, presumably because of surface back-filling from the bulk reservoir. Chemical evidence for membrane degradation was obtained by mass spectrometry and Fourier transform infrared spectroscopy. Further evidence for the formation of hydrophilic channels was furnished by fluorescence microscopy and imaging ellipsometry data.

Continuous and discontinuous volume-phase transitions in surface-tethered, photo-crosslinked poly(N-isopropylacrylamide) networks

The water–polymer demixing behavior of surface-tethered poly(N-isopropylacrylamide) networks copolymerized with x mol% of photo-crosslinkable methacryloyloxybenzophenone (MaBP) (x = 1, 3, 5, 10%) was characterized with neutron reflection and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Neutron reflection revealed that water is expelled discontinuously at low crosslink densities and continuously at high crosslink densities. The demarcation between the two behaviors occurred roughly at the critical point as measured by cloud point experiments. The neutron reflection experiments further revealed that the discontinuous concentration jump at low crosslink densities takes place in the presence of significant amounts of water and that water is not completely expelled in the process, with 2–3 water molecules remaining per polymer segment after the collapse of the network, independent of crosslink density. Parallel measurements with ATR-FTIR confirm that the transition is driven by dehydration of the isopropyl groups, with water remaining confined between the amide groups even at temperatures well above the demixing temperature. The internal water, however, is readily exchanged with deuterium oxide at temperatures up to 100 °C. This exchange points to the absence of a hydrophobic skin or physical barrier that would prevent water from completely leaving the film above the demixing temperature.

Mouse fibroblast cell adhesion studied by neutron reflectometry.

Neutron reflectometry (NR) was used to examine live mouse fibroblast cells adherent on a quartz substrate in a deuterated phosphate-buffered saline environment at room temperature. These measurements represent the first, to our knowledge, successful visualization and quantization of the interface between live cells and a substrate with subnanometer resolution using NR. NR data, attributable to the adhesion of live cells, were observed and compared with data from pure growth medium. Independently of surface cell density, the average distance between the center of the cell membrane region and the quartz substrate was determined to be approximately 180 A. The membrane region ( approximately 80 A thick) contains the membranes of cells that are inhomogeneously distributed or undulating, likely conforming to the nonplanar geometry of the supporting adherence proteins. A second region of cell membranes at a greater distance from the substrate was not detectable by NR due to the resolution limits of the technique employed. Attachment of the live cell samples was confirmed by interaction with both distilled water and trypsin. Distinct changes in the NR data after exposure indicate the removal of cells from the substrate.

Analysis of biosurfaces by neutron reflectometry: From simple to complex interfaces

Because of its high sensitivity for light elements and the scattering contrast manipulation via isotopic substitutions, neutron reflectometry (NR) is an excellent tool for studying the structure of soft-condensed material. These materials include model biophysical systems as well as in situ living tissue at the solid-liquid interface. The penetrability of neutrons makes NR suitable for probing thin films with thicknesses of 5-5000 Å at various buried, for example, solid-liquid, interfaces [J. Daillant and A. Gibaud, Lect. Notes Phys. 770, 133 (2009); G. Fragneto-Cusani, J. Phys.: Condens. Matter 13, 4973 (2001); J. Penfold, Curr. Opin. Colloid Interface Sci. 7, 139 (2002)]. Over the past two decades, NR has evolved to become a key tool in the characterization of biological and biomimetic thin films. In the current report, the authors would like to highlight some of our recent accomplishments in utilizing NR to study highly complex systems, including in-situ experiments. Such studies will result in a much better understanding of complex biological problems, have significant medical impact by suggesting innovative treatment, and advance the development of highly functionalized biomimetic materials.

Investigations of surrogate cellular membranes using neutron reflectometry.

The nonperturbative nature of neutron reflectometry (NR) coupled with its isotopic sensitivity has made it an ideal candidate for the study of model biological membranes at the solid-liquid interface. In this article, methods are presented for the creation and characterization of supported model membranes which can mimic many of the critical attributes of cell membranes. It is demonstrated that NR can characterize the structure, composition and organization of model membranes deposited on solid, nanoporous and polymer supports. Additionally, in situ NR measurements of the interactions between model membranes and external stimuli are presented. Finally, an investigation of the adherence region of live mouse fibroblast cells is described.

Understanding dynamic changes in live cell adhesion with neutron reflectometry.

Neutron reflectometry (NR) was used to examine various live cells adhesion to quartz substrates under different environmental conditions, including flow stress. To the best of our knowledge, these measurements represent the first successful visualization and quantization of the interface between live cells and a substrate with sub-nanometer resolution. In our first experiments, we examined live mouse fibroblast cells as opposed to past experiments using supported lipids, proteins, or peptide layers with no associated cells. We continued the NR studies of cell adhesion by investigating endothelial monolayers and glioblastoma cells under dynamic flow conditions. We demonstrated that neutron reflectometry is a powerful tool to study the strength of cellular layer adhesion in living tissues, which is a key factor in understanding the physiology of cell interactions and conditions leading to abnormal or disease circumstances. Continuative measurements, such as investigating changes in tumor cell - surface contact of various glioblastomas, could impact advancements in tumor treatments. In principle, this can help us to identify changes that correlate with tumor invasiveness. Pursuit of these studies can have significant medical impact on the understanding of complex biological problems and their effective treatment, e.g. for the development of targeted anti-invasive therapies.