Paola Brocca

Bacteria-derived lipopeptides are immunogenic triggers of host defences in metazoans and plants. Root-associated rhizobacteria produce cyclic lipopeptides that activate induced systemic resistance against microbial infection in various plant species. Whether and how these molecules are perceived at the plant cell surface remains elusive. Here we reveal that immune activation in Arabidopsis thaliana by the lipopeptide elicitor surfactin is mediated via a specific interaction with membrane sphingolipids. It relies on host membrane remodelling and subsequent activation of mechanosensitive ion channels. This mechanism leads to host defence potentiation and resistance to the necrotrophic fungus Botrytis cinerea and appears distinct from pattern-triggered immunity induced by classical host pattern recognition receptors. These results reveal a previously uncharacterized mechanism through which lipopeptides derived from non-pathogenic bacteria activate plant immune responses.

We report a new interferometric method to study the interactions between two gas bubbles undergoing small-amplitude oscillations in a liquid, based on the extension of a previously developed one-bubble set-up. Nanometric oscillations of millimetre-sized supported bubbles are excited acoustically; the response of each bubble is recorded interferometrically, as a function of the mutual distance (from quasi-contact to greater than the bubbles radii). The interferometric nature of the technique and the resonant nature of the vibration modes enable the accurate measurement of the amplitude (with sub-nanometric sensitivity), frequency and mutual phase of oscillation, whose variations over the bubble–bubble distance range allow the interactions to be probed. The bubbles oscillate at the same frequencies, exhibiting a low-frequency, in-phase and a high-frequency, out-of-phase resonance peak, whose separation is a function of distance, in good agreement with the theory for free interacting bubbles. The technique, here demonstrated for the volume modes of air bubbles in water, can be extended to other gas–liquid and liquid–liquid interfaces, bare or adsorbate-covered, as well as to shape oscillations.

Inhaled siRNA therapy has a unique potential for treatment of severe lung diseases, such as cystic fibrosis (CF). Nevertheless, a drug delivery system tackling lung barriers is mandatory to enhance gene silencing efficacy in the airway epithelium. We recently demonstrated that lipid-polymer hybrid nanoparticles (hNPs), comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell of dipalmitoyl phosphatidylcholine (DPPC), may assist the transport of the nucleic acid cargo through mucus-covered human airway epithelium. To study in depth the potential of hNPs for siRNA delivery to the lungs and to investigate the hypothesized benefit of PEGylation, here, an siRNA pool against the nuclear factor-κB (siNFκB) was encapsulated inside hNPs, endowed with a non-PEGylated (DPPC) or a PEGylated (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) or DSPE-PEG) lipid shell. Resulting hNPs were tested for their stability profiles and transport properties in artificial CF mucus, mucus collected from CF cells, and sputum samples from a heterogeneous and representative set of CF patients. Initial information on hNP properties governing their interaction with airway mucus was acquired by small-angle X-ray scattering (SAXS) studies in artificial and cellular CF mucus. The diffusion profiles of hNPs through CF sputa suggested a crucial role of lung colonization of the corresponding donor patient, affecting the mucin type and content of the sample. Noteworthy, PEGylation did not boost mucus penetration in complex and sticky samples, such as CF sputa from patients with polymicrobial colonization. In parallel, in vitro cell uptake studies performed on mucus-lined Calu-3 cells grown at the air–liquid interface (ALI) confirmed the improved ability of non-PEGylated hNPs to overcome mucus and cellular lung barriers. Furthermore, effective in vitro NFκB gene silencing was achieved in LPS-stimulated 16HBE14o- cells. Overall, the results highlight the potential of non-PEGylated hNPs as carriers for pulmonary delivery of siRNA for local treatment of CF lung disease. Furthermore, this study provides a detailed understanding of how distinct models may provide different information on nanoparticle interaction with the mucus barrier.

Extracellular vesicles (EVs)-mediated communication relies not only on the delivery of complex molecular cargoes as lipids, proteins, genetic material, and metabolites to their target cells but also on the modification of the cell surface local properties induced by the eventual fusion of EVs’ membranes with the cells’ plasma membrane. Here we applied scanning calorimetry to study the phase transition of single phospholipid (DMPC) monolamellar vesicles, investigating the thermodynamical effects caused by the fusion of doping amounts of mesenchymal stem cells-derived EVs. Specifically, we studied EVs-induced consequences on the lipids distributed in the differently curved membrane leaflets, having different density and order. The effect of EV components was found to be not homogeneous in the two leaflets, the inner (more disordered one) being mainly affected. Fusion resulted in phospholipid membrane flattening associated with lipid ordering, while the transition cooperativity, linked to membrane domains’ coexistence during the transition process, was decreased. Our results open new horizons for the investigation of the peculiar effects of EVs of different origins on target cell membrane properties and functionality.

Correction for ‘Structural insights into fusion mechanisms of small extracellular vesicles with model plasma membranes’ by Fabio Perissinotto et al., Nanoscale, 2021, 13, 5224–5233, DOI: 10.1039/D0NR09075A.

Extracellular vesicles (EVs) represent a potent intercellular communication system. Within a lipid bilayer such small vesicles transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. Due to their specific biological functions they have been proposed as biomarkers for various diseases and as optimal candidates for therapeutic applications. Despite of their extreme biological relevance, the small size (30 to a few hundred nanometers in diameter) of EVs still poses a great challenge for their isolation, quantification and biophysical/biochemical characterization, therefore the complex network of EVs and cells as well as their interaction remains to be further revealed. Here we propose a multiscale platform based on Atomic Force Microscopy, Small Angle X-ray Scattering, Small Angle Neutron Scattering and Neutron Reflectometry to reveal structure-function correlations of purified EVs through the analysis of their interaction with model membrane systems, in form of both supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. The analysis reveals a strong interaction of EVs with the model membranes and preferentially with liquid ordered raft-like lipid domains, and opens the way to understand uptake mechanisms in different vesicle to cell membrane relative compositions.

Protein uptake at the interface of a millimeter-sized air bubble in water, is investigated by a recently developed differential interferometric technique. The technique allows the study of capillary waves with amplitudes in the range of 10-9 m, excited at the surface of the bubble by an electric field of intensity in the order of 10 V/cm. By studying the resonant modes of the bubble (radial and shape modes) it is possible to assess variations of interfacial properties and, in particular, of the net surface charge as a function of bulk protein concentration. Sensing the interfacial charge, the technique enables us to follow the absorption process in condition of low concentrations, not easily assessable by other methods. We focus on bovine serum albumin (BSA) and Lysozyme, as representatives of typical globular proteins. To provide a comprehensive insight into the novelty of the technique we also investigated the equilibrium adsorption of SDS ionic surfactant for bulk concentrations hundreds times lower than the CMC. Results unveil how the absorption of charged molecules affects the amplitudes of the bubble resonant modes even before affecting the frequencies in a transition-like fashion. Different adsorption models are proposed and developed. They are validated against the experimental findings by comparing frequency and amplitude data. By measuring the charging rate of the bubble interface, we have followed the absorption kinetics of BSA and Lysozyme recognizing a slow, energy barrier limited, phenomena, with characteristic times in agreement with data in the literature. The evaluation of the surface excess concentration () of BSA and SDS at equilibrium is obtained by monitoring charge uptake. At the investigated low bulk concentrations, reliable comparisons with literature data from equilibrium surface tension isotherm models, are reported.