Publications

Curcumin, the active compound in turmeric, is renowned for its anti-inflammatory, antioxidant, and antimicrobial properties, making it beneficial for treating conditions like arthritis, neurodegenerative diseases, and various cancers. Despite its promising therapeutic potential, curcumin's poor bioavailability-due to its rapid metabolism and low solubility-limits its clinical efficacy. To address this, recent research has focused on enhancing curcumin delivery using nanoparticles, liposomes, and novel nanomaterials. Among these, laponite, a synthetic nanoclay, has shown promise in improving curcumin delivery due to its unique properties, including large surface area, dual charge, and stability in solution. This study explores the use of curcumin-laponite nanoparticles as carrier vehicles for controlled delivery to in vitro model membranes. Utilizing advanced techniques such as neutron reflectometry, atomic force microscopy, quartz crystal microbalance with dissipation, and infrared spectroscopy, the interaction between curcumin-laponite nanoparticles and solid-supported lipid bilayers is monitored, revealing enhanced stability and controlled release of curcumin across the membrane. These findings pave the way for the development of curcumin-based therapies targeting cardiovascular, neurological, and oncological diseases, leveraging the synergistic effects of curcumin's biological activity and laponite's delivery capabilities.

JTD Keywords: Curcumin, Drug delivery, Laponite, Neutron reflectivity, Supported lipid bilayers

Mesenchymal stem cells (MSCs) interact with their surroundings via integrins, which link to the actin cytoskeleton and translate physical cues into biochemical signals through mechanotransduction. N-cadherins enable cell-cell communication and are also linked to the cytoskeleton. This crosstalk between integrins and cadherins modulates MSC mechanotransduction and fate. Here we show the role of this crosstalk in the mechanosensing of viscosity using supported lipid bilayers as substrates of varying viscosity. We functionalize these lipid bilayers with adhesion peptides for integrins (RGD) and N-cadherins (HAVDI), to demonstrate that integrins and cadherins compete for the actin cytoskeleton, leading to an altered MSC mechanosensing response. This response is characterised by a weaker integrin adhesion to the environment when cadherin ligation occurs. We model this competition via a modified molecular clutch model, which drives the integrin/cadherin crosstalk in response to surface viscosity, ultimately controlling MSC lineage commitment. The crosstalk between cell-cell and cell-matrix adhesions regulates stem cell fate. Here, the authors reveal a critical role for matrix viscosity in controlling this crosstalk, which they explain via a modified molecular clutch model.

JTD Keywords: Actin cytoskeleton, Adhesion, Animals, Arginyl-glycyl-aspartic acid, Cadherins, Cell adhesion, Cell communication, Fibronectin, Force transmission, Humans, Hydrogel, Integrins, Lipid bilayers, Matrix, Mechanotransduction, Mechanotransduction, cellular, Mesenchymal stem cells, Mobility, Oligopeptides, Osteogenic differentiation, Substrate stiffness, Vinculin, Viscosity

The accurate spatial segregation into distinct phases within cell membranes coordinates vital biochemical processes and functionalities in living organisms. One of nature's strategies to localize reactivity is the formation of dynamic raft domains. Most raft models rely on liquid-ordered L-0 phases in a liquid-disordered L-d phase lacking correlation and remaining static, often necessitating external agents for phase separation. Here, we introduce a synthetic system of bicomponent glycodendrimersomes coassembled from Janus dendrimers and Janus glycodendrimers (JGDs), where lactose-lactose interactions exclusively drive lateral organization. This mechanism results in modulated phases across two length scales, yielding raft-like microdomains featuring nanoarrays at the nanoscale. By varying the density of lactose and molecular architecture of JGDs, the nanoarray type and size, shape, and spacing of the domains were controlled. Our findings offer insight into the potential primordial origins of rudimentary raft domains and highlight the crucial role of glycans within the glycocalyx.

JTD Keywords: Article, Artificial cells, Atomic force microscopy, Bicomponents, Bilayer, Bilayer membrane, Biochemical functionality, Biochemical process, Biological-membranes, Cell component, Cell membrane, Cellular parameters, Chemical interaction, Chemical structure, Chemistry, Cytology, Defined janus glycodendrimers, Dehydration, Dendrimer, Dendrimers, Dilution, Dimer, External agents, Fourier transform, Giant vesicles, Glycan, Glycans, Glycocalyx, Glycodendrimers, Janus dendrimer, Janus glycodendrimer, Lactose, Lateral organization, Lectin, Lipid rafts, Living organisms, Membrane damage, Membrane microdomain, Membrane microdomains, Membrane structure, Metabolism, Modulated phases, Molecule, Monomer, Nanoarrays, Oligosaccharide, Organization, Periodicity, Phase separation, Phase-separation, Phospholipids, Polysaccharide, Polysaccharides, Raft like domain, Relative humidity, Spatial segregation, Structure analysis, Sugars, Synthetic systems, Tetramer, Unclassified drug, Unilamellar vesicles, Water

Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD+, as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research. This article details current knowledge and major unknowns in brain energy metabolism and lays out a roadmap for future research.image

JTD Keywords: Acetate, Acetyl-coa, Aerobic glycolysis, Atp-citrate lyase, Extracellular glutamate concentration, Fatty-acid transport, Glucose-metabolism, Glut4, In-vivo, Insulin, Lipid droplet accumulation, Nicotinamide adenine-dinucleotide, Noradrenaline, Obese zucker rats, Rat cerebral-cortex

The immobilization of vesicles has been conceptualized as a method to functionalize biointerfaces. However, the preservation of their integrity post immobilization remains a considerable challenge. Interfacial interactions can cause vesicle rupture upon close surface contact and non-specific protein adsorption impairing surface functions. To date, immobilization of vesicles has relied solely on either entrapment or prior modification of vesicles, both of which require laborious preparation and limit their applications. This work develops a bioinspired strategy to pin vesicles without prior modification while preserving their intact shape. This work introduces antifouling diblock copolymers and ultrathin surface-attached hydrogels containing a brush-like interface consisting of a bottle brush copolymer of N-(2-hydroxypropyl) methacrylamide (HPMA) and N-(3-methacrylamidopropyl)-N,N-dimethyldodecan-1-aminiumiodide (C12+). The presence of positive charges generates an attractive force that pulls vesicles toward the surface. At the surface, the amphiphilic properties of the combs facilitate their insertion into the membrane, mimicking the harpooning mechanism observed in antimicrobial peptides. Importantly, the antifouling poly(HPMA) backdrop serves to safeguard the vesicles by preventing deformation and breakage. Using a combination of thermodynamic analysis, surface plasmon resonance, and confocal laser scanning microscopy, this work demonstrates the efficiency of this biomimetic system to capture vesicles while maintaining an antifouling interface necessary for bioapplications. This work presents a novel supramolecular approach that combines three key elements: long-range attraction, vesicle pinning, and short-range repulsion to attract and harpoon vesicles, while protecting them at the surface. This work envisions these coatings as universal and biocompatible platforms that can be used not only to study vesicle interactions, but also as tools for biomedical applications.image

JTD Keywords: Antifouling coatings, Coatings, Delivery, Extracellular vesicles, Fabrication, Hydrogel, Janus dendrimers, Lipid vesicles, Liposomes, Membrane insertion, Polymer brushes, Proteins, Surface-energy components, Ultrathin surface-attached hydrogels, Vesicle pinning

The use of nanocarriers in medicine, so-called nanomedicine, is one of the most innovative strategies for targeting drugs at the action site and increasing their activity index and effectiveness. Phytomedicine is the oldest traditional method used to treat human diseases and solve health problems. The recent literature on the treatment of malaria infections using nanodelivery systems and phytodrugs or supplements has been analyzed. For the first time, in the present review, a careful look at the considerable potential of nanomedicine in promoting phytotherapeutic efficacy was done, and its key role in addressing a translation through a significant reduction of the current burden of malaria in many parts of the world has been underlined.

JTD Keywords: antiplasmodial activity, bioavailability, chloroquine, combination therapy, discovery, drug-delivery, drug-delivery systems, nanocapsules, nanomedicine, natural molecules, pharmacokinetics, phytomedicine, plasmodium-falciparum, Artemisinin-based combination therapy, Drug-delivery systems, Nanomedicine, Natural molecules, Phytomedicine, Solid lipid nanoparticles

Nanoparticles (NPs) are commonly functionalized using targeting ligands to drive their selective uptake in cells of interest. Typical target cell types are cancer cells, which often overexpress distinct surface receptors that can be exploited for NP therapeutics. However, these targeted receptors are also moderately expressed in healthy cells, leading to unwanted off-tumor toxicities. Multivalent interactions between NP ligands and cell receptors have been investigated to increase the targeting selectivity towards cancer cells due to their non-linear response to receptor density. However, to exploit the multivalent effect, multiple variables have to be considered such as NP valency, ligand affinity, and cell receptor density. Here, we synthesize a panel of aptamer-functionalized silica-supported lipid bilayers (SSLB) to study the effect of valency, aptamer affinity, and epidermal growth factor receptor (EGFR) density on targeting specificity and selectivity. We show that there is an evident interplay among those parameters that can be tuned to increase SSLB selectivity towards high-density EGFR cells and reduce accumulation at non-tumor tissues. Specifically, the combination of high-affinity aptamers and low valency SSLBs leads to increased high-EGFR cell selectivity. These insights provide a better understanding of the multivalent interactions of NPs with cells and bring the nanomedicine field a step closer to the rational design of cancer nanotherapeutics.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: aptamer avidity and affinity, delivery, microscopy, multivalency, multivalent, nanoparticle targeting, silica -supported lipid bilayers, Aptamer avidity and affinity, Multivalency, Nanoparticle targeting, Silica-supported lipid bilayers, Supported lipid-bilayers, Tumor targeting

The mechanism of action of intravesical Mycobacterium bovis BCG immunotherapy treatment for bladder cancer is not completely known, leading to misinterpretation of BCG-unresponsive patients, who have scarce further therapeutic options. BCG is grown under diverse culture conditions worldwide, which can impact the antitumor effect of BCG strains and could be a key parameter of treatment success. Here, BCG and the nonpathogenic Mycobacterium brumae were grown in four culture media currently used by research laboratories and BCG manufacturers: Sauton-A60, -G15 and -G60 and Middlebrook 7H10, and used as therapies in the orthotopic murine BC model. Our data reveal that each mycobacterium requires specific culture conditions to induce an effective antitumor response. since higher survival rates of tumor-bearing mice were achieved using M. brumae-A60 and BCG-G15 than the rest of the treatments. M. brumae-A60 was the most efficacious among all tested treatments in terms of mouse survival, cytotoxic activity of splenocytes against tumor cells, higher systemic production of IL-17 and IFN-gamma, and bladder infiltration of selected immune cells such as ILCs and CD4(TEM). BCG-G15 triggered an antitumor activity based on a massive infiltration of immune cells, mainly CD3(+) (CD4(+) and CD8(+)) T cells, together with high systemic IFN-gamma release. Finally, a reduced variety of lipids was strikingly observed in the outermost layer of M. brumae-A60 and BCG-G15 compared to the rest of the cultures, suggesting an influence on the antitumor immune response triggered. These findings contribute to understand how mycobacteria create an adequate niche to help the host subvert immunosuppressive tumor actions.

JTD Keywords: bcg, innate immune response, innate-lymphoid cells, lipid, non-muscle invasive, Bcg, Calmette-guerin bcg, Glycerol, Identification, Immune-response, Innate immune response, Innate-lymphoid cells, Lipid, Lipids, Mycolic acids, Neutral-red, Non-muscle invasive, Phenolic glycolipids, Tuberculosis, Tumor microenvironment, Virulence

Building functional mimics of cell membranes is an important task toward the development of synthetic cells. So far, lipid and amphiphilic block copolymers are the most widely used amphiphiles with the bilayers by the former lacking stability while membranes by the latter are typically characterized by very slow dynamics. Herein, we introduce a new type of Janus dendrimer containing a zwitterionic phosphocholine hydrophilic headgroup (JDPC ) and a 3,5-substituted dihydrobenzoate-based hydrophobic dendron. JDPC self-assembles in water into zwitterionic dendrimersomes (z-DSs) that faithfully recapitulate the cell membrane in thickness, flexibility, and fluidity, while being resilient to harsh conditions and displaying faster pore closing dynamics in the event of membrane rupture. This enables the fabrication of hybrid DSs with components of natural membranes, including pore-forming peptides, structure-directing lipids, and glycans to create raft-like domains or onion vesicles. Moreover, z-DSs can be used to create active synthetic cells with life-like features that mimic vesicle fusion and motility as well as environmental sensing. Despite their fully synthetic nature, z-DSs are minimal cell mimics that can integrate and interact with living matter with the programmability to imitate life-like features and beyond. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

JTD Keywords: biological-membranes, bottom-up synthetic biology, chain, hybrid vesicles, hydroethidine, organization, polymersome, proteins, stability, synthetic cells, thickness, vesicle fusion, vesicle motility, vesicles, zwitterionic dendrimersomes, Biosensor, Biosensors, Bottom-up synthetic biology, Hybrid vesicles, Lipid-bilayers, Synthetic cells, Vesicle fusion, Vesicle motility, Zwitterionic dendrimersomes

The immunomodulatory potential of mycobacteria to be used for therapeutic purposes varies by species and culture conditions and is closely related to mycobacterial lipid composition. Although the lipids present in the mycobacterial cell wall are relevant, lipids are mainly stored in intracellular lipid inclusions (ILIs), which have emerged as a crucial structure in understanding mycobacteria-host interaction. Little is known about ILI ultrastructure, production, and composition in nonpathogenic species. In this study, we compared the lipid profiles of the nonpathogenic immunomodulatory agent Mycobacterium brumae during pellicle maturation under different culture conditions with qualitative and quantitative approaches by using high-resolution imaging and biochemical and composition analyses to understand ILI dynamics. The results showed wax esters, mainly in early stages of development, and acylglycerols in mature ILI composition, revealing changes in dynamics, amount, and morphometry, depending on pellicle maturation and the culture media used. Low-glycerol cultures induced ILIs with lower molecular weights which were smaller in size in comparison with the ILIs produced in glycerol-enriched media. The data also indicate the simple metabolic plasticity of lipid synthesis in M. brumae, as well as its high versatility in generating different lipid profiles. These findings provide an interesting way to enhance the production of key lipid structures via the simple modulation of cell culture conditions.

JTD Keywords: cell wall, electron microscopy, intrabacterial, lipid inclusions, mycobacterium, Bodies, Cell wall, Electron microscopy, Growth, In-vitro, Intrabacterial, Lipid inclusions, Mycobacterium, Prokaryotes, Triacylglycerol, Tuberculosis, Ultrastructural imaging, Virulence, Wax esters

Increased liver glycogen content has been shown to reduce food intake, attenuate obesity, and improve glucose tolerance in a mouse model of high-fat diet (HFD)-induced obesity. Here we studied the contribution of liver glycogen to the regulation of obesity and glucose metabolism in a model of type 2 diabetes and obesity, namely the db/db mouse. To this end, we crossed db/db mice with animals overexpressing protein targeting to glycogen (PTG) in the liver to generate db/db mice with increased liver glycogen content (db/db-PTG). Hepatic PTG overexpression reduced food intake and fat weight and attenuated obesity and hyperglycemia in db/db mice. Db/db-PTG mice showed similar energy expenditure and physical activity to db/db mice. PTG overexpression reduced liver phosphoenolpyruvate carboxykinase (PEPCK) protein levels and repressed hepatic glucose production in db/db mice. Moreover, increased liver glycogen elevated hepatic ATP content in these animals. However, lipid metabolism was not modified by PTG overexpression. In conclusion, increased liver glycogen content ameliorates the diabetic and obesity phenotype in db/db mice.Copyright © 2022 López-Soldado, Guinovart and Duran.

JTD Keywords: atp, db, dyslipidemia, food intake, glucose, homeostasis, liver, metabolism, mouse, receptor, Atp, Db/db, Food intake, Food-intake, Glucose, Glycogen, Liver

According to circular economy, wine-making by-products represent a fascinating biomass, which can be used for the sustainable exploitation of polyphenols and the development of new nanotechnological health-promoting products. In this study, polyphenols contained in the grape pomace were extracted by maceration with ethanol in an easy and low dissipative way. The obtained extract, rich in malvidin-3-glucoside, quercetin, pro-cyanidin B2 and gallic acid, was incorporated into phospholipid vesicles tailored for intestinal delivery. To improve their performances, vesicles were enriched with gelatine or a maltodextrin (Nutriose (R)), or their com-bination (gelatine-liposomes, nutriosomes and gelatine-nutriosomes). The small (-147 nm) and negatively charged (--50mV) vesicles were stable at different pH values mimicking saliva (6.75), gastric (1.20) and intestinal (7.00) environments. Vesicles effectively protected intestinal cells (Caco-2) from the oxidative stress and promoted the biofilm formation by probiotic bacteria. A preliminary evaluation of the vesicle feasibility at industrial levels was also performed, analysing the economic and energetic costs needed for their production.

JTD Keywords: Adhesion, Antioxidant activity, Caco-2 cells, Dextrin, Grape pomace extract, Lactobacillus-reuteri, Manufacturing costs, Oxidative stress, Ph, Phospholipid vesicles, Polyphenols, Probiotic bacteria, Protein

Quantum dots (QDs) are semiconductor nanoparticles with unique optical and electronic properties, whose interest as potential nano-theranostic platforms for imaging and sensing is increasing. The design and use of QDs requires the understanding of cell-nanoparticle interactions at a microscopic and nanoscale level. Model systems such as supported lipid bilayers (SLBs) are useful, less complex platforms mimicking physico-chemical properties of cell membranes. In this work, we investigated the effect of topographical homogeneity of SLBs bearing different surface charge in the adsorption of hydrophilic QDs. Using quartz-crystal microbalance, a label-free surface sensitive technique, we show significant differences in the interactions of QDs onto homogeneous and inhomogeneous SLBs formed following different strategies. Within short time scales, QDs adsorb onto topographically homogeneous, defect-free SLBs is driven by electrostatic interactions, leading to no layer disruption. After prolonged QD exposure, the nanomechanical stability of the SLB decreases suggesting nanoparticle insertion. In the case of inhomogeneous, defect containing layers, QDs target preferentially membrane defects, driven by a subtle interplay of electrostatic and entropic effects, inducing local vesicle rupture and QD insertion at membrane edges. © 2021

JTD Keywords: adsorption, atomic force microscopy, bilayer formation, gold nanoparticles, hydrophilic quantum dots, lipid membrane defects, model, nanomechanics, quartz crystal microbalance with dissipation, size, supported lipid bilayers, surfaces, Atomic force microscopy, Atomic-force-microscopy, Cytology, Defect-free, Electronic properties, Electrostatics, Hydrophilic quantum dot, Hydrophilic quantum dots, Hydrophilicity, Hydrophilics, Lipid bilayers, Lipid membrane defect, Lipid membrane defects, Lipid membranes, Lipids, Nanocrystals, Nanomechanics, Optical and electronic properties, Quartz, Quartz crystal microbalance with dissipation, Quartz crystal microbalances, Quartz-crystal microbalance, Semiconductor nanoparticles, Semiconductor quantum dots, Supported lipid bilayers

Mycobacterium bovis bacillus Calmette-Guérin (BCG) efficacy as an immunotherapy tool can be influenced by the genetic background or immune status of the treated population and by the BCG substrain used. BCG comprises several substrains with genetic differences that elicit diverse phenotypic characteristics. Moreover, modifications of phenotypic characteristics can be influenced by culture conditions. However, several culture media formulations are used worldwide to produce BCG. To elucidate the influence of growth conditions on BCG characteristics, five different substrains were grown on two culture media, and the lipidic profile and physico-chemical properties were evaluated. Our results show that each BCG substrain displays a variety of lipidic profiles on the outermost surface depending on the growth conditions. These modifications lead to a breadth of hydrophobicity patterns and a different ability to reduce neutral red dye within the same BCG substrain, suggesting the influence of BCG growth conditions on the interaction between BCG cells and host cells.

JTD Keywords: cell wall, efficacy, glycerol, hydrophobicity, lipid, neutral red, pdim, pgl, protein, strains, viability, virulence, Acylglycerol, Albumin, Article, Asparagine, Bacterial cell wall, Bacterial gene, Bacterium culture, Bcg vaccine, Catalase, Cell wall, Chloroform, Controlled study, Escherichia coli, Gene expression, Genomic dna, Glycerol, Glycerol monomycolate, Hexadecane, Housekeeping gene, Hydrophobicity, Immune response, Immunogenicity, Immunotherapy, Lipid, Lipid fingerprinting, Magnesium sulfate, Mercaptoethanol, Methanol, Methylglyoxal, Molybdatophosphoric acid, Mycobacterium bovis bcg, Neutral red, Nonhuman, Pdim, Petroleum ether, Pgl, Phenotype, Physical chemistry, Real time reverse transcription polymerase chain reaction, Rna 16s, Rna extraction, Rv0577, Staining, Thin layer chromatography, Unclassified drug

Tau hyperphosphorylation is the first step of neurofibrillary tangle (NFT) formation. In the present study, samples of the entorhinal cortex (EC) and frontal cortex area 8 (FC) of cases with NFT pathology classified as stages I–II, III–IV, and V–VI without comorbidities, and of middle-aged (MA) individuals with no NFT pathology, were analyzed by conventional label-free and SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 214 in the EC, 65 of which were dysregulated at the first stages (I–II) of NFT pathology; 167 phosphoproteins were dysregulated in the FC, 81 of them at stages I–II of NFT pathology. A large percentage of dysregulated phosphoproteins were identified in the two regions and at different stages of NFT progression. The main group of dysregulated phosphoproteins was made up of components of the membranes, cytoskeleton, synapses, proteins linked to membrane transport and ion channels, and kinases. The present results show abnormal phosphorylation of proteins at the first stages of NFT pathology in the elderly (in individuals clinically considered representative of normal aging) and sporadic Alzheimer's disease (sAD). Dysregulated protein phosphorylation in the FC precedes the formation of NFTs and SPs. The most active period of dysregulated phosphorylation is at stages III–IV when a subpopulation of individuals might be clinically categorized as suffering from mild cognitive impairment which is a preceding determinant stage in the progression to dementia. Altered phosphorylation of selected proteins, carried out by activation of several kinases, may alter membrane and cytoskeletal functions, among them synaptic transmission and membrane/cytoskeleton signaling. Besides their implications in sAD, the present observations suggest a molecular substrate for “benign” cognitive deterioration in “normal” brain aging.

JTD Keywords: (phospho)proteomics, alzheimer's disease, amyloid-beta, association guidelines, brain aging, cytoskeleton, frontal-cortex, kinases, lipid rafts, membranes, national institute, neuropathologic assessment, pathological process, protein phosphorylation, synapse pathology, synapses, tau, tau pathology, (phospho)proteomics, Age-related tauopathy, Alzheimer's disease, Brain aging, Cytoskeleton, Kinases, Membranes, Protein phosphorylation, Synapses, Tau

Membrane fission triggered by the endosomal sorting complex required for transport (ESCRT) is an important process observed in several pathogenic and non-pathogenic cellular events. From a synthetic-biology viewpoint, ESCRT proteins represent an interesting machinery for the construction of cell mimetic sub-compartments produced by fission. Since their discovery, the studies on ESCRT-III-mediated action, have mainly focused on protein dynamics, ignoring the role of lipid organization and membrane phase state. Recently, it has been suggested that membrane buds formed by the action of ESCRT-III are generated from transient microdomains in endosomal membranes. However, the interplay between membrane domain formation and ESCRT remodeling pathways has not been investigated. Here, giant unilamellar vesicles made of ternary lipid mixtures, either homogeneous in phase or exhibiting liquid-ordered/liquid-disordered phase coexistence, were employed as a model membrane system. These vesicles were incubated with purified recombinant ESCRT-III proteins from the parasite Entamoeba histolytica. In homogeneous membranes, we observe that EhVps32 can trigger domain formation while EhVps20 preferentially co-localizes in the liquid disordered phase. The addition of EhVps24 appears to induce the formation of intraluminal vesicles produced from the liquid-ordered phase. In phase separated membranes, the intraluminal vesicles are also generated from the liquid-ordered phase and presumably emerge from the phase boundary region. Our findings reinforce the hypothesis that ESCRT-mediated remodeling depends on the membrane phase state. Furthermore, the obtained results point to a potential synthetic biology approach for establishing eukaryotic mimics of artificial cells with microcompartments of specific membrane composition, which can also differ from that of the mother vesicle.

JTD Keywords: cell-membranes, coexistence, complex, escrt-iii, fission, guvs, lipid domains, lipid rafts, membrane fission, microcompartments, microscopy, phase separation, plasma-membrane, protein microarrays, structural basis, ternary mixtures, Escrt-iii, Giant unilamellar vesicles, Guvs, Lipid domains, Membrane fission, Microcompartments, Phase separation, Ternary mixtures

In this work beclomethasone dipropionate was loaded into liposomes and hyalurosomes modified with mucin to improve the ability of the payload to counteract the oxidative stress and involved damages caused by cigarette smoke in the airway. The vesicles were prepared by dispersing all components in the appropriate vehicle and sonicating them, thus avoiding the use of organic solvents. Unilamellar and bilamellar vesicles small in size (~117 nm), homogeneously dispersed (polydispersity index lower than 0.22) and negatively charged (~−11 mV), were obtained. Moreover, these vesicle dispersions were stable for five months at room temperature (~25 C). In vitro studies performed using the Next Generation Impactor confirmed the suitability of the formulations to be nebulized as they were capable of reaching the last stages of the impactor that mimic the deeper airways, thus improving the deposition of beclomethasone in the target site. Further, biocompatibility studies performed by using 16HBE bronchial epithelial cells confirmed the high biocompatibility and safety of all the vesicles. Among the tested formulations, only mucin-hyalurosomes were capable of effectively counteracting the production of reactive oxygen species (ROS) induced by cigarette smoke extract, suggesting that this formulation may represent a promising tool to reduce the damaging effects of cigarette smoke in the lung tissues, thus reducing the pathogenesis of cigarette smoke-associated diseases such as chronic obstructive pulmonary disease, emphysema, and cancer. ◦

JTD Keywords: 16hbe cells, beclomethasone, cigarette smoke extract, mucin, oxidative stress, phospholipid vesicles, pulmonary delivery, 16hbe cells, Beclomethasone, Cigarette smoke extract, Mucin, Oxidative stress, Phospholipid vesicles, Pulmonary delivery

Grape extract-loaded fibre-enriched vesicles, nutriosomes, were prepared by combining antioxidant extracts obtained from grape pomaces and a prebiotic, soluble fibre (Nutriose®FM06). The nutriosomes were small in size (from ∼140 to 260 nm), homogeneous (polydispersity index < 0.2) and highly negative (∼ −79 mV). The vesicles were highly stable during 12 months of storage at 25 °C. When diluted with warmed (37 °C) acidic medium (pH 1.2) of high ionic strength, the vesicles only displayed an increase of the mean diameter and a low release of the extract, which were dependent on Nutriose concentration. The formulations were highly biocompatible and able to protect intestinal cells (Caco-2) from oxidative stress damage. In vivo results underlined that the composition of mouse microbiota was not affected by the vesicular formulations. Overall results support the potential application of grape nutriosomes as an alternative strategy for the protection of the intestinal tract.

JTD Keywords: Antioxidant activity, Grape pomace, Gut microbiota, In vivo studies, Intestinal cells, Nutriosomes, Phospholipid vesicles, Prebiotic activity

Alcoholism is one of the leading and increasingly prevalent reasons of liver associated morbidity and mortality worldwide. Alcoholic hepatitis (AH) constitutes a severe disease with currently no satisfying treatment options. Lipoxin A4 (LXA4), a 15-lipoxygenase (ALOX15)-dependent lipid mediator involved in resolution of inflammation, showed promising pre-clinical results in the therapy of several inflammatory diseases. Since inflammation is a main driver of disease progression in alcoholic hepatitis, we investigated the impact of endogenous ALOX15-dependent lipid mediators and exogenously applied LXA4 on AH development. A mouse model for alcoholic steatohepatitis (NIAAA model) was tested in Alox12/15+/+ and Alox12/15−/− mice, with or without supplementation of LXA4. Absence of Alox12/15 aggravated parameters of liver disease, increased hepatic immune cell infiltration in AH, and elevated systemic neutrophils as a marker for systemic inflammation. Interestingly, i.p. injections of LXA4 significantly lowered transaminase levels only in Alox12/15−/− mice and reduced hepatic immune cell infiltration as well as systemic inflammatory cytokine expression in both genotypes, even though steatosis progressed. Thus, while LXA4 injection attenuated selected parameters of disease progression in Alox12/15−/− mice, its beneficial impact on immunity was also apparent in Alox12/15+/+ mice. In conclusion, pro-resolving lipid mediators may be beneficial to reduce inflammation in alcoholic hepatitis.

JTD Keywords: Alcoholic hepatitis, Arachidonate 12/15-lipoxygenase (Alox12/15), Lipoxin A4, Resolution of inflammation, Specialized pro-resolving lipid mediators (SPMs)

Lipid membranes are involved in many physiological processes like recognition, signaling, fusion or remodeling of the cell membrane or some of its internal compartments. Within the cell, they are the ultimate barrier, while maintaining the fluidity or flexibility required for a myriad of processes, including membrane protein assembly. The physical properties of in vitro model membranes as model cell membranes have been extensively studied with a variety of techniques, from classical thermodynamics to advanced modern microscopies. Here we review the nanomechanics of solid-supported lipid membranes with a focus in their phase behavior. Relevant information obtained by quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM) as complementary techniques in the nano/mesoscale interface is presented. Membrane morphological and mechanical characterization will be discussed in the framework of its phase behavior, phase transitions and coexistence, in simple and complex models, and upon the presence of cholesterol.

JTD Keywords: Lipid phase behavior, Phase transition, Phase coexistence, Nanomechanics, Thermodynamics, Atomic force microscopy (AFM), Quartz crystal microbalance with dissipation monitoring (QCM-D)

Background: Severe hepatic steatosis shows a high prevalence and contributes to morbidity and mortality in human immunodeficiency virus (HIV) infected patients. Known risk factors include obesity, dyslipidaemia and features of metabolic syndrome. Fibroblast growth factor 21 (FGF-21) is involved with hepatic glucose and lipid metabolism. This study aimed to evaluate FGF-21 as a biomarker for severe hepatic steatosis in non-obese HIV-infected patients. Methods: This is a prospective, cross-sectional, monocentric study including HIV-infected out-patients. Hepatic steatosis was measured via controlled attenuation parameter (CAP) using FibroScan 502 touch (ECHOSENS, France). Severe hepatic steatosis was defined at CAP ≥ 253 dB/m. Peripheral blood samples were collected and plasma was analysed for FGF-21. Demographic and clinical characteristics were collected from patient's health records. Results: In total, 73 non-obese HIV-monoinfected patients were included in this study. Prevalence of severe hepatic steatosis was 41%. Patients with severe hepatic steatosis showed significantly higher levels of FGF-21. Univariate analysis revealed FGF-21, BMI, hyperlipidaemia, ALT levels and arterial hypertension as significant, while multivariate analysis showed only FGF-21, arterial hypertension and ALT levels as significant independent risk factors for severe hepatic steatosis. Conclusion: This study presents FGF-21 as an independent and stronger predictor of severe hepatic steatosis than blood lipids in HIV-infected patients. Moreover, arterial hypertension and ALT levels predict severe steatosis even in non-obese HIV-monoinfected patients. Furthermore, this study supports existing metabolic risk factors and expands them to non-obese HIV-infected patients.

JTD Keywords: BMI, CAP, Dyslipidaemia, FGF-21, Fibroscan, HIV, Hyperlipidaemia, Liver, NAFLD, NASH, Steatosis

In the present work curcumin loaded hyalurosomes were proposed as innovative systems for the treatment of rheumatoid arthritis. Vesicles were prepared using a one-step and environmentally friendly method. Aiming at finding the most suitable formulation in terms of size, surface charge and stability on storage, an extensive pre-formulation study was performed using different type and amount of phospholipids. Curcumin loaded vesicles prepared with 180 mg/ml of Phospholipon 90G (P90G) and immobilized with sodium hyaluronate (2 mg/ml) were selected because of their small size (189 nm), homogeneous dispersion (PI 0.24), negative charge (−35 mV), suitable ability to incorporate high amount of curcumin (E% 88%) and great stability on storage. The in vitro study using fibroblast-like synovial cells cultured in synovial fluid, demonstrated the ability of these vesicles to downregulate the production of anti-apoptotic proteins IAP1 and IAP2 and stimulate the production of IL-10, while the production of IL-6 and IL-15 and reactive oxygen species was reduced, confirming their suitability in counteracting pathogenesis of rheumatoid arthritis.

JTD Keywords: Curcumin, IL-6 and IL-15, In vitro inflammation, Oxidative stress, Phospholipid vesicles, Synoviocytes

The function of biological membranes is controlled by the interaction of the fluid lipid bilayer with various proteins, some of which induce or react to curvature. These proteins can preferentially bind or diffuse towards curved regions of the membrane, induce or stabilize membrane curvature and sequester membrane area into protein-rich curved domains. The resulting tight interplay between mechanics and chemistry is thought to control organelle morphogenesis and dynamics, including traffic, membrane mechanotransduction, or membrane area regulation and tension buffering. Despite all these processes are fundamentally dynamical, previous work has largely focused on equilibrium and a self-consistent theoretical treatment of the dynamics of curvature sensing and generation has been lacking. Here, we develop a general theoretical and computational framework based on a nonlinear Onsager's formalism of irreversible thermodynamics for the dynamics of curved proteins and membranes. We develop variants of the model, one of which accounts for membrane curving by asymmetric crowding of bulky off-membrane protein domains. As illustrated by a selection of test cases, the resulting governing equations and numerical simulations provide a foundation to understand the dynamics of curvature sensing, curvature generation, and more generally membrane curvature mechano-chemistry.

JTD Keywords: Curvature generation, Curvature sensing, Lipid bilayers, Membrane proteins

Several cellular processes, including adhesion, signaling and transcription, endocytosis, and membrane resealing, among others, involve conformational changes such as bending, vesiculation, and tubulation. These mechanisms generally involve membrane separation from the cytoskeleton as well as strong bending, for which the membrane chemical composition and physicochemical properties, often highly localized and dynamic, are key players. The mechanical role of the lipid membrane in force triggered (or sensing) mechanisms in cells is important, and understanding the lipid bilayers’ physical and mechanical properties is essential to comprehend their contribution to the overall membrane. Atomic force microscopy (AFM)-based experimental approaches have been to date very valuable to deepen into these aspects. As a stand-alone, high-resolution imaging technique and force transducer with the possibility to operate in aqueous environment, it defies most other surface instrumentation in ease of use, sensitivity and versatility. In this chapter, we introduce the different AFM-based methods to assess topological and nanomechanical information on model membranes, specifically to supported lipid bilayers (SLBs), including several examples ranging from pure phospholipid homogeneous bilayers to multicomponent and phase-separated SLBs, increasing the bilayer complexity, in the direction of mimicking biological membranes.

JTD Keywords: Atomic force microscopy, Force spectroscopy, Model membranes, Nanomechanics, Supported lipid bilayers

Curcumin is an antimalarial compound easy to obtain and inexpensive, having shown little toxicity across a diverse population. However, the clinical use of this interesting polyphenol has been hampered by its poor oral absorption, extremely low aqueous solubility and rapid metabolism. In this study, we have used the anionic copolymer Eudragit® S100 to assemble liposomes incorporating curcumin and containing either hyaluronan (Eudragit-hyaluronan liposomes) or the water-soluble dextrin Nutriose® FM06 (Eudragit-nutriosomes). Upon oral administration of the rehydrated freeze-dried nanosystems administered at 25/75 mg curcumin·kg−1·day−1, only Eudragit-nutriosomes improved the in vivo antimalarial activity of curcumin in a dose-dependent manner, by enhancing the survival of all Plasmodium yoelii-infected mice up to 11/11 days, as compared to 6/7 days upon administration of an equal dose of the free compound. On the other hand, animals treated with curcumin incorporated in Eudragit-hyaluronan liposomes did not live longer than the controls, a result consistent with the lower stability of this formulation after reconstitution. Polymer-lipid nanovesicles hold promise for their development into systems for the oral delivery of curcumin-based antimalarial therapies.

JTD Keywords: Malaria, Curcumin, Nanomedicine, Oral administration, Lipid nanovesicles, Eudragit, Nutriose, Hyaluronan, Plasmodium yoelii

In this study, we show that dry saturated phospholipid layers prepared by the spin-coating technique could present thinner regions associated to interdigitated phases under some conditions. The morphological characteristics of lipid layers of saturated phosphocholines, such as dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), have been measured by Atomic Force Microscopy and revealed that the presence of interdigitated regions is not induced by the same parameters that induce them in hydrated samples. To achieve these results the effect of the lipid hidrocabonated chain length, the presence of alcohol in the coating solution, the spinning velocity and the presence of cholesterol were tested. We showed that DPPC and DSPC bilayers, on the one side, can show structures with similar height than interdigitated regions observed in hydrated samples, while, on the other side, DLPC and DMPC tend to show no evidence of interdigitation. Results indicate that the presence of interdigitated areas is due to the presence of lateral tensions and, hence, that they can be eliminated by releasing these tensions by, for instance, the addition of cholesterol. These results demonstrate that interdigitation in lipid layers is a rather general phenomena and can be observed in lipid bilayers in dry conditions.

JTD Keywords: Spin-coating, Lipid layers, Atomic Force Microscopy, Interdigitation

Casein micelles are ~200 nm electronegative particles that constitute 80 wt% of the milk proteins. During synthesis in the lactating mammary cells, caseins are thought to interact in the form of ~20 nm assemblies, directly with the biological membranes of the endoplasmic reticulum and/or the Golgi apparatus. However, conditions that drive this interaction are not yet known. Atomic force microscopy imaging and force spectroscopy were used to directly observe the adsorption of casein particles on supported phospholipid bilayers with controlled compositions to vary their phase state and surface charge density, as verified by X-ray diffraction and zetametry. At pH 6.7, the casein particles adsorbed onto bilayer phases with zwitterionic and liquid-disordered phospholipid molecules, but not on phases with anionic or ordered phospholipids. Furthermore, the presence of adsorbed caseins altered the stability of the yet exposed bilayer. Considering their respective compositions and symmetry/asymmetry, these results cast light on the possible interactions of casein assemblies with the organelles’ membranes of the lactating mammary cells.

JTD Keywords: Casein proteins, Phospholipid membrane, Supported lipid bilayer, Atomic force microscopy

Resveratrol and gallic acid, a lipophilic and a hydrophilic phenol, were co-loaded in innovative, biocompatible nanovesicles conceived for ensuring the protection of the skin from oxidative- and inflammatory-related affections. The basic vesicles, liposomes and glycerosomes, were produced by a simple, one-step method involving the dispersion of phospholipid and phenols in water or water/glycerol blend, respectively. Liposomes and glycerosomes were modified by the addition of poloxamer, a stabilizer and viscosity enhancer, thus obtaining viscous or semisolid dispersions of structured vesicles. The vesicles were spherical, unilamellar and small in size (~70 nm in diameter). The superior ability of the poloxamer-structured vesicles to promote the accumulation of both phenols in the skin was demonstrated, as well as their low toxicity and great ability to protect fibroblasts from chemically-induced oxidative damage. The in vivo administration of the vesicular phenols on TPA (phorbol ester)-exposed skin led to a significant reduction of oedema and leukocyte infiltration.

JTD Keywords: Fibroblasts, Mice, Phenol, Phospholipid vesicle, Poloxamer, Skin inflammation

Resveratrol and gallic acid were co-loaded in phospholipid vesicles aiming at protecting the skin from external injuries, such as oxidative stress and microbial infections. Liposomes were prepared using biocompatible phospholipids dispersed in water. To improve vesicle stability and applicability, the phospholipids and the phenols were dispersed in water/propylene glycol or water/glycerol, thus obtaining PEVs and glycerosomes, respectively. The vesicles were characterized by size, morphology, physical stability, and their therapeutic efficacy was investigated in vitro. The vesicles were spherical, unilamellar and small in size: liposomes and glycerosomes were around 70 nm in diameter, while PEVs were larger (∼170 nm). The presence of propylene glycol or glycerol increased the viscosity of the vesicle systems, positively affecting their stability. The ability of the vesicles to promote the accumulation of the phenols (especially gallic acid) in the skin was demonstrated, as well as their low toxicity and great ability to protect keratinocytes and fibroblasts from oxidative damage. Additionally, an improvement of the antimicrobial activity of the phenols was shown against different skin pathogens. The co-loading of resveratrol and gallic acid in modified phospholipid vesicles represents an innovative, bifunctional tool for preventing and treating skin affections.

JTD Keywords: Fibroblasts, Keratinocytes, Phenol, Phospholipid vesicle, Skin pathogens

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information

JTD Keywords: Atomic force microscopy, Force spectroscopy, Lipid membranes, Supported lipid bilayers, Nanomechanics, Cholesterol, Sphingolipids, Membrane structure, XR-AFM combination

In the present work new highly biocompatible nanovesicles were developed using polyanion sodium hyaluronate to form polymer immobilized vesicles, so called hyalurosomes. Curcumin, at high concentration was loaded into hyalurosomes and physico-chemical properties and in vitro/in vivo performances of the formulations were compared to those of liposomes having the same lipid and drug content. Vesicles were prepared by direct addition of dispersion containing the polysaccharide sodium hyaluronate and the polyphenol curcumin to a commercial mixture of soy phospholipids, thus avoiding the use of organic solvents. An extensive study was carried out on the physico-chemical features and properties of curcumin-loaded hyalurosomes and liposomes. Cryogenic transmission electron microscopy and small-angle X-ray scattering showed that vesicles were spherical, uni- or oligolamellar and small in size (112-220 nm). The in vitro percutaneous curcumin delivery studies on intact skin showed an improved ability of hyalurosomes to favour a fast drug deposition in the whole skin. Hyalurosomes as well as liposomes were biocompatible, protected in vitro human keratinocytes from oxidative stress damages and promoted tissue remodelling through cellular proliferation and migration. Moreover, in vivo tests underlined a good effectiveness of curcumin-loaded hyalurosomes to counteract 12-O-tetradecanoilphorbol (TPA)-produced inflammation and injuries, diminishing oedema formation, myeloperoxydase activity and providing an extensive skin reepithelization. Thanks to the one-step and environmentally-friendly preparation method, component biocompatibility and safety, good in vitro and in vivo performances, the hyalurosomes appear as promising nanocarriers for cosmetic and pharmaceutical applications.

JTD Keywords: Cell oxidative stress, Hyaluronic acid/Hyaluronan, Phospholipid vesicles, Polyphenols, Skin inflammation, Wound healing

Biocompatible quercetin nanovesicles were developed by coating polyethylene glycol-containing vesicles with chitosan and nutriose, aimed at targeting the colon. Uncoated and coated vesicles were prepared using hydrogenated soy phosphatidylcholine and quercetin, a potent natural anti-inflammatory and antioxidant drug. Physicochemical characterization was carried out by light scattering, cryogenic microscopy and X-ray scattering, the results showing that vesicles were predominantly multilamellar and around 130 nm in size. The in vitro release of quercetin was investigated under different pH conditions simulating the environment of the gastrointestinal tract, and confirmed that the chitosan/nutriose coating improved the gastric resistance of vesicles, making them a potential carrier system for colon delivery. The preferential localization of fluorescent vesicles in the intestine was demonstrated using the In Vivo FX PRO Imaging System. Above all, a marked amelioration of symptoms of 2,4,6-trinitrobenzenesulfonic acid-induced colitis was observed in animals treated with quercetin-loaded coated vesicles, favoring the restoration of physiological conditions. Therefore, quercetin-loaded chitosan/nutriose-coated vesicles can represent a valuable therapeutic tool for the treatment of chronic intestinal inflammatory diseases, and presumably a preventive system, due to the synergic action of antioxidant quercetin and beneficial prebiotic effects of the chitosan/nutriose complex.

JTD Keywords: Chitosan/nutriose complex, Colon targeting, Phospholipid vesicles, Quercetin, Rat colitis

A fast atomic force microscope (AFM) has been developed that can be installed as a sample holder for grazing-incidence X-ray experiments at solid/gas or solid/liquid interfaces. It allows a wide range of possible investigations, including soft and biological samples under physiological conditions (hydrated specimens). The structural information obtained using the X-rays is combined with the data gathered with the AFM (morphology and mechanical properties), providing a unique characterization of the specimen and its dynamics in situ during an experiment. In this work, lipid monolayers and bilayers in air or liquid environment have been investigated by means of AFM, both with imaging and force spectroscopy, and X-ray reflectivity. In addition, this combination allows the radiation damage induced by the beam on the sample to be studied, as has been observed on DOPC and DPPC supported lipid bilayers under physiological conditions.

JTD Keywords: In situ atomic force microscopy, Grazing-incidence scattering and reflectivity, Radiation damage, Model lipid membranes

JTD Keywords: antimalarial, heparin, magic bullet, malaria, nanomedicine, nanotechnology, nanovector, Plasmodium, polymers, targeted drug delivery, chloroquine, immunoliposome, liposome, nanoparticle, solid lipid nanoparticle, Anopheles, antimalarial activity, drug delivery system, drug efficacy, erythrocyte, human, IC50, malaria, malaria control, nanoencapsulation, nonhuman, pathophysiology, Plasmodium, Review

Study Objectives: To test the hypotheses that brain oxygen partial pressure (PtO2) in response to obstructive apneas changes with age and that it might lead to different levels of cerebral tissue oxidative stress. Design: Prospective controlled animal study. Setting: University laboratory. Participants: Sixty-four male Wistar rats: 32 young (3 mo old) and 32 aged (18 mo). Interventions: Protocol 1: Twenty-four animals were subjected to obstructive apneas (50 apneas/h, lasting 15 sec each) or to sham procedure for 50 min. Protocol 2: Forty rats were subjected to obstructive apneas or sham procedure for 4 h. Measurements and Results: Protocol 1: Real-time PtO2 measurements were performed using a fast-response oxygen microelectrode. During successive apneas cerebral cortex PtO2 presented a different pattern in the two age groups; there was a fast increase in young rats, whereas it remained without significant changes between the beginning and the end of the protocol in the aged group. Protocol 2: Brain oxidative stress assessed by lipid peroxidation increased after apneas in young rats (1.34 ± 0.17 nmol/mg of protein) compared to old ones (0.63 ± 0.03 nmol/mg), where a higher expression of antioxidant enzymes was observed. Conclusions: The results suggest that brain oxidative stress in aged rats is lower than in young rats in response to recurrent apneas, mimicking obstructive sleep apnea. This could be due to the different PtO2 response observed between age groups and the increased antioxidant expression in aged rats.

JTD Keywords: Aging, Animal model, Obstructive apnea, Oxidative stress, Tissue oxygenation, antioxidant, glutathione disulfide, aged, animal experiment, animal model, animal tissue, apnea, arterial oxygen saturation, article, brain cortex, brain oxygen tension, brain tissue, controlled study, groups by age, hypoxia, lipid peroxidation, male, nonhuman, oxidative stress, pressure, priority journal, rat

We investigate the effects of Cholesterol (Chol) in the structural and nanomechanical properties of binary and ternary spin-coated single lipid bilayers made of Dioleoylphosphatidylcholine (DOPC) and Sphingomyelin (SM) in dry conditions. We show that for the DOPC/Chol bilayers, Chol induces an initial increase of the bilayer thickness, followed by decrease for concentrations above 30% Chol. The mechanical properties, instead, appear practically insensitive to the Chol content. For the SM/Chol bilayers we have observed both the thinning of the bilayer and the decrease of the force necessary to break it for Chol content above 40. mol%. In both binary mixtures phase separation is not observed. For ternary single bilayers of DOPC/SM/Chol, Chol induces phase segregation and the formation of domains resembling lipid rafts. The domains show a thickness and mechanical response clearly distinct from the surrounding phase and dependent on the relative Chol content. Based on the results obtained for the binary mixtures, DOPC- and SM-enriched domains can be identified. We highlight that many of the effects of Chol reported here for the dry multicomponent single lipid bilayers resemble closely those observed in hydrated bilayers, thus offering an additional insight into their properties.

JTD Keywords: AFM, Air-stable lipid layer, Force spectroscopy, Lipid raft, Spin-coating

Atomic Force Microscopy (AFM) has become an invaluable tool for studying the micro-and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. The main strength of AFM relies on the possibility to operate in an aqueous environment on a wide variety of biological samples, from single molecules-DNA or proteins-to macromolecular assemblies like biological membranes. Understanding the effect of mechanical stress on membranes is of primary importance in biophysics, since cells are known to perform their function under a complex combination of forces. In the later years, AFM-based Force-Spectroscopy (AFM-FS) has provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Lipid membranes are electrostatically charged entities that physiologically coexist with electrolyte solutions. Thus, specific interactions with ions are a matter of considerable interest. The distribution of ions in the solution and their interaction with the membranes are factors that substantially modify the structure and dynamics of the cell membranes. Furthermore, signaling processes are modified by the membrane capability of retaining ions. Supported Lipid Bilayers (SLBs) are a versatile tool to investigate phospholipid membranes mimicking biological surfaces. In the present contribution, we review selected experiments on the mechanical stability of SLBs as models of lipid membranes by means of AFM-FS, with special focus on the effect of cations and ionic strength in the overall nanomechanical stability.

JTD Keywords: Atomic force microscopy, Cations, Force spectroscopy, Lipid bilayer, Mechanical stability

Ubiquinone (UQ) is one of the main electron and proton shuttle molecules in biological systems, and dipalmitoylphosphatidylcholine (DPPC) is one of the most used model lipids. Supported planar bilayers (SPBs) are extensively accepted as biological model membranes. In this study, SPBs have been deposited on ITO, which is a semiconductor with good electrical and optical features. Specifically, topographic atomic force microscopy (AFM) images and force curves have been performed on SPBs with several DPPC:UQ ratios to study the location and the interaction of UQ in the SPB. Additionally, cyclic voltammetry has been used to understand the electrochemical behavior of DPPC:UQ SPBs. Obtained results show that, in our case, UQ is placed in two main different positions in SPBs. First, between the DPPC hydrophobic chains, fact that originates a decrease in the breakthrough force of the bilayer, and the second between the two leaflets that form the SPBs. This second position occurs when increasing the UQ content, fact that eventually forms UQ aggregates at high concentrations. The formation of aggregates produces an expansion of the SPB average height and a bimodal distribution of the breakthrough force. The voltammetric response of UQ depends on its position on the bilayer.

JTD Keywords: Bimodal distribution, Biological models, Dipalmitoyl phosphatidylcholine, Electrochemical behaviors, Hydrophobic chains, Supported lipid bilayers, Supported planar bilayers, Voltammetric response

How do metal cations affect the stability and structure of phospholipid bilayers? What role does ion binding play in the insertion of proteins and the overall mechanical stability of biological membranes? Investigators have used different theoretical and microscopic approaches to study the mechanical properties of lipid bilayers. Although they are crucial for such studies, molecular-dynamics simulations cannot yet span the complexity of biological membranes. In addition, there are still some experimental difficulties when it comes to testing the ion binding to lipid bilayers in an accurate way. Hence, there is a need to establish a new approach from the perspective of the nanometric scale, where most of the specific molecular phenomena take place. Atomic force microscopy has become an essential tool for examining the structure and behavior of lipid bilayers. In this work, we used force spectroscopy to quantitatively characterize nanomechanical resistance as a function of the electrolyte composition by means of a reliable molecular fingerprint that reveals itself as a repetitive jump in the approaching force curve. By systematically probing a set of bilayers of different composition immersed in electrolytes composed of a variety of monovalent and divalent metal cations, we were able to obtain a wealth of information showing that each ion makes an independent and important contribution to the gross mechanical resistance and its plastic properties. This work addresses the need to assess the effects of different ions on the structure of phospholipid membranes, and opens new avenues for characterizing the (nano)mechanical stability of membranes.

JTD Keywords: Molecular-dynamics simulation, Liquid expanded monolayers, Lipid-bilayers, Hofmeister series, Monovalent salt, Phosphatidylcholine, Microscopy, Binding, Surfaces, NaCl

The structure and the electrochemical behaviour of Langmuir and Langmuir-Blodgett (LB) films of the biological ubiquinone-10 (UQ) and a mixture of dipalmytoilphosphatidylcholine (DPPC) and UQ at the molar ratios DPPC:UQ 5:1 and 10:1 have been investigated. The surface pressure-area isotherms of the Langmuir films and the AFM images of the LB films show the formation of a monolayer in the DPPC:UQ mixture till a certain surface pressure is attained, and then at higher surface pressures the UQ is progressively expelled. The cyclic voltammograms of DPPC:UQ LB films formed on indium tin oxide, ITO, at different surface pressures show one reduction and one oxidation peak at low surface pressures, but two or even more reduction and oxidations peaks at medium and high surface pressures. The electrochemical behaviour is correlated with the film structure.

JTD Keywords: Cyclic voltammetry, Electron transfer, Langmuir-Blodgett, Lipid monolayer, Modified ITO electrode, Ubiquinone

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JTD Keywords: AFM, Force spectroscopy, Breakthrough force, Supported lipid bilayers, Lipid membranes, (nano)mechanics

Near-field optical microscopy is a technique not limited by the laws of diffraction that enables simultaneous high-resolution fluorescence and topographic measurements at the nanometer scale. This chapter highlights the intrinsic advantages of near-field optics in the study of cellular structures. The first part of the chapter lays the foundations of the near-field concept and technical implementation of near-field scanning optical microscopy (NSOM), whereas the second part of the chapter focuses on applications of NSOM to the study of model membranes and cellular structures on the plasma membrane. The last part of the chapter discusses further directions of near-field optics, including optical antennas and fluorescence correlation spectroscopy approaches in the near-field regime.

JTD Keywords: Biological membranes, Cell membrane nanoscale compartmentalization, Cellular nanodomains, Fluorescence correlation spectroscopy in reduced volumes, Immunoreceptor imaging, Lipid rafts, Near-field scanning optical microscopy, Optical nano-antennas, Shear force imaging, Single molecule detection, Super-resolution microscopy

Characterization of molecular dynamics on living cell membranes at the nanoscale is fundamental to unravel the mechanisms of membrane organization and compartmentalization. Here we demonstrate the feasibility of fluorescence correlation spectroscopy (FCS) based on the nanometric illumination of near-field scanning optical microscopy (NSOM) probes on intact living cells. NSOM-FCS applied to fluorescent lipid analogs allowed us to reveal details of the diffusion hidden by larger illumination areas. Moreover, the technique offers the unique, advantages of evanescent axial illumination and straightforward implementation of multiple color excitation. As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale on cell membranes.

JTD Keywords: Mode wave-guides, Lipid rafts, Difussion, Organization, Aperture

Understanding the effect of mechanical stress on membranes is of primary importance in biophysics. Here we use force spectroscopy AFM to quantitatively characterize the nanomechanical stability of supported lipid bilayers as a function of their chemical composition. The onset of plastic deformation reveals itself as a repetitive jump in the approaching force curve, which represents a molecular fingerprint for the bilayer mechanical stability. By systematically probing a set of chemically distinct supported lipid bilayers (SLBs), we first show that both the headgroup and tail have a decisive effect on their mechanical properties. While the mechanical stability of the probed SLBs linearly increases by 3.3 nN upon the introduction of each additional -CH2- in the chain, it exhibits a significant dependence on the phospholipid headgroup, ranging from 3 nN for DPPA to 66 nN for DPPG. Furthermore, we also quantify the reduction of the membrane mechanical stability as a function of the number of unsaturations and molecular branching in the chemical structure of the apolar tails. Finally, we demonstrate that, upon introduction of cholesterol and ergosterol, contrary to previous belief the mechanical stability of membranes not only increases linearly in the liquid phase (DLPC) but also for phospholipids present in the gel phase (DPPC). Our results are discussed in the framework of the continuum nucleation model. This work highlights the compelling effect of subtle variations in the chemical structure of phospholipid molecules on the membrane response when exposed to mechanical forces, a mechanism of common occurrence in nature.

JTD Keywords: Atomic-force microscopy, Molecular-dynamics simulation, Aqueous-electrolyte solutions, Supported planar membranes, Phospholipid-bilayers, Biological-membranes, Physical-properties, Fluid membranes, Model membranes, Chain-length

Lipid bilayers determine the architecture of cell membranes and regulate a myriad of distinct processes that are highly dependent on the lateral organization of the phospholipid molecules that compose the membrane. Indeed, the mechanochemical properties of the membrane are strongly correlated with the function of several membrane proteins, which demand a very specific, highly localized physicochemical environment to perform their function. Several mesoscopic techniques have been used in the past to investigate the mechanical properties of lipid membranes. However, they were restricted to the study of the ensemble properties of giant bilayers. Force spectroscopy with AFM has emerged as a powerful technique able to provide valuable insights into the nanomechanical properties of supported lipid membranes at the nanometer/nanonewton scale in a wide variety of systems. In particular, these measurements have allowed direct measurement of the molecular interactions arising between neighboring phospholipid molecules and between the lipid molecules and the surrounding solvent environment. The goal of this review is to illustrate how these novel experiments have provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Here we report in detail the main discoveries achieved by force spectroscopy with AFM on supported lipid bilayers, and we also discuss on the exciting future perspectives offered by this growing research field.

JTD Keywords: Force spectroscopy, Atomic force microscopy, Lipid bilayer, Nanomechanics

For many years, it was believed that the laws of diffraction set a fundamental limit to the spatial resolution of conventional light microscopy. Major developments, especially in the past few years, have demonstrated that the diffraction barrier can be overcome both in the near- and far-field regime. Together with dynamic measurements, a wealth of new information is now emerging regarding the compartmentalization of cell membranes. In this review we focus on optical methods designed to explore the nanoscale architecture of the cell membrane, with a focal point on near-field optical microscopy (NSOM) as the first developed technique to provide truly optical super-resolution beyond the diffraction limit of light. Several examples illustrate the unique capabilities offered by NSOM and highlight its usefulness on cell membrane studies, complementing the palette of biophysical techniques available nowadays.

JTD Keywords: Membrane nanodomain, Lipid raft, Single molecule detection, Near-field scanning optical microscopy, Super-resolution optical microscopy

In this study, we compare for the first time the nanomechanical properties of lipid bilayer islands on flat and porous surfaces. 1,2-Dimyzistoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers were deposited on flat (silicon and mica) and porous silicon (pSi) substrate surfaces and examined using atomic force spectroscopy and force volume imaging. Force spectroscopy measurements revealed the effects of the underlying substrate and of the lipid phase on the nanomechanical properties of bilayers islands. For mica and silicon, significant differences in breakthrough force between the center and the edges of bilayer islands were observed for both phospolipids. These differences were more pronounced for DMPC than for DPPC, presumably due to melting effects at the edges of DMPC bilayers. In contrast, bilayer islands deposited on pSi yielded similar breakthrough forces in the central region and along the perimeter of the islands, and those values in turn were similar to those measured along the perimeter of bilayer islands deposited on the flat substrates. The study also demonstrates that pSi is suitable solid support for the formation of pore-spanning phospholipid bilayers with potential applications in transmembrane protein studies, drug delivery, and biosensing.

JTD Keywords: Black lipid-membranes, Gold surfaces, Supported bilayers, Channel activity, Micro-BLMS, Silicon, Proteins, Vesicles, AFM, Temperature measurement

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (A beta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, A beta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for A beta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different A beta(1-42) species on Ca2+ homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca2+ that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca2+ increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

JTD Keywords: AFM, Alzheimers-disease, Chondroitin sulfate, Heparan-sulfate, Lipid-bilayers, Beta-peptide, In-vitro, Neurodegenerative diseases, Extracellular-matrix, Prion protein

The nanomechanical properties of Langmuir-Blodgett monolayers of arachidic acid extracted at surface pressures of 1, 15, and 35 mN/m and deposited on mica were investigated by atomic force microscopy, force spectroscopy, and lateral force microscopy. It was experimentally demonstrated that the arachidic acid molecular orientation depends on the extraction pressure. According to this, tilting angles of 50, 34, and 22 degrees with respect to the surface perpendicular were detected and identified as conformations that maximize van der Waals interactions between the arachidic acid alkyl chains. The vertical force needed to puncture the monolayers with the AFM tip strongly depends on the molecular tilting angles attained at different monolayer extraction surface pressures, obtaining values that range from 13.07 +/- 3.24 nN for 50 degrees to 22.94 +/- 5.49 nN for 22 degrees tilting angles. The different molecular interactions involved in the monolayer cohesion are discussed and quantitatively related to the experimental monolayer breakthrough forces. The friction measurements performed from low vertical forces up to monolayer disruption reveal the existence of three well-defined regimes: first, a low friction response due to the elastic deformation of the monolayer, which is followed by a sharp increase in the friction force due to the onset of a sudden plastic deformation. The last regime corresponds to the monolayer rupture and the contact between tip and substrate. The friction coefficient of the substrate is seen to depend on the monolayer extraction pressure, a fact that is discussed in terms of the relationship between the sample compactness and its rupture mechanism.

JTD Keywords: AFM, SAM, Reflection-absortion spectroscopy, Lipid-bilayers, Frictional-properies, Molecular-structure, Thermal behavior, Nanometer-scale, Chain-length, LB films