Activation energy for pore opening in lipid membranes under an electric field.

The standard model of pore formation was introduced more than fifty years ago, and it has been since, despite some refinements, the cornerstone for interpreting experiments related to pores in membranes. A central prediction of the model concerning pore opening under an electric field is that the activation barrier for pore formation is lowered proportionally to the square of the electric potential. However, this has only been scarcely and inconclusively confronted to experiments. In this paper, we study the electropermeability of model lipid membranes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) containing different fractions of POPC-OOH, the hydroperoxidized form of POPC, in the range 0 to 100 mol %. By measuring ion currents across a 50-µm-diameter black lipid membrane (BLM) with picoampere and millisecond resolution, we detect hydroperoxidation-induced changes to the intrinsic bilayer electropermeability and to the probability of opening angstrom-size or larger pores. Our results over the full range of lipid compositions show that the energy barrier to pore formation is lowered linearly by the absolute value of the electric field, in contradiction with the predictions of the standard model.

Negative lipid membranes enhance the adsorption of TAT-decorated elastin-like polypeptide micelles.

A cell-penetrating peptide (CPP) is a short amino-acid sequence capable of efficiently translocating across the cellular membrane of mammalian cells. However, the potential of CPPs as a delivery vector is hampered by the strong reduction of its translocation efficiency when it bears an attached molecular cargo. To overcome this problem, we used previously developed diblock copolymers of elastin-like polypeptides (ELPBCs), which we end functionalized with TAT (transactivator of transcription), an archetypal CPP built from a positively charged amino acid sequence of the HIV-1 virus. These ELPBCs self-assemble into micelles at a specific temperature and present the TAT peptide on their corona. These micelles can recover the lost membrane affinity of TAT and can trigger interactions with the membrane despite the presence of a molecular cargo. Herein, we study the influence of membrane surface charge on the adsorption of TAT-functionalized ELP micelles onto giant unilamellar vesicles (GUVs). We show that the TAT-ELPBC micelles show an increased binding constant toward negatively charged membranes compared to neutral membranes, but no translocation is observed. The affinity of the TAT-ELPBC micelles for the GUVs displays a stepwise dependence on the lipid charge of the GUV, which, to our knowledge, has not been reported previously for interactions between peptides and lipid membranes. By unveiling the key steps controlling the interaction of an archetypal CPP with lipid membranes, through regulation of the charge of the lipid bilayer, our results pave the way for a better design of delivery vectors based on CPPs.

Accumulation of styrene oligomers alters lipid membrane phase order and miscibility.

Growth of plastic waste in the natural environment, and in particular in the oceans, has raised the accumulation of polystyrene and other polymeric species in eukyarotic cells to the level of a credible and systemic threat. Oligomers, the smallest products of polymer degradation or incomplete polymerization reactions, are the first species to leach out of macroscopic or nanoscopic plastic materials. However, the fundamental mechanisms of interaction between oligomers and polymers with the different cell components are yet to be elucidated. Simulations performed on lipid bilayers showed changes in membrane mechanical properties induced by polystyrene, but experimental results performed on cell membranes or on cell membrane models are still missing. We focus here on understanding how embedded styrene oligomers affect the phase behavior of model membranes using a combination of scattering, fluorescence, and calorimetric techniques. Our results show that styrene oligomers disrupt the phase behavior of lipid membranes, modifying the thermodynamics of the transition through a spatial modulation of lipid composition.

Tear of lipid membranes by nanoparticles.

Health concerns associated with the advent of nanotechnologies have risen sharply when it was found that particles of nanoscopic dimensions reach the cell lumina. Plasma and organelle lipid membranes, which are exposed to both the incoming and the engulfed nanoparticles, are the primary targets of possible disruptions. However, reported adhesion, invagination and embedment of nanoparticles (NPs) do not compromise the membrane integrity, precluding direct bilayer damage as a mechanism for toxicity. Here it is shown that a lipid membrane can be torn by small enough nanoparticles, thus unveiling mechanisms for how lipid membrane can be compromised by tearing from nanoparticles. Surprisingly, visualization by cryo transmission electron microscopy (cryo-TEM) of liposomes exposed to nanoparticles revealed also that liposomal laceration is prevented by particle abundance. Membrane destruction results thus from a subtle particle-membrane interplay that is here elucidated. This brings into a firmer molecular basis the theorized mechanisms of nanoparticle effects on lipid bilayers and paves the way for a better assessment of nanoparticle toxicity.

Active colloids orbiting giant vesicles.

Living or artificial self-propelled colloidal particles show original dynamics when they interact with other objects like passive particles, interfaces or membranes. These active colloids can transport small cargos or can be guided by passive objects, performing simple tasks that could be implemented in more complex systems. Here, we present an experimental investigation at the single particle level of the interaction between isolated active colloids and giant unilamellar lipid vesicles. We observed a persistent orbital motion of the active particle around the vesicle, which is independent of both the particle and the vesicle sizes. Force and torque transfers between the active particle and the vesicle is also described. These results differ in many aspects from recent theoretical and experimental reports on active particles interacting with solid spheres or liquid drops, and may be relevant for the study of swimming particles interacting with cells in biology or with microplastics in environmental science.

A machine learning study of the two states model for lipid bilayer phase transitions.

We have adapted a set of classification algorithms, also known as machine learning, to the identification of fluid and gel domains close to the main transition of dipalmitoyl-phosphatidylcholine (DPPC) bilayers. Using atomistic molecular dynamics conformations in the low and high temperature phases as learning sets, the algorithm was trained to categorise individual lipid configurations as fluid or gel, in relation with the usual two-states phenomenological description of the lipid melting transition. We demonstrate that our machine can learn and sort lipids according to their most likely state without prior assumption regarding the nature of the order parameter of the transition. Results from our machine learning study provide strong support in favour of a two-states model approach of membrane fluidity.

Interaction of Giant Unilamellar Vesicles with the Surface Nanostructures on Dragonfly Wings.

The waxy epicuticle of dragonfly wings contains a unique nanostructured pattern that exhibits bactericidal properties. In light of emerging concerns of antibiotic resistance, these mechano-bactericidal surfaces represent a particularly novel solution by which bacterial colonization and the formation of biofilms on biomedical devices can be prevented. Pathogenic bacterial biofilms on medical implant surfaces cause a significant number of human deaths every year. The proposed mechanism of bactericidal activity is through mechanical cell rupture; however, this is not yet well understood and has not been well characterized. In this study, we used giant unilamellar vesicles (GUVs) as a simplified cell membrane model to investigate the nature of their interaction with the surface of the wings of two dragonfly species, Austrothemis nigrescens and Trithemis annulata, sourced from Victoria, Australia, and the Baix Ebre and Terra Alta regions of Catalonia, Spain. Confocal laser scanning microscopy and cryo-scanning electron microscopy techniques were used to visualize the interactions between the GUVs and the wing surfaces. When exposed to both natural and gold-coated wing surfaces, the GUVs were adsorbed on the surface, exhibiting significant deformation, in the process of membrane rupture. Differences between the tensile rupture limit of GUVs composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine and the isotropic tension generated from the internal osmotic pressure were used to indirectly determine the membrane tensions, generated by the nanostructures present on the wing surfaces. These were estimated as being in excess of 6.8 mN m-1, the first experimental estimate of such mechano-bactericidal surfaces. This simple model provides a convenient bottom-up approach toward understanding and characterizing the bactericidal properties of nanostructured surfaces.

The role of shape disorder in the collective behaviour of aligned fibrous matter.

We study the compression of bundles of aligned macroscopic fibers with intrinsic shape disorder, as found in human hair and in many other natural and man-made systems. We show by a combination of experiments, numerical simulations and theory how the statistical properties of the shapes of the fibers control the collective mechanical behaviour of the bundles. This work paves the way for designing aligned fibrous matter with purposed-designed properties from large numbers of individual strands of selected geometry and rigidity.

DPPC Bilayers in Solutions of High Sucrose Content.

The properties of lipid bilayers in sucrose solutions have been intensely scrutinized over recent decades because of the importance of sugars in the field of biopreservation. However, a consensus has not yet been formed on the mechanisms of sugar-lipid interaction. Here, we present a study on the effect of sucrose on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers that combines calorimetry, spectral fluorimetry, and optical microscopy. Intriguingly, our results show a significant decrease in the transition enthalpy but only a minor shift in the transition temperature. Our observations can be quantitatively accounted for by a thermodynamic model that assumes partial delayed melting induced by sucrose adsorption at the membrane interface.

Kinetic evolution of DOPC lipid bilayers exposed to α-cyclodextrins.

Cyclodextrins are cyclic oligosaccharides capable of forming inclusion complexes with a variety of molecules, and as such have been recognized as a pharmaceutical and biotechnological asset. Cyclodextrins are known to interact with the components of cell membranes, and this correlates with a significant degree of cytotoxicity. In this work, we report on the mechanism of degradation of a model dioleoyl-phosphatidylcholine (DOPC) bilayer exposed to a solution with increasing concentrations of α-cyclodextrins. By combining optical fluorescence microscopy and quartz-crystal microbalance experiments, we study the evolution of supported lipid bilayers (SLBs) and giant unilamellar vesicles (GUVs). The rate of lipid removal is found to display a strong nonlinear dependence on the cyclodextrin concentration. A mechanism involving lipid aggregates is proposed.

Molecular dynamics study of the LCST transition in aqueous poly(N-n-propylacrylamide).

The breadth of technological applications of smart polymers relies on the possibility of tuning their molecular structure to respond to external stimuli. In this context, N-substituted acrylamide-based polymers are widely studied thermoresponsive polymers. Poly(N-n-propylacrylamide) (PNnPAm), which is a structural isomer of the poly(N-isopropylacrylamide) (PNIPAm) exhibits however, a lower phase transition in aqueous solution. In this work, we use all-atom molecular dynamics simulations of PNnPAm in aqueous solutions to study, from a microscopic point-of-view, the influence of chain size and concentration on the LCST of PNnPAm. Our analysis shows that the collapse of a single oligomer of PNnPAm upon heating is dependent on the chain length and corresponds to a complex interplay between hydration and intermolecular interactions. Analysis of systems with multiple chains shows an aggregation of PNnPAm chains above the LCST.

Reply to the 'Comment on "Relating side chain organization of PNIPAm with its conformation in aqueous methanol"' by N. van der Vegt and F. Rodriguez-Ropero, Soft Matter, 2017, 13, DOI: 10.1039/C6SM02139E.

In a comment van der Vegt and Rodriguez-Ropero (vdVRR) challenged our explanation of the co-non-solvency effect of PNIPAm in aqueous methanol solutions. They argue, based on a careful selection of published studies including some of their own, that direct repulsions between the different constituents are sufficient to understand this phenomenon. According to vdVRR, the emerging view of entropic collapse, put forward by Flory (1910-1985) to explain common polymers in poor solvents, would be enough to explain co-non-solvency. In this reply we attempt to bring this discussion into firmer grounds. We provide a more comprehensive view of available experimental, numerical and theoretical results and review basic concepts of physical chemistry and of statistical mechanics of polymer collapse that show how methanol mediated attractions between chain monomers are required to understand this fascinating behavior.

Reply to the 'Comment on "Relating side chain organization of PNIPAm with its conformation in aqueous methanol"' by A. Pica and G. Graziano, Soft Matter, 2017, 13, DOI: 10.1039/C7SM01065F.

We have recently proposed preferential binding by a cosolvent as the mechanism for chain collapse under co-non-solvency. Here we summarise our earlier works and provide further evidence that alcohol preferentially binds to PNIPAm, forming cosolvent bridges, and thus drives the transition. We also clarify some of the common misconceptions evoked in this debate with Pica and Graziano (PG), reinforcing the arguments of our earlier reply-comment [Soft Matter, 2017, 13, 2292] and published works.

Influence of a pH-sensitive polymer on the structure of monoolein cubosomes.

Cubosomes consist in submicron size particles of lipid bicontinuous cubic phases stabilized by surfactant polymers. They provide an appealing road towards the practical use of lipid cubic phases for pharmaceutical and cosmetic applications, and efforts are currently being made to control the encapsulation and release properties of these colloidal objects. We overcome in this work the lack of sensitivity of monoolein cubosomes to pH conditions by using a pH sensitive polymer designed to strongly interact with the lipid structure at low pH. Our cryo-transmission electron microscope (cryo-TEM) and small-angle X-ray scattering (SAXS) results show that in the presence of the polymer the cubic phase structure is preserved at neutral pH, albeit with a larger cell size. At pH 5.5, in the presence of the polymer, the nanostructure of the cubosome particles is significantly altered, providing a pathway to design pH-responsive cubosomes for applications in drug delivery.

Electroformation of Giant Unilamellar Vesicles: Investigating Vesicle Fusion versus Bulge Merging.

Partially ordered stacks of phospholipid bilayers on a flat substrate can be obtained by the evaporation of a spread droplet of phospholipid-in-chloroform solution. When exposed to an aqueous buffer, numerous micrometric buds populate the bilayers, grow in size over minutes, and eventually detach, forming the so-called liposomes or vesicles. While observation of vesicle growth from a hydrated lipid film under an optical microscope suggests numerous events of vesicle fusion, there is little experimental evidence for discriminating between merging of connected buds, i.e., a shape transformation that does not imply bilayer fusion and real membrane fusion. Here, we use electroformation to grow giant unilamellar vesicles (GUVs) from a stack of lipids in a buffer containing either (i) nanometric liposomes or (ii) previously prepared GUVs. By combining different fluorescent labels of the lipids in the substrate and in the solution, and by performing a fluorescence analysis of the resulting GUVs, we clearly demonstrate that merging of bulges is the essential pathway for vesicle growth in electroformation.

Relating side chain organization of PNIPAm with its conformation in aqueous methanol.

Combining nuclear magnetic resonance (NMR), dynamic light scattering (DLS), and µs long all-atom simulations with two million particles, we establish a delicate correlation between increased side chain organization of PNIPAm and its collapse in aqueous methanol mixtures. We find that the preferential binding of methanol with PNIPAm side chains, bridging distal monomers along the polymer backbone, results in increased organization. Furthermore, methanol-PNIPAm preferential binding is dominated by hydrogen bonding. Our findings reveal that the collapse of PNIPAm is dominated by enthalpic interactions and that the standard poor solvent (entropic) effects play no major role.

Bilayer properties of 1,3-diamidophospholipids.

A series of 1,3-diamido phosphocholines was synthesized, and their potential to form stable bilayers was investigated. Large and giant unilamellar vesicles produced from these new lipids form a wide variety of faceted liposomes. Factors such as cooling rates and the careful choice of the liposome preparation method influence the formation of facets. Interdigitation was hypothesized as a main factor for the stabilization of facets and effectively monitored by small-angle X-ray scattering measurements.

Dielectric discontinuity in equilibrium block copolymer micelles.

The surface tension between the hydrophobic core and the solvent is known to play a major role in the self-assembly of diblock copolymer micelles in solution. Coulombic forces are also very important in the case of aggregates with weakly charged coronas. The aggregation number and morphology are often tuned by the addition of electrolytes to the solution via electrostatic screening and an eventual change in solvent quality. However, when the surface tension is low enough, dielectric discontinuity between the core and the solvent becomes important enough in comparison to other mechanisms, driving the surface tension at the same time it screens electrostatic interactions in the corona. Below, we demonstrate the importance of this effect for micelles with neutral and weakly charged coronas.

Co-non-solvency: mean-field polymer theory does not describe polymer collapse transition in a mixture of two competing good solvents.

Smart polymers are a modern class of polymeric materials that often exhibit unpredictable behavior in mixtures of solvents. One such phenomenon is co-non-solvency. Co-non-solvency occurs when two (perfectly) miscible and competing good solvents, for a given polymer, are mixed together. As a result, the same polymer collapses into a compact globule within intermediate mixing ratios. More interestingly, polymer collapses when the solvent quality remains good and even gets increasingly better by the addition of the better cosolvent. This is a puzzling phenomenon that is driven by strong local concentration fluctuations. Because of the discrete particle based nature of the interactions, Flory-Huggins type mean field arguments become unsuitable. In this work, we extend the analysis of the co-non-solvency effect presented earlier [D. Mukherji et al., Nat. Commun. 5, 4882 (2014)]. We explain why co-non-solvency is a generic phenomenon, which can only be understood by the thermodynamic treatment of the competitive displacement of (co)solvent components. This competition can result in a polymer collapse upon improvement of the solvent quality. Specific chemical details are not required to understand these complex conformational transitions. Therefore, a broad range of polymers are expected to exhibit similar reentrant coil-globule-coil transitions in competing good solvents.

Physical damage on giant vesicles membrane as a result of methylene blue photoirradiation.

In this study we pursue a closer analysis of the photodamage promoted on giant unilamellar vesicles membranes made of dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), by irradiating methylene blue present in the giant unilamellar vesicles solution. By means of optical microscopy and electro-deformation experiments, the physical damage on the vesicle membrane was followed and the phospholipids oxidation was evaluated in terms of changes in the membrane surface area and permeability. As expected, oxidation modifies structural characteristics of the phospholipids that lead to remarkable membrane alterations. By comparing DOPC- with POPC-made membranes, we observed that the rate of pore formation and vesicle degradation as a function of methylene blue concentration follows a diffusion law in the case of DOPC and a linear variation in the case of POPC. We attributed this scenario to the nucleation process of oxidized species following a diffusion-limited growth regime for DOPC and in the case of POPC a homogeneous nucleation process. On the basis of these premises, we constructed models based on reaction-diffusion equations that fit well with the experimental data. This information shows that the outcome of the photosensitization reactions is critically dependent on the type of lipid present in the membrane.