Yuri Gerelli

Lipid Exchange and Flip-Flop in Solid Supported Bilayers

Inter- and intrabilayer transfer of phospholipid molecules was investigated by neutron reflectometry. The structure of solid supported lipid bilayers exposed to a solution of isotopically labeled vesicles was monitored as a function of temperature, time, and vesicle concentration. Lipid interbilayer exchange was shown to be the time limiting process, while lipid intrabilayer movement, the so-called flip-flop, was too fast to be visualized within the experimental acquisition time. The exchange process was characterized by an Arrhenius-like behavior and the activation energy of the process was concentration-independent. The results are discussed and compared extensively with the literature available on the topic.

Lipid Rearrangement in DSPC/DMPC Bilayers: A Neutron Reflectometry Study

Lipid translocation in membranes is still far from being understood and well characterized for natural cell membranes as well as for simpler bilayer model systems. Several discrepancies with respect to its occurrence and its characteristic time scale are present in the literature. In the current work, the structural changes induced by lipid rearrangement in a distearoyl-/dimyristoyl-phosphocholine binary lipid system have been addressed by means of neutron reflectivity. It has been shown that a fast, spontaneous compositional reorganization with lipid transfer between the two leaflets of the bilayer takes place only when the lipid species are both in the fluid phase. This process has been identified as the so-called lipid flip-flop. Moreover, the influence of the preparation protocol on the structural properties of the system has been investigated.

Neutron Reflectometry reveals the interaction between functionalized SPIONs and the surface of lipid bilayers

The safe application of nanotechnology devices in biomedicine requires fundamental understanding on how they interact with and affect the different components of biological systems. In this respect, the cellular membrane, the cell envelope, certainly represents an important target or barrier for nanosystems. Here we report on the interaction between functionalized SuperParamagnetic Iron Oxide Nanoparticles (SPIONs), promising contrast agents for Magnetic Resonance Imaging (MRI), and lipid bilayers that mimic the plasma membrane. Neutron Reflectometry, supported by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) experiments, was used to characterize this interaction by varying both SPION coating and lipid bilayer composition. In particular, the interaction of two different SPIONs, functionalized with a cationic surfactant and a zwitterionic phospholipid, and lipid bilayers, containing different amount of cholesterol, were compared. The obtained results were further validated by Dynamic Light Scattering (DLS) measurements and Cryogenic Transmission Electron Microscopy (Cryo-TEM) images. None of the investigated functionalized SPIONs were found to disrupt the lipid membrane. However, in all case we observed the attachment of the functionalized SPIONs onto the surface of the bilayers, which was affected by the bilayer rigidity, i.e. the cholesterol concentration.

Nucleolipid bilayers: A quartz crystal microbalance and neutron reflectometry study.

POP-Ade (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyladenosine) is a biocompatible anionic nucleolipid with the DNA nucleoside, Adenosine, in the polar headgroup. We have studied the affinity of nucleic acids of different contour length, composition and structure toward supported lipid bilayers (SLB) composed of POP-Ade mixed with the zwitterionic phospholipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) using quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). In order to highlight the specificity of the nucleic acid interaction, the results were compared with data obtained for SLB containing the anionic phospholipid POPG (1-palmitoyl-2-oleoyl-sn-phosphatidyl-glycerol) replacing POP-Ade. Our results demonstrate that the presence of a nucleobase headgroup provides the bilayers with the ability to bind single stranded nucleic acids in a selective fashion, according to a Watson-Crick pattern. In addition the interaction with double stranded nucleic acids was strengthened. Overall, these findings represent fundamental information for the design of biocompatible DNA vectors with DNA-RNA-based amphiphiles.

Multi-lamellar organization of fully deuterated lipid extracts of yeast membranes

Neutron scattering studies on mimetic biomembranes are currently limited by the low availability of deuterated unsaturated lipid species. In the present work, results from the first neutron diffraction experiments on fully deuterated lipid extracts from the yeast Pichia pastoris are presented. The structural features of these fully deuterated lipid stacks are compared with those of their hydrogenous analogues and with other similar synthetic systems. The influence of temperature and humidity on the samples has been investigated by means of small momentum-transfer neutron diffraction. All of the lipid extracts investigated self-assemble into multi-lamellar stacks having different structural periodicities; the stacking distances are affected by temperature and humidity without altering the basic underlying arrangement. At high relative humidity the deuterated and hydrogenous samples are similar in their multi-lamellar arrangement, being characterized by two main periodicities of ∼75 and ∼110 Å reflecting the presence of a large number of polar phospholipid molecules. Larger differences are found at lower relative humidity, where hydrogenous lipids are characterized by a larger single lamellar structure than that observed in the deuterated samples. In both cases the heterogeneity in composition is reflected in a wide structural complexity. The different behaviour upon dehydration can be related to compositional differences in the molecular composition of the two samples, which is attributed to metabolic effects related to the use of perdeuterated growth media.

Biogenic Supported Lipid Bilayers from Nanosized Extracellular Vesicles

Fabrication of synthetic surfaces that reproduce structure and function of biological membranes is an open challenge. This work reports the first example of supported lipid bilayers obtained from extracellular vesicles (EVSLBs). EVSLBs harness and pattern in two dimensions key properties of extracellular vesicle (EV) membranes, which present intermediate complexity between synthetic mimics and natural membranes and innate link to phenotype and function of the originating cells. Silica‐supported EVSLBs are formed from nanosized EVs—separated from culture media of prostate cancer model TRAMP‐C2 murine cells—following a characteristic crowding‐fusion pathway. They display peculiar properties at different length scales, such as 2.5 nm roughness, lipid‐raft‐like domains, and cushioned patches (with the cushion filled with the native EV biomolecular cargo), which preserve the native EV membrane orientation with the proto‐oncogene tyrosine‐protein kinase Src (c‐Src) accessible to antibody recognition.

Disruption of Asymmetric Lipid Bilayer Models Mimicking the Outer Membrane of Gram-Negative Bacteria by an Active Plasticin

The outer membrane (OM) of Gram-negative bacteria is a complex and asymmetric bilayer that antimicrobial peptides must disrupt in order to provoke the cell lysis. The inner and external leaflets of the OM are mainly composed of phospholipids (PL), and lipopolysaccharide (LPS), respectively. Supported lipid bilayers are interesting model systems to mimic the lipid asymmetric scaffold of the OM and determine the quantitative and mechanistic effect of antimicrobial agents, using complementary physicochemical techniques. We report the formation of asymmetric PL/LPS bilayers using the Langmuir-Blodgett/Langmuir-Schaefer technique on two different surfaces (sapphire and mica) with synthetic phospholipids constituting the inner leaflet and bacteria-extracted mutant LPS making up the outer one. The combination of neutron reflectometry and atomic force microscopy techniques allowed the examination of the asymmetric scaffold structure along the normal to the interface and its surface morphology in buffer conditions. Our results allow discrimination of two structurally related peptides, one neutral and inactive, and the other cationic and active. The active cationic plasticin PTCDA1-KF disrupts the asymmetric OM at relevant concentrations through a carpeting scenario characterized by a dramatic removal of lipid molecules from the surface.

Structure of surfactant and phospholipid monolayers at the air/water interface modeled from neutron reflectivity data

Specular neutron reflectometry is a powerful technique to resolve interfacial compositions and structures in soft matter. Surprisingly however, even after several decades, a universal modeling approach for the treatment of data of surfactant and phospholipid monolayers at the air/water interface has not yet been established. To address this shortcoming, first a systematic evaluation of the suitability of different models is presented. The result is a comprehensive validation of an optimum model, which is evidently much needed in the field, and which we recommend as a starting point for future data treatment. While its limitations are openly discussed, consequences of failing to take into account various key aspects are critically examined and the systematic errors quantified. On the basis of this physical framework, we go on to show for the first time that neutron reflectometry can be used to quantify directly in situ at the air/water interface the extent of acyl chain compaction of phospholipid monolayers with respect to their phase. The achieved precision of this novel quantification is ∼10%. These advances together enhance significantly the potential for exploitation in future studies data from a broad range of systems including those involving synthetic polymers, proteins, DNA, nanoparticles and drugs.

Structure and Composition of Native Membrane Derived Polymer-Supported Lipid Bilayers

Over the last two decades, supported lipid bilayers (SLBs) have been extensively used as model systems to study cell membrane structure and function. While SLBs have been traditionally produced from simple lipid mixtures, there has been a recent surge in compositional complexity to better mimic cellular membranes and thereby bridge the gap between classic biophysical approaches and cell experiments. To this end, native cellular membrane derived SLBs (nSLBs) have emerged as a new category of SLBs. As a new type of biomimetic material, an analytical workflow must be designed to characterize its molecular composition and structure. Herein, we demonstrate how a combination of fluorescence microscopy, neutron reflectometry, and secondary ion mass spectrometry offers new insights on structure, composition, and quality of nSLB systems formed using so-called hybrid vesicles, which are a mixture of native membrane material and synthetic lipids. With this approach, we demonstrate that the nSLB formed a continuous structure with complete mixing of the synthetic and native membrane components and a molecular stoichiometry that essentially mirrors that of the hybrid vesicles. Furthermore, structural investigation of the nSLB revealed that PEGylated lipids do not significantly thicken the hydration layer between the bilayer and substrate when on silicon substrates; however, nSLBs do have more topology than their simpler, purely synthetic counterparts. Beyond new insights regarding the structure and composition of nSLB systems, this work also serves to guide future researchers in producing and characterizing nSLBs from their cellular membrane of choice.