Sandrine Morandat

Nanoscale analysis of supported lipid bilayers using atomic force microscopy

During the past 15 years, atomic force microscopy (AFM) has opened new opportunities for imaging supported lipid bilayers (SLBs) on the nanoscale. AFM offers a means to visualize the nanoscale structure of SLBs in physiological conditions. A unique feature of AFM is its ability to monitor dynamic events, like bilayer alteration, remodelling or digestion, upon incubation with various external agents such as drugs, detergents, proteins, peptides, nanoparticles, and solvents. Here, we survey recent progress made in the area.

The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ

Exogenous molecules from dietary sources such as polyphenols are very efficient in preventing the alteration of lipid membranes by oxidative stress. Among the polyphenols, we have chosen to study rosmarinic acid (RA). We investigated the efficiency of RA in preventing lipid peroxidation and in interacting with lipids. We used liposomes of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) to show that RA was an efficient antioxidant. By HPLC, we determined that the maximum amount of RA associated with the lipids was ~1 mol%. Moreover, by using Langmuir monolayers, we evidenced that cholesterol decreases the penetration of RA. The investigation of transferred lipid/RA monolayers by atomic force microscopy revealed that 1 mol% of RA in the membrane was not sufficient to alter the membrane structure at the nanoscale. By fluorescence, we observed no significant modification of membrane permeability and fluidity caused by the interaction with RA. We also deduced that RA molecules were mainly located among the polar headgroups of the lipids. Finally, we prepared DLPC/RA vesicles to evidence for the first time that up to 1 mol% of RA inserts spontaneously in the membrane, which is high enough to fully prevent lipid peroxidation without any noticeable alteration of the membrane structure due to RA insertion.

Topological Effects and Binding Modes Operating with Multivalent Iminosugar-Based Glycoclusters and Mannosidases

Multivalent iminosugars have been recently explored for glycosidase inhibition. Affinity enhancements due to multivalency have been reported for specific targets, which are particularly appealing when a gain in enzyme selectivity is achieved but raise the question of the binding mode operating with this new class of inhibitors. Here we describe the development of a set of tetra- and octavalent iminosugar probes with specific topologies and an assessment of their binding affinities toward a panel of glycosidases including the Jack Bean α-mannosidase (JBαMan) and the biologically relevant class II α-mannosidases from Drosophila melanogaster belonging to glycohydrolase family 38, namely Golgi α-mannosidase ManIIb (GM) and lysosomal α-mannosidase LManII (LM). Very different inhibitory profiles were observed for compounds with identical valencies, indicating that the spatial distribution of the iminosugars is critical to fine-tune the enzymatic inhibitory activity. Compared to the monovalent reference, the best multivalent compound showed a dramatic 800-fold improvement in the inhibitory potency for JBαMan, which is outstanding for just a tetravalent ligand. The compound was also shown to increase both the inhibitory activity and the selectivity for GM over LM. This suggests that multivalency could be an alternative strategy in developing therapeutic GM inhibitors not affecting the lysosomal mannosidases. Dynamic light scattering experiments and atomic force microscopy performed with coincubated solutions of the compounds with JBαMan shed light on the multivalent binding mode. The multivalent compounds were shown to promote the formation of JBαMan aggregates with different sizes and shapes. The dimeric nature of the JBαMan allows such intermolecular cross-linking mechanisms to occur.

Atomic force microscopy of model lipid membranes

AbstractSupported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles. FigureThe experimental atomic force microscopy (AFM) setup used to examine supported lipid bilayers (SLBs) under physiological conditions.

Preparation of an electrochemical biosensor based on lipid membranes in nanoporous alumina.

Model lipid bilayers are versatile tools to investigate the molecular processes occurring at the membrane level. Among the model membranes, substrate supported bilayers have attracted much interest because they are robust and they can be investigated by powerful surface sensitive techniques such as electrochemical measurements. In a biosensor, lipid films can be used not only as a support for the biological sensing elements but also as sensing elements themselves to detect molecules that are able to alter the structure and the properties of biomembranes. In this work, we have prepared a tethered lipid membrane-based biosensor able to detect the alterations of membrane structure and fluidity. This tethered lipid membrane was prepared in a nanoporous aluminium oxide that provides a high surface area and a protective environment against dewetting. The membrane contained PEG-PE lipids as hydrating, protective and tethering agents and ubiquinone which is a redox lipophilic mediator embedded within the acyl chains of the lipid bilayer. The lipid membrane was prepared inside the pores of the nanoporous support by a PEG-triggered fusion of liposomes. This sensing system was efficient to detect the alterations of lipid membranes that are induced by the addition of a commonly used non-ionic detergent: Triton X-100.

The potent antimalarial drug cyclosporin A preferentially destabilizes sphingomyelin-rich membranes.

Cyclosporin A (CsA) is a hydrophobic cyclic peptide produced by a fungus. CsA is widely used as an immunosuppressive agent to inhibit the rejection of transplanted organs. CsA also exhibits an antiparasitic activity against Plasmodium, the microorganism responsible for malaria disease. This antimalarial activity is not completely understood yet. In this study, we have used Langmuir monolayers and atomic force microscopy to investigate the interaction of CsA with different lipids: phosphatidylcholines with different molecular packing, cholesterol, and sphingomyelin. We have shown that CsA inserts in all kinds of lipid monolayers but it has a marked preference for sphingomyelin monolayers. This preferential insertion of CsA within sphingomyelin-enriched membranes could explain the antimalarial activity of CsA. Indeed, the parasites need to produce a membrane network inside the erythrocytes, which allows for their proper development/multiplication by exchanging nutrients with the external medium. This membrane network is particularly enriched in sphingomyelin, so the preferential insertion of CsA in these bilayers may destabilize them, thereby inhibiting the development of the parasite.

Preosteoblasts and fibroblasts respond differently to anatase titanium dioxide nanoparticles: A cytotoxicity and inflammation study

There is a bundle of proofs suggesting that some industrial nanoparticles (NPs) can provoke diseases and pollute the environment durably. However, these issues still remain controversial. In the biomedical field, TiO(2) NPs were recently proposed to serve as fillers in polymeric materials to improve bone prostheses and scaffolds. Submicrometer TiO(2) particles could also result from wear debris of prostheses. Thus, it appears to be of the highest importance to elucidate the effects of well-characterized TiO(2) NPs on the behaviour of osteoblasts. In this work, we have measured the toxicity of anatase TiO(2) NPs with two different cell types, on L929 fibroblasts and for the first time on MC-3T3 pre-osteoblasts, with the aim to determine the level of cellular toxicity and inflammation. Our results clearly show that these NPs provoke different dose-response effects, with the pre-osteoblasts being much more sensitive than fibroblasts. Furthermore, we observed that anatase TiO(2) NPs had no effect on cell adhesion. By contrast, both cell types had their morphology and LDH release modified in the presence of NPs. Their DNA was also found to be fragmented as analyzed by quantifying the sub-G1 cell population with flow cytometry. By measuring the production of IL-6 and TNF-α proinflammatory cytokines, we have shown that TNF-α was never produced and that MC-3T3 cells were secreting IL-6. Most importantly, our results highlight the necessity of evaluating the toxicity of prostheses wear debris, and of NP coatings of medical implants, to determine if they can possibly provoke inflammation and inhibit bone reconstruction.

Cytochrome c interaction with neutral lipid membranes: influence of lipid packing and protein charges

The interaction of cytochrome c (cyt c) with fluid/gel neutral supported lipid membranes was investigated by time-lapse atomic force microscopy (AFM). AFM revealed the random formation of depressed areas in fluid membranes promoted by cyt c. These depressions corresponded to the desorption of fluid bilayer patches induced by cyt c. By contrast, the gel domains were never desorbed but they were progressively thickened in the presence of the protein. These results suggest that cyt c molecules might intercalate between the mica and the lipid bilayer. Although the interaction of cyt c with the mica surface is likely to be an artifact, this work is the first direct observation of cyt c ability to cross membranes. Furthermore, our data show that the net positive charge of cyt c molecules plays a pivotal role but it is not the sole factor responsible for cyt c insertion in the membrane.

Cytochrome c provokes the weakening of zwitterionic membranes as measured by force spectroscopy.

Cytochrome c (cyt c) is a small soluble protein from the intermembrane space of mitochondria. This protein is essential because it transfers electrons between two membrane complexes of the respiratory chain. In fact, during this transfer, the positively charged amino-acid residues surrounding the heme in the protein structure allow the cyt c to interact properly with the anionic part of other molecules: mainly the cardiolipin-rich membrane of mitochondria and respiratory complexes. We have previously shown that besides its interaction with anionic lipids, the cyt c is also able to cross neutral lipid membranes. In this work, with the help of AFM and punch-through experiments, we have measured the force required to penetrate the membrane in the fluid and in the gel phases with or without cyt c molecules. In the presence of cyt c molecules, the structures generated by the interaction with the protein were considerably weakened, which led to the desorption of the fluid bilayer and to a considerable loss of cohesion of the gel phase. These results show the usefulness of punch-through experiments in determining the changes of membrane properties in the presence of external agents.

Development of immobilization technique for liver microsomes.

In the present report, physically adsorbed rat liver microsomes were used in order to optimize the immobilization of membrane proteins on solid surfaces for use in biosensing and microreactor applications. Physical adsorption was used to form thin films on solid supports (gold, mica, macroporous aluminum oxide membrane). The characterization of the films was performed by surface plasmon resonance (SPR), atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM). Commercially available macroporous aluminium oxide membranes with a high surface area, allow the retention of a high amount of microsomal membranes in the form of a thin film. Microsomal film functionality was tested by monitoring the activities of several enzymes of phases I and II. Microsomal modified supports can be re-utilized for the same or different substrate after washing with appropriate buffer.