1O2 is the first excited state of molecular oxygen and the key intermediate in photosensitized oxidation reactions. Although 1O2 emits near-infrared (NIR) phosphorescence, direct microscopic observation of its emission is severely hampered by its low emission quantum yield, the competitive kinetics between its decay and diffusion, and the limited efficiency of NIR detectors. Here, we describe a 1O2 phosphorescence lifetime imaging microscope (1O2-PLIM) that allows acquiring lifetime and intensity profiles of 1O2 phosphorescence emission with improved resolution. Calibration carried out with photosensitizer solutions returned the expected lifetimes for 1O2 generation and decay. Nanometer-sized beads allowed the reconstruction of the excitation volume and the estimation of detection limit as 1 million 1O2 molecules in 60 fL of ethanol. Scanning samples in a confocal configuration provided intensity and lifetime image reconstruction of 1O2 emission from complex systems such as micrometer-sized polymer beads bound to photosensitizers, as well as from HaCaT keratynocytes previously incubated with a photosensitizer. Raw image data was corrected for the 1O2 emission lifetime and diffusion pathway within the confocal volume, using a mathematical model specifically developed for this purpose. This advancement in 1O2 imaging enables a better understanding and control of light-mediated reactions across chemistry, biomedicine, and environmental science. Obtaining precise values of 1O2 lifetimes in complex environments has remained a challenge due to a low emission quantum yield and competitive decay and diffusion kinetics. Here, the authors report a 1O2 phosphorescence lifetime imaging microscope (1O2-PLIM) that facilitates the acquisition of lifetime and intensity profiles of 1O2 phosphorescence emission with improved resolution, demonstrating its application to the study of micrometer-sized polymer beads bound to photosensitizers and HaCaT keratynocytes previously incubated with a photosensitizer.

Lipid hydroperoxidation significantly changes membrane biophysical properties, yet its effect on the main phase transition of unsaturated phospholipids remains unclear. Here, we investigate how hydroperoxidation of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) influences its phase behavior. Bilayers composed of SOPC and increasing fractions of hydroperoxidized SOPC (SOPC-OOH) were analyzed using differential scanning calorimetry, the band shift of the new solvatochromic probe A10, and Laurdan generalized polarization fluorescence. The incorporation of hydroperoxidized acyl chains perturbed membrane thermodynamics, revealing a complex phase-transition landscape governed by the interplay between acyl-chain ordering and the spatial distribution of -OOH groups. These results highlight the sensitivity of lipid phase behavior to oxidative modifications at the molecular level and provide new insights into how lipid peroxidation modulates membrane organization in cellular and biomimetic systems.

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.

As the primary products of lipid oxidation, lipid hydroperoxides constitute an important class of lipids generated by aerobic metabolism. However, despite several years of effort, the structure of the hydroperoxidized bilayer has not yet been observed under electron microscopy. Here we use a 200 kV Cryo-TEM to image small unilamellar vesicles (SUVs) made (i) of pure POPC or SOPC, (ii) of their pure hydroperoxidized form, and (iii) of their equimolar mixtures. We show that the challenges posed by the determination of the thickness of the hydroperoxidized bilayers under these observation conditions can be addressed by an image analysis method that we developed and describe here.

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.

Hypothesis: Measuring rotational and translational Brownian motion of single spherical particles reveals dissipations due to the interaction between the particle and the environment. Experiments: In this article, we show experiments where the in-plane translational and the two rotational drag coefficients of a single spherical Brownian particle can be measured. These particle drags are functions of the particle size and of the particle-wall distance, and of the viscous dissipations at play. We measure drag coefficients for Janus particles close to a solid wall and close to a lipid bilayer membrane. Findings: For a particle close to a wall, we show that according to hydrodynamic models, particle-wall distance and particle size can be determined. For a particle partially wrapped by lipid membranes, in absence of strong binding interactions, translational and rotational drags are significantly larger than the ones of non-wrapped particles. Beside the effect of the membrane viscosity, we show that dissipations in the deformed membrane cap region strongly contribute to the drag coefficients.

Lipid peroxidation is a process which is key in cell signaling and disease, it is exploited in cancer therapy in the form of photodynamic therapy. The appearance of hydrophilic moieties within the bilayer’s hydrocarbon core will dramatically alter the structure and mechanical behavior of membranes. Here, we combine viscosity sensitive fluorophores, advanced microscopy, and X-ray diffraction and molecular simulations to directly and quantitatively measure the bilayer’s structural and viscoelastic properties, and correlate these with atomistic molecular modelling. Our results indicate an increase in microviscosity and a decrease in the bending rigidity upon peroxidation of the membranes, contrary to the trend observed with non-oxidized lipids. Fluorescence lifetime imaging microscopy and MD simulations give evidence for the presence of membrane regions of different local order in the oxidized membranes. We hypothesize that oxidation promotes stronger lipid-lipid interactions, which lead to an increase in the lateral heterogeneity within the bilayer and the creation of lipid clusters of higher order.

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.

The interaction between Janus colloids and giant lipid vesicles was experimentally investigated to elucidate the dynamics and mechanisms related to microparticle engulfment by lipid vesicles. Janus (Pt–SiO2 and Pt–MF, where MF is melamine formaldehyde) colloids do not spontaneously adhere to POPC or DOPC bilayers, but by applying external forces via centrifugation we were able to force the contact between the particles and the membranes, which may result in a partial engulfment state of the particle. Surface properties of the Janus colloids play a crucial role in the driven particle engulfment by vesicles. Engulfment of the silica and platinum regions of the Janus particles can be observed, whereas the polymer (MF) region does not show any affinity towards the lipid bilayer. By using fluorescence microscopy, we were able to monitor the particle orientation and measure the rotational dynamics of a single Janus particle engulfed by a vesicle. By adding hydrogen peroxide to the solution, particle self-propulsion was used to perform an active transport of a giant vesicle by a single active particle. Finally, we observe that partially engulfed particles experience a membrane curvature-induced force, which pushes the colloids towards the bottom where the membrane curvature is the lowest.

Lipid hydroperoxides are the primary reaction products of lipid oxidation, a natural outcome of life under oxygen. While playing a major role in cell metabolism, the microscopic origins of the effects of lipid hydroperoxidation on biomembranes remain elusive. Here we probe the polar structure of partially to fully hydroperoxidized bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by a combination of environment-sensitive fluorescent probes and coarse-grained Martini numerical simulations. We find that the inserted organic hydroperoxide group -OOH migrates preferentially to the surface for bilayers with small fractions of hydroperoxidized lipids, but populates also significantly the bilayer interior for larger fractions. Our findings suggest that by modifying the intimate polarity of biomembranes, lipid peroxidation will have a significant impact on the activity of transmembrane proteins and on the bio-medical efficiency of membrane active molecules such as cell-penetrating and antimicrobial peptides.

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.

Atomistic molecular dynamics simulations have reached a degree of maturity that makes it possible to investigate the lipid polymorphism of model bilayers over a wide range of temperatures. However if both the fluid Lα and tilted gel Lβ′ states are routinely obtained, the Pβ′ ripple phase of phosphatidylcholine lipid bilayers is still unsatifactorily described. Performing simulations of lipid bilayers made of different numbers of DPPC (1,2-dipalmitoylphosphatidylcholine) molecules ranging from 32 to 512, we demonstrate that the tilted gel phase Lβ′ expected below the pretransition cannot be obtained for large systems (equal or larger than 94 DPPC molecules) through common simulations settings or temperature treatments. Large systems are instead found in a disordered gel phase which display configurations, topography and energies reminiscent from the ripple phase Pβ′ observed between the pretransition and the main melting transition. We show how the state of the bilayers below the melting transition can be controlled and depends on thermal history and conditions of preparations. A mechanism for the observed topographic instability is suggested.

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.

The $L_\alpha$ phase of lipid bilayers is a fluid self-assembled state, key to the formulation of cosmetics, detergents and pharmaceutics. Despite having been extensively scrutinized in self-assembled phospholipid or surfactant bilayers, the formation of a fluid $L_\alpha$ state has defied understanding in mixtures of fatty alcohols, surfactants and water, where is viewed as the essential step for the preparation of creamy dispersions. Here, atomistic molecular dynamics simulations show the existence of a fluid bilayer in aqueous mixtures of cetyl (C$_{16}$OH) and stearyl (C$_{18}$OH) alcohols, and cetyl-trimethylammonium chloride (CTAC). These simulated bilayer systems display not only a rich temperature phase diagram with many of the features seen in experiments but carry also the unambigous signature of fluid bilayer behavior.

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.

In this review, we summarize recent theoretical and computational developments in the field of smart responsive materials, together with complementary experimental data. A material is referred to as smart responsive when a slight change in external stimulus can drastically alter its structure, function, or stability. Because of this smart responsiveness, these systems are used for the design of advanced functional materials. The most characteristic properties of smart polymers are discussed, especially polymer properties in solvent mixtures. We show how multiscale simulation approaches can shed light on the intriguing experimental observations. Special emphasis is given to two symmetric phenomena: co-non-solvency and co-solvency. The first phenomenon is associated with the collapse of polymers in two miscible good solvents, whereas the latter is associated with the swelling of polymers in poor solvent mixtures. Furthermore, we discuss when the standard Flory–Huggins-type mean-field polymer theory can (or cannot) be applied to understand these complex solution properties. We also sketch a few examples to highlight possible future directions, that is, how smart polymer properties can be used for the design principles of advanced functional materials.

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. Here, we present a study on the effect of sucrose on 1,2-dipalmitoyl-sn-glycero-3-phos- phocholine bilayers that combines calorimetry, spectral fluorimetry, and optical microscopy. 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 [1]. The influence on melting of short hydrophobic oligomers inserted into the bilayer will also be discussed, and compared with our results for sucrose. [1] Morandi et al., Biophysical Journal 114, 2165-2173, 2018

We investigate the consequences of lipid peroxidation on the permeation properties of membranes comprising unsaturated lipid molecules by means of coarse-grained molecular simulations. After discussion on the impact of peroxidation on the properties of lipid bilayers such as stretching modulus, area per lipid, water permeation, and the distributions of various lipid components across the membrane, we focus in particular on the effect of peroxidation on the passive translocation of small nanoparticles of varying hydrophobicity across lipid bilayers. We consider two types of oxidized lipid bilayers which differ in their degree of peroxidation using a schematic model for the oxidized beads. Consistently with our previous work for nonoxidized lipid bilayers, we find a narrow window of translocation of the nanoparticles when their hydrophobicity is varied. Our studies suggest that oxidized lipid bilayers feature a more hydrophilic environment and strongly enhance the translocation rate of small nanoparticles without the formation of pores. Furthermore, an optimal permeation rate is found for peroxidized membranes, associated with more hydrophilic nanoparticles than that in the case of nonoxidized lipid bilayers. Strategies for targeting peroxidized lipid membranes in a more specific way are discussed in the light of our findings.

Polymers, also known as macromolecules, are high molecular weight species made by the covalent binding of units called monomers; they are ubiquitous in natural and industrial systems of self-assembled bilayers. In a living cell, macromolecules are by weight the most abundant carbon-containing molecules. In cosmetics, pharmaceutics or detergency most formulations of membrane solutions have polymers added for performance, processing, conditioning or delivery. The presence of polymers in, on, or in the vicinity of a membrane changes not only the structure and viscosity of the liquid media where the membranes evolve: it modifies the properties of the membrane itself and its interactions with the environment. Giant unilamellar vesicles are systems of choice for studying such changes. In this chapter we introduce basic theoretical concepts for understanding, quantifying and predicting membrane transformations induced by the presence of macromolecules.

Giant unilamellar vesicles (GUVs) are model membrane systems consisting of a single lipid bilayer separating an inner lumen from the outer solution, with dimensions comparable to that of eukaryotic cells. The importance of these biomimetic systems has recently grown with the development of easy and safe methods to assemble GUVs from complex biorelevant compositions. However, size and position control is still a key challenge for GUV formation and manipulation. Here, a gel-assisted formation method is introduced, able to produce arrays of giant unilamellar anchored vesicles (GUAVs) with a predetermined narrow size distribution. The approach based on micropatterned gel substrates of cross-linked poly(N-isopropylacrylamide) allows performing parallel measurements on thousands of immobile unilamellar vesicles. Such power and flexibility will respond to the growing need for developing platforms of biomimetic constructs from cell-sized single bilayers.