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Microfluidic mixing is recognized as a convenient method to produce liposomes for its scalability and reproducibility. Numerous studies have described the effect of process parameters such as flow rate ratios and total flow rate on size and size distribution of vesicles. In this work, we focused our attention on the effect of flow rate ratios on the encapsulation efficiency of liposomes, as we hypothesized that different amount of residual organic solvent could affect the retention of lipophilic drug molecules within the bilayer. In a further step, we investigated how the liposomes integrity and loading were impacted by different methods of solvent removal: direct dialysis and dilution & dialysis. Liposomes were prepared by rapidly mixing an ethanolic solution of lipids and a model drug with buffer in a herringbone micromixer, employing four different flow rate ratios (FRR, 4:1, 7:3, 3:2, 1:1). Quercetin, resveratrol and ascorbyl palmitate were used as model antioxidant drugs with different lipophilicity. Data showed that liposomes produced using lower flow rate ratios (i.e., with more residual ethanol) had lower encapsulation efficiencies as well as a more prominent loss of lipids from the bilayer following purification with direct dialysis. If the amount of residual ethanol was reduced to 5% (dilution & dialysis method), the lipids and drug leakage was prevented. Such effect was correlated with the drug aggregation propensity in different ethanol/water mixtures measured by molecular dynamics simulations. Overall, these results highlight the need to tailor the purification method basing on the molecular properties of the loaded drug to ensure high encapsulation and limit the waste of material., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In the development of therapeutic extracellular vesicles (EVs), drug encapsulation efficiencies are significantly lower when compared with synthetic nanomedicines. This is due to the hierarchical structure of the EV membrane and the physicochemical properties of the candidate drug (molecular weight, hydrophilicity, lipophilicity, and so on). As a proof of concept, here we demonstrated the importance of drug compartmentalization in EVs as an additional parameter affecting the therapeutic potential of drug-loaded EVs. In human adipose mesenchymal stem cell (hADSC) derived EVs, we performed a comparative drug loading analysis using two formulations of the same chemotherapeutic molecule - free doxorubicin (DOX) and 1,2-distearoyl- sn-glycero -3-phosphoethanolamine (DSPE) lipid-conjugated doxorubicin (L-DOX) - to enhance the intracellular uptake and therapeutic efficacy. By nano surface energy transfer (NSET) and molecular simulation techniques, along with cryo-TEM analysis, we confirmed the differential compartmentalization of these two molecules in hADSC EVs. L-DOX was preferentially adsorbed onto the surface of the EV, due to its higher lipophilicity, whereas free DOX was mostly encapsulated within the EV core. Also, the L-DOX loaded EV (LDOX@EV) returned an almost three-fold higher DOX content as compared to the free DOX loaded EV (DOX@EV), for a given input mass of drug. Based on the cellular investigations, L-DOX@EV showed higher cell internalization than DOX@EV. Also, in comparison with free L-DOX, the magnitude of therapeutic potential enhancement displayed by the surface compartmentalized L-DOX@EV is highly promising and can be exploited to overcome the sensitivity of many potential drugs, which are impermeable in nature. Overall, this study illustrates the significance of drug compartmentalization in EVs and how this could affect intracellular delivery, loading efficiency, and therapeutic effect. This will further lay the foundation for the future systematic investigation of EV-based biotherapeutic delivery platforms for personalized medicine., Competing Interests: All authors declared no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Aβ (1-40) can transfer from the aqueous phase to the bilayer and thus form stable ion-channel-like pores where the protein has alpha-helical conformation. The stability of the pores is due to the presence of the GXXXG motif. It has been reported that these ion-channel-like pores are stabilized by a Cα-H···O hydrogen bond that is established between a glycine of the GXXXG sequence of an alpha-helix and another amino acid of a vicinal alpha-helix. However, conflicting data are reported in the literature. Some authors have suggested that hydrogen bonding does not have a stabilizing function. Here we synthesized pentapeptides having a GXXXG motif to explore its role in pore stability. We used molecular dynamics simulations, quantum mechanics, and experimental biophysical techniques to determine whether hydrogen bonding was formed and had a stabilizing function in ion-channel-like structures. Starting from our previous molecular dynamics data, molecular quantum mechanics simulations, and ATR data showed that a stable ion-channel-like pore formed and a band centered at 2910 cm -1 was attributed to the interaction between Gly 7 of an alpha-helix and Asp 23 of a vicinal alpha-helix.

Despite the extensive use of poly-lactic-glycolic-acid (PLGA) in biomedical applications, computational research on the mesoscopic characterization of PLGA-based delivery systems is limited. In this study, a computational model for PLGA is proposed, developed, and validated for the reproducibility of transport properties that can influence drug release, the rate of which remains difficult to control. For computational efficiency, coarse-grained (CG) models of the molecular components under consideration were built using the MARTINI force field version 2.2. The translocation free energy barrier Δ G t * across the PLGA matrix in the aqueous phase of docetaxel and derivatives of varying sizes and solubilities was predicted via molecular dynamics (MD) simulations and compared with experimental release data. The thermodynamic quantity Δ G t * anticipates and can help explain the release kinetics of hydrophobic compounds from the PLGA matrix, albeit within the limit of a drug concentration below a critical aggregation concentration. The proposed computational framework would allow one to predict the pharmacological behavior of polymeric implants loaded with a variety of payloads under different conditions, limiting the experimental workload and associated costs.

Coarse-grained (CG) modelling with the Martini force field has come of age. By combining a variety of bead types and sizes with a new mapping approach, the newest version of the model is able to accurately simulate large biomolecular complexes at millisecond timescales. In this perspective, we discuss possible applications of the Martini 3 model in drug discovery and development pipelines and highlight areas for future development. Owing to its high simulation efficiency and extended chemical space, Martini 3 has great potential in the area of drug design and delivery. However, several aspects of the model should be improved before Martini 3 CG simulations can be routinely employed in academic and industrial settings. These include the development of automatic parameterisation protocols for a variety of molecule types, the improvement of backmapping procedures, the description of protein flexibility and the development of methodologies enabling efficient sampling. We illustrate our view with examples on key areas where Martini could give important contributions such as drugs targeting membrane proteins, cryptic pockets and protein-protein interactions and the development of soft drug delivery systems., Competing Interests: The authors declare no conflicts of interest., (© The Author(s) 2022.)

With the change in lifestyle and aging of the population, osteoarthritis (OA) is emerging as a major medical burden globally. OA is a chronic inflammatory and degenerative disease initially manifesting with joint pain and eventually leading to permanent disability. To date, there are no drugs available for the definitive treatment of osteoarthritis and most therapies have been palliative in nature by alleviating symptoms rather than curing the disease. This coupled with the vague understanding of the early symptoms and methods of diagnosis so that the disease continues as a global problem and calls for concerted research efforts. A cascade of events regulates the onset and progression of osteoarthritis starting with the production of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α; catabolic enzymes, such as matrix metalloproteinases (MMPs)-1, -3, and -13, culminating into cartilage breakdown, loss of lubrication, pain, and inability to load the joint. Although intra-articular injections of small and macromolecules are often prescribed to alleviate symptoms, low residence times within the synovial cavity severely impair their efficacy. This review will briefly describe the factors dictating the onset and progression of the disease, present the current clinically approved methods for its treatment and diagnosis, and finally elaborate on the main challenges and opportunities for the application of nano/micromedicines in the treatment of osteoarthritis. Thus, future treatment regimens will benefit from simultaneous consideration of the mechanobiological, the inflammatory, and tissue degradation aspects of the disease. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement., (© 2022 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals LLC.)

Over the past thirty years, researchers have highlighted the role played by a class of proteins or polypeptides that forms pathogenic amyloid aggregates in vivo, including i) the amyloid Aβ peptide, which is known to form senile plaques in Alzheimer's disease; ii) α-synuclein, responsible for Lewy body formation in Parkinson's disease and iii) IAPP, which is the protein component of type 2 diabetes-associated islet amyloids. These proteins, known as intrinsically disordered proteins (IDPs), are present as highly dynamic conformational ensembles. IDPs can partially (mis) fold into (dys) functional conformations and accumulate as amyloid aggregates upon interaction with other cytosolic partners such as proteins or lipid membranes. In addition, an increasing number of reports link the toxicity of amyloid proteins to their harmful effects on membrane integrity. Still, the molecular mechanism underlying the amyloidogenic proteins transfer from the aqueous environment to the hydrocarbon core of the membrane is poorly understood. This review starts with a historical overview of the toxicity models of amyloidogenic proteins to contextualize the more recent lipid-chaperone hypothesis. Then, we report the early molecular-level events in the aggregation and ion-channel pore formation of Aβ, IAPP, and α-synuclein interacting with model membranes, emphasizing the complexity of these processes due to their different spatial-temporal resolutions. Next, we underline the need for a combined experimental and computational approach, focusing on the strengths and weaknesses of the most commonly used techniques. Finally, the last two chapters highlight the crucial role of lipid-protein complexes as molecular switches among ion-channel-like formation, detergent-like, and fibril formation mechanisms and their implication in fighting amyloidogenic diseases., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Despite tremendous worldwide efforts, clinical trials assessing Alzheimer's disease (AD)-related therapeutics have been relentlessly unsuccessful. Hence, there is an urgent need to challenge old hypotheses with novel paradigms. An emerging concept is that the amyloid-beta (Aβ) peptide, which was until recently deemed a major player in the cause of AD, may instead modulate synaptic plasticity and protect against excitotoxicity. The link between Aβ-mediated synaptic plasticity and Aβ trafficking is central for understanding AD pathogenesis and remains a perplexing relationship. The crossover between Aβ pathological and physiological roles is subtle and remains controversial. Based on existing literature, as a signaling molecule, Aβ is proposed to modulate its own turnover and synaptic plasticity through what is currently believed to be the cause of AD: the transient formation of pore-like oligomers. A change of perspective regarding how Aβ pores exert a protective function will unavoidably revolutionize the entire field of anti-amyloid drug development., (© 2021 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Ionizable lipid nanoparticles (LNPs) are the most clinically advanced nano-delivery system for therapeutic nucleic acids. The great effort put in the development of ionizable lipids with increased in vivo potency brought LNPs from the laboratory benches to the FDA approval of patisiran in 2018 and the ongoing clinical trials for mRNA-based vaccines against SARS-CoV-2. Despite these success stories, several challenges remain in RNA delivery, including what is known as "endosomal escape." Reaching the cytosol is mandatory for unleashing the therapeutic activity of RNA molecules, as their accumulation in other intracellular compartments would simply result in efficacy loss. In LNPs, the ability of ionizable lipids to form destabilizing non-bilayer structures at acidic pH is recognized as the key for endosomal escape and RNA cytosolic delivery. This is motivating a surge in studies aiming at designing novel ionizable lipids with improved biodegradation and safety profiles. In this work, we describe the journey of RNA-loaded LNPs across multiple intracellular barriers, from the extracellular space to the cytosol. In silico molecular dynamics modeling, in vitro high-resolution microscopy analyses, and in vivo imaging data are systematically reviewed to distill out the regulating mechanisms underlying the endosomal escape of RNA. Finally, a comparison with strategies employed by enveloped viruses to deliver their genetic material into cells is also presented. The combination of a multidisciplinary analytical toolkit for endosomal escape quantification and a nature-inspired design could foster the development of future LNPs with improved cytosolic delivery of nucleic acids., (© 2021 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of The American Institute of Chemical Engineers.)

Many PEGylated nanoparticles activate the complement system, which is an integral component of innate immunity. This is of concern as uncontrolled complement activation is potentially detrimental and contributes to disease pathogenesis. Here, it is demonstrated that, in contrast to carboxyPEG 2000 -stabilized poly(lactic- co -glycolic acid) nanoparticles, surface camouflaging with appropriate combinations and proportions of carboxyPEG 2000 and methoxyPEG 550 can largely suppress nanoparticle-mediated complement activation through the lectin pathway. This is attributed to the ability of the short, rigid methoxyPEG 550 chains to laterally compress carboxyPEG 2000 molecules to become more stretched and assume an extended, random coil configuration. As supported by coarse-grained molecular dynamics simulations, these conformational attributes minimize statistical protein binding/intercalation, thereby affecting sequential dynamic processes in complement convertase assembly. Furthermore, PEG pairing has no additional effect on nanoparticle longevity in the blood and macrophage uptake. PEG pairing significantly overcomes nanoparticle-mediated complement activation without the need for surface functionalization with complement inhibitors.

The addition of polyethylene glycol (PEG) chains to poly(lactic- co -glycolic acid) (PLGA) matrices is extensively used to modulate the biodegradation, drug loading and release, mechanical properties, and chemical stability of the original system. Multiple parameters, including the molecular weight, relative concentration, polarity, and solubility, affect the physicochemical properties of the polymer blend. Here, molecular dynamics simulations with the united-atom 2016H66 force field are used to model the behavior of PLGA and PEG chains and thus predict the overall physicochemical features of the resulting blend. First, the model accuracy is validated against fundamental properties of pure PLGA and PEG samples. In agreement with previous experimental and theoretical observations, the PLGA solubility results to be higher in acetonitrile than in water, with Flory parameters ν ACN = 0.63 ± 0.01 and ν W = 0.21 ± 0.02, and the Young's modulus of PLGA and PEG equal to Y = 2.0 ± 0.43 and 0.32 ± 0.34 GPa, respectively. Next, four PEG/PLGA blending regimes are identified by varying the relative concentrations and molecular weights of the individual polymers. The computational results demonstrate that at low PEG concentrations (<8% w/w), homogeneous blends are generated for both low and high PEG molecular weights. In contrast, at comparable PEG and PLGA concentrations (∼50% w/w), short PEG chains are only partially miscible whereas long PEG chains segregate within the PLGA matrix. This behavior has been confirmed experimentally via differential scanning calorimetry and is in agreement with previous observations. Finally, the computed Young's modulus of PLGA/PEG blends is observed to decrease with the PEG content returning the lowest values for the partial and fully segregated regimens ( Y ≈ 1.3 GPa). This work proposes a computational scheme for predicting the physicochemical properties of PLGA/PEG blends paving the way toward the rational design of polymer mixtures for biomedical applications., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Over the years, nanoparticles, microparticles, implants of poly(D,l-lactide-co-glycolide) (PLGA) have been demonstrated for diverse biomedical applications. Yet, initial burst release and optimal modulation of the release profiles limit their clinical use. Here, shape-defined PLGA microPlates (μPLs) were realized for the sustained release of two anti-inflammatory molecules, the natural polyphenol curcumin (CURC) and the corticosteroid dexamethasone (DEX). Under the electron microscope, μPLs appeared as square prisms with an edge length of 20 μm. The top-down fabrication process allowed the authors to vary, readily and systematically, the μPL height from 5 to 10 μm and the PLGA mass from 1 to 5, 10 and 20 mg. 'Taller' particles realized with higher PLGA concentrations encapsulated more drug reaching on average values of about 150 pg/μPL, for both CURC and DEX. The μPL height and PLGA concentration had major effects on drug release, too. Under sink conditions, DEX release from tall μPLs at 1 h reduced from 50% to 10% and 2% for the 5, 10 and 20 mg PLGA configurations, respectively. Also, DEX was released more slowly from taller as compared to short μPLs. The opposite trend was observed for CURC, possibly for its lower hydrophobicity and molecular weight as compared to DEX. This was also confirmed by quantifying the free energy of translocation for the two drugs via molecular dynamics simulations. Finally, the anti-inflammatory activity of μPLs was tested in vitro on LPS-stimulated rat monocytes and in vivo on a murine model of UVB-induced skin burns. Both in vitro and in vivo, the expression of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) was significantly reduced by the application of μPLs as compared to the free compounds. In vivo, one single topical deposition of CURC-μPLs outperformed multiple, free CURC applications. This work demonstrates that geometry and polymer density can be effectively used to modulate the pharmacological performance of microparticles and mitigate the initial burst release., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer's disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly β-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aβ(1-40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.

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 around 10 -9 m, excited at the surface of the bubble by an electric field of intensity on the order of 10 V/cm. When one studies 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 conditions 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 comprehensive insight into the novelty of the technique, we also investigated the equilibrium adsorption of sodium dodecyl sulfate (SDS) ionic surfactant for bulk concentrations at hundreds of times lower than the Critical Micelle Concentration (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 (Γ) 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.

The lipid bilayer is a flexible matrix that is able to adapt in response to the perturbation induced by inclusions, such as peptides and proteins. Here we use molecular dynamics simulations with a coarse-grained model to investigate the effect of a helical inclusion on a lipid bilayer in the liquid disordered phase. We show that the helical inclusion induces a collective tilt of acyl chains, with a small, yet unambiguous difference between a right- and a left-handed inclusion. This behavior is rationalized using the elastic continuum theory: The magnitude of the chiral (twist) deformation of the bilayer is determined by the interaction at the lipid/inclusion interface, and the decay length is controlled by the elastic properties of the bilayer. The lipid reorganization can thus be identified as a generic mechanism that, together with specific interactions, contributes to chiral recognition in phospholipid bilayers. An enhanced response is expected in highly ordered environments, such as rafts in biomembranes, with a potential impact on membrane-mediated interactions between inclusions.

The large GTPase dynamin catalyzes membrane fission in eukaryotic cells, but despite three decades of experimental work, competing and partially conflicting models persist regarding some of its most basic actions. Here we investigate the mechanical and functional consequences of dynamin scaffold shape changes and disassembly with the help of a geometrically and elastically realistic simulation model of helical dynamin-membrane complexes. Beyond changes of radius and pitch, we emphasize the crucial role of a third functional motion: an effective rotation of the filament around its longitudinal axis, which reflects alternate tilting of dynamin's PH binding domains and creates a membrane torque. We also show that helix elongation impedes fission, hemifission is reached via a small transient pore, and coat disassembly assists fission. Our results have several testable structural consequences and help to reconcile mutual conflicting aspects between the two main present models of dynamin fission-the two-stage and the constrictase model., Competing Interests: MP, ZM, MD No competing interests declared, (© 2018, Pannuzzo et al.)

Smart polymer hydrogels, which can undergo structural and volume phase transitions in response to external stimuli, have gained much attention for their widespread technological applications. Compared to linear polymers, branched chains offer more extensive opportunities to rationally design functional materials, since they permit more extensive structural tunability-for instance by adjusting the balance between hydrophobic and hydrophilic units, the grafting fraction of backbone monomers, or the side chain length, topology, and solubility. Here we conduct coarse-grained molecular dynamics simulations to assess how well generic physical principles capture this complex interplay of tuning parameters, specifically when building networks from complex branched chains with a hydrophobic backbone. Swollen chains collapse upon reducing side chain solubility, length, and grafting density, but neither the sharpness of this transition nor its dynamic range, if measured via chain extension, depends monotonically on these parameters. Networks comprising such chains are more swollen and exhibit even sharper transitions, but their higher responsiveness goes along with a swelling ratio that falls behind that of single chains.

Krabbe's disease is a neurodegenerative disorder caused by deficiency of galactocerebrosidase activity that affects the myelin sheath of the nervous system, involving dysfunctional metabolism of sphingolipids. It has no cure. Because substrate inhibition therapy has been shown to be effective in some human lysosomal storage diseases, we hypothesize that a substrate inhibition therapeutic approach might be appropriate to allow correction of the imbalance between formation and breakdown of glycosphingolipids and to prevent pathological storage of psychosine. The enzyme responsible for the biosynthesis of galactosylceramide and psychosine is uridine diphosphate-galactose ceramide galactosyltransferase (2-hydroxyacylsphingosine 1-β-galactosyltransferase; UGT8; EC 2.4.1.45), which catalyzes the transferring of galactose from uridine diphosphate-galactose to ceramide or sphingosine, an important step of the biosynthesis of galactosphingolipids. Because some bisphosphonates have been identified as selective galactosyltransferase inhibitors, we verify the binding affinity to a generated model of the enzyme UGT8 and investigate the molecular mechanisms of UGT8-ligand interactions of the bisphosphonate zoledronate by a multistep framework combining homology modeling, molecular docking, and molecular dynamics simulations. From structural information on UGTs' active site stereochemistry, charge density, and access through the hydrophobic environment, the molecular docking procedure allowed us to identify zoledronate as a potential inhibitor of human ceramide galactosyltransferase. More importantly, zoledronate derivates were designed through computational modeling as putative new inhibitors. Experiments in vivo and in vitro have been planned to verify the possibility of using zoledronate and/or the newly identified inhibitors of UGT8 for a substrate inhibition therapy useful for treatment of Krabbe's disease and/or other lysosomal disorders. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

We investigate, both theoretically and experimentally, the role played by the oscillations of the cell membrane on the capture rate of substances freely diffusing around the cell. To obtain quantitative results, we propose and build up a reproducible and tunable biomimetic experimental model system to simulate the phenomenon of an oscillation-enhanced (or depressed) capture rate (chemoreception) of a diffusant. The main advantage compared to real biological systems is that the different oscillation parameters (type of deformation, frequencies, and amplitudes) can be finely tuned. The model system that we use is an anchored gas drop submitted to a diffusive flow of charged surfactants. When the surfactant meets the surface of the bubble, it is reversibly adsorbed. Bubble oscillations of the order of a few nanometers are selectively excited, and surfactant transport is accurately measured. The surfactant concentration past the oscillating bubbles was detected by conductivity measurements. The results highlight the role of surface oscillations on the diffusant capture rate. Particularly unexpected is the onset of intense overshoots during the adsorption process. The phenomenon is particularly relevant when the bubbles are exposed to intense forced oscillations near resonance.

The dynamic interplay between cholesterol, asymmetrically (at physiological condition) or symmetrically (hallmark of aging) distributed in membrane, and β amyloid peptides is investigated by a computational approach. The drawn overall picture, starting from the very appearance of β amyloid peptides and going through their self-assembling into potentially toxic oligomeric species, reinforces some of the experimental and theoretical shots recently reported in literature, while new important molecular hints on the physiological role played by the β amyloid peptide are proposed. The so dreaded formation of amyloid pores selective for the passage of calcium ions could in fact explain their physiological concomitant recruitment in the regulation of synaptic plasticity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The protein transport inside a cell is a complex phenomenon that goes through several difficult steps. The facilitated transport requires sophisticated machineries involving protein assemblies. In this work, we developed a diffusion-reaction model to simulate co-transport kinetics of proteins and lipids. We assume the following: (a) there is always a small lipid concentration of order of the Critical Micellar Concentration (CMC) in equilibrium with the membrane; (b) the binding of lipids to proteins modulates the hydrophobicity of the complexes and, therefore, their ability to interact and merge with the bilayer; and (c) some lipids leave the bilayer to replenish those bound to proteins. The model leads to a pair of integral equations for the time-evolution of the adsorbed proteins in the lipid bilayer. Relationships between transport kinetics, CMC, and lipid-protein binding constants were found. Under particular conditions, a perturbation analysis suggests the onset of kinks in the protein adsorption kinetics. To validate our model, we performed leakage measurements of vesicles composed by either high or low CMC lipids interacting with Islet Amyloid PolyPeptide (IAPP) and Aβ (1-40) used as sample proteins. Since the lipid-protein complex stoichiometry is not easily accessible, molecular dynamics simulations were performed using monomeric IAPP interacting with an increasing number of phospholipids. Main results are the following: (a) 1:1 lipid-protein complexes generally show a faster insertion rate proportional to the complex hydrophobicity and inversely related to lipid CMC; (b) on increasing the number of bound lipids, the protein insertion rate decreases; and, ((c) at slow lipids desorption rate, the lipid-assisted proteins transport might exhibit a discontinuous behavior and does non-linearly depend on protein concentration.)

Glycolipids are important components of biological membranes. High concentrations of glycolipids are particularly found in lipid rafts, which take part in many physiological phenomena. This different partitioning and interaction pattern of glycolipids in the membrane as compared to those of phospholipids are likely due to their different chemical structures: the polar regions of glycosphingolipids can be even larger than for their hydrophobic moieties, giving rise to a rich conformational landscape. Here we study the influence of glycosphingolipids galactosylceramide (GCER) and monosialotetrahexosylganglioside (GM1) on the structural and thermodynamic properties of a phospholipid (DPPC) bilayer. Using the method of coarse-grained molecular dynamics simulation we show that both glycolipids increase the phase-transition temperature of phospholipid membranes and that the extent of this increase depends on the headgroup size and structure. GM1 shows a strong tendency to form mixed clusters with phospholipids, thereby stabilizing the membrane. In contrast, GCER is dispersed in the membrane. By occupying the interstitial space between phospholipids it causes a tighter packing of the lipids in the membrane.

We report that at very low initial bulk concentrations, a couple of hundred times below the critical micellar concentration (CMC), anionic surfactant sodium dodecyl sulfate (SDS) adsorbed at the air-water interface of a gas bubble cannot be removed, on the time scale of the experiment (hours), when the surrounding solution is gently replaced by pure water. Extremely sensitive interferometric measurements of the resonance frequency of the bubble-forced oscillations give precise access to the concentration of the surfactant monolayer. The bulk-interface dynamic exchange of SDS molecules is shown to be inhibited below a concentration which we believe refers to a kind of gas-liquid phase transition of the surface monolayer. Above this threshold we recover the expected concentration-dependent desorption. The experimental observations are interpreted within simple energetic considerations supported by molecular dynamics (MD) calculations.

Periodic variation of the distance between two weakly adhering bodies gives rise to a huge tangential motions of the sandwiched solvent layer (squeezing flow). Oscillations either can be induced by an external applied field or can spontaneously arise from the coupling with the solvent heat bath. First we calculated by the Navier-Stokes equation the components of the fluid velocity near two oscillating juxtaposed plates. Then we evaluated the influence of plate oscillations on the transport properties of a trace diffusant dissolved at t = 0 in the outer medium for both deterministic and stochastic excitations. By employing both analytical (Fokker-Planck) and coarse-grained molecular dynamics (MD) simulations, we proved that the entry and migration rates of the diffusant sharply increases with the oscillation amplitudes. Enhancement was related to relevant parameters like oscillation frequency, fluid layer thickness, fluid viscosity, and temperature. An extension to the case of oscillating multistacked lamellae has been also made. Theoretical and MD results suggest a significant enhancement of the diffusant flux even in the worse situation of thermally excited small amplitude fluctuations. Excitation arising from other sources (e.g., microwave or ultrasound irradiation of solid-fluid layered systems) could have a dramatic effect on the transport phenomena. Possible implications to relevant biological problems have been discussed.

Peptide- or protein-induced curvatures of lipid membranes may be studied in molecular dynamics (MD) simulations. In these, membranes are usually modeled as infinitely extended bilayers by using periodic boundary conditions. However, the enforced periodicity results in an underestimation of the bending power of peptides, unless the patch size is much larger than the induced curvature radii. In this letter, we propose a novel approach to evaluate the bending power of a given distribution and/or density of peptides based on the use of flat open-edged lipid patches. To ensure long-lived metastable structures, the patch rim is stabilized in MD simulations by a local enrichment with short-chain lipids. By combining the theory of continuum elastic media with MD simulations, we prove that open-edged patches evolve from a planar state to a closed vesicle, with a transition rate that strongly depends on the concentration of lipid soluble peptides. For close-to-critical values for the patch size and edge energy, the response to even small changes in peptide concentration adopts a transition-like behavior (buckling instability). The usage of open-edged membrane patches amplifies the bending power of peptides, thereby enabling the analysis of the structural properties of membrane-peptide systems. We applied the presented method to investigate the curvature induced by aggregating β -amyloid peptides, unraveling a strong sensitivity of membrane deformation to the peptide concentration.

We report on the mechanism of membrane fusion mediated by polyethylene glycol (PEG) and Ca(2+) by means of a coarse-grained molecular dynamics simulation approach. Our data provide a detailed view on the role of cations and polymer in modulating the interaction between negatively charged apposed membranes. The PEG chains cause a reduction of the inter-lamellar distance and cause an increase in concentration of divalent cations. When thermally driven fluctuations bring the membranes at close contact, a switch from cis to trans Ca(2+)-lipid complexes stabilizes a focal contact acting as a nucleation site for further expansion of the adhesion region. Flipping of lipid tails induces subsequent stalk formation. Together, our results provide a molecular explanation for the synergistic effect of Ca(2+) and PEG on membrane fusion.

We report measurements of the relaxation and resonance frequency of forced oscillating bubbles covered by a layer of surface-active molecules, the anionic surfactant sodium dodecyl sulfate (SDS). Less systematic investigations have been also carried out on neutral and cationic surfactants. A divergence of the viscous damping is observed at a very low bulk concentration. Subtle variations in the resonance peak are also measured. Bubble oscillations are driven by an electric field and measured with a sensitive interferometric technique. Results are interpreted with a model which takes care of the coupling between the dynamics of fluid surface oscillations and the properties of a surfactant monolayer in the vicinity of the phase transition from a gas-like distribution to a liquid-like assembly (the so-called gas-LE transition). Important charge effects are also considered. The basic assumptions of the model (cooperative adsorption of the surfactant at the air-water interface and coupling between the shape of the deformed surface and the local surfactant concentration) have been fully confirmed by extensive coarse-grained molecular dynamics simulations on model systems.

The human islet amyloid polypeptide (hIAPP) is the primary component in the toxic islet amyloid deposits in type-2 diabetes. hIAPP self-assembles to aggregates that permeabilize membranes and constitutes amyloid plaques. Uncovering the mechanisms of amyloid self-assembly is the key to understanding amyloid toxicity and treatment. Although structurally similar, hIAPP's rat counterpart, the rat islet amyloid polypeptide (rIAPP), is non-toxic. It has been a puzzle why these peptides behave so differently. We combined multiscale modelling and theory to explain the drastically different dynamics of hIAPP and rIAPP: The differences stem from electrostatic dipolar interactions. hIAPP forms pentameric aggregates with the hydrophobic residues facing the membrane core and stabilizing water-conducting pores. We give predictions for pore sizes, the number of hIAPP peptides, and aggregate morphology. We show the importance of curvature-induced stress at the early stages of hIAPP assembly and the α-helical structures over β-sheets. This agrees with recent fluorescence spectroscopy experiments.

The effect of viscosity on the encounter rate of two interacting membranes was investigated by combining a non-equilibrium Fokker-Planck model together with extensive Molecular Dynamics (MD) calculations. The encounter probability and stabilization of transient contact points represent the preliminary steps toward short-range adhesion and fusion of lipid leaflets. To strengthen our analytical model, we used a Coarse Grained MD method to follow the behavior of two charged palmitoyl oleoyl phosphatidylglycerol membranes embedded in a electrolyte-containing box at different viscosity regimes. Solvent friction was modulated by varying the concentration of a neutral, water-soluble polymer, polyethylene glycol, while contact points were stabilized by divalent ions that form bridges among juxtaposed membranes. While a naïve picture foresees a monotonous decrease of the membranes encounter rate with solvent viscosity, both the analytical model and MD simulations show a complex behavior. Under particular conditions, the encounter rate could exhibit a maximum at a critical viscosity value or for a critical concentration of bridging ions. These results seem to be confirmed by experimental observations taken from the literature.

The mechanisms underlying the formation of extracellular amyloid plaques on neuronal membranes, a major hallmark of Alzheimer's disease, are the subject of intense debate. Here we use multiscale simulations and analytical theory to unveil the early steps of the spontaneous self-assembly of membrane-embedded α-helical Aβ (1-40) peptides. Based on a simple analytical model describing the electrostatic repulsions among water-exposed charged residues, the presence of distorted structures called "frustrated helices" is predicted. Large scale (20 μs) Coarse Grained simulations of 36 replicas of Aβ (1-40) performed within a POPC lipid matrix confirmed the formation of supramolecular assemblies which resemble a twisted ribbon. Fully atomistic simulations have demonstrated the stability of these helical structures. Concomitant to the formation of these large assemblies, CG simulations evidenced membrane curvature and substantiate the view that these assemblies may entail mechanical stress on membrane structure. We think that these findings provide an alternative view to the traditional models that consider a conformational transition towards β-sheet rich structures as a prerequisite for triggering membrane damage and, eventually, neurotoxicity.

A simple hydrodynamic model has been developed to explain the experimentally observed chirality selection in stirred solutions of self-assembling achiral dyes. Selection depends on the stirring direction: the dichroic signal reverses its shape in clockwise or anti-clockwise rotations. Our model investigates the possible role of the liquid-solid interface in nucleating, growing, and transferring to the bulk of chiral seeds. The nucleation step requires a double modulation of the hydrodynamic field exhibiting different velocity along two orthogonal axes. Under a series of restrictions, such a condition is easily met at the solid-liquid interface and it is dictated by the boundary conditions and geometry of stirring. In stagnant conditions, growing helices made-up of self-assembled achiral dyes have no chiral preference forming a racemic mixture that contains identical amount of right-handed (R) and left-handed (L) configurations. The application of a hydrodynamic torque (related to the velocity gradient and width of the helix) breaks down the original symmetry, a further velocity gradient perpendicular to the first one ensures, after averaging, a slightly different population of R and L conformations. The yields of the hydrodynamic-induced chirality excess are extremely tiny, hence the suggested mechanism is significant only if next chirality amplification processes are efficient. Again, hydrodynamics provides a tool for the detachment of weakly bound aggregates once they have reached a critical length. Aggregates are transported in the bulk where the ripening process goes to completion. The efficiency of the surface catalytic effect strongly depends on the aggregate-surface sticking energy, reaching a maximum at intermediate sticking energies (of order of 10 kT). Numerical estimates show that the proposed mechanism is rather efficient, giving rise to entatiomeric excesses near (but smaller than) those experimentally found.

We followed the process of enzymatic digestion of ganglioside GD1a, operated by sialidase on aggregated micelles. The product is the ganglioside GM1, lacking the external sialic acid. The structural aspects and the kinetics connected to the process occurring on a fragmented-condensed substrate, the ganglioside micelles, are investigated by small angle X-ray scattering (SAXS). Observed at short times, the kinetics of the reaction shows a transient step-like decay, while it tends to a smooth Michaelis-Menten kinetics in the late stages. We propose a model, based on the fragmented-condensed nature of the substrate, that well reproduces the experimental observation without invoking any feedback mechanism in the reaction, usually required for an oscillatory behavior. The model predicts an initial regime dominated by the strict enzyme-substrate interaction, with a step-like appearance.

We develop a semi-quantitative analytical theory to describe adhesion between two identical planar charged surfaces embedded in a polymer-containing electrolyte solution. Polymer chains are uncharged and differ from the solvent by their lower dielectric permittivity. The solution mimics physiological fluids: It contains 0.1 M of monovalent ions and a small number of divalent cations that form tight bonds with the headgroups of charged lipids. The components have heterogeneous spatial distributions. The model was derived self-consistently by combining: (a) a Poisson-Boltzmann like equation for the charge densities, (b) a continuum mean-field theory for the polymer profile, (c) a solvation energy forcing the ions toward the polymer-poor regions, and (d) surface interactions of polymers and electrolytes. We validated the theory via extensive coarse-grained Molecular Dynamics (MD) simulations. The results confirm our analytical model and reveal interesting details not detected by the theory. At high surface charges, polymer chains are mainly excluded from the gap region, while the concentration of ions increases. The model shows a strong coupling between osmotic forces, surface potential and salting-out effects of the slightly polar polymer chains. It highlights some of the key differences in the behaviour of monomeric and polymeric mixed solvents and their responses to Coulomb interactions. Our main findings are: (a) the onset of long-ranged ion-induced polymer depletion force that increases with surface charge density and (b) a polymer-modified repulsive Coulomb force that increases with surface charge density. Overall, the system exhibits homeostatic behaviour, resulting in robustness against variations in the amount of charges. Applications and extensions of the model are briefly discussed.

Insight into the forces governing a system is essential for understanding its behavior and function. Thermodynamic investigations provide a wealth of information that is not, or is hardly, available from other methods. This article reviews thermodynamic approaches and assays to measure collective properties such as heat adsorption / emission and volume variations. These methods can be successfully applied to the study of lipid vesicles (liposomes) and biological membranes. With respect to instrumentation, differential scanning calorimetry, pressure perturbation calorimetry, isothermal titration calorimetry, dilatometry, and acoustic techniques aimed at measuring the isothermal and adiabatic processes, two- and three-dimensional compressibilities are considered. Applications of these techniques to lipid systems include the measurement of different thermodynamic parameters and a detailed characterization of thermotropic, barotropic, and lyotropic phase behavior. The membrane binding and / or partitioning of solutes (proteins, peptides, drugs, surfactants, ions, etc.) can also be quantified and modeled. Many thermodynamic assays are available for studying the effect of proteins and other additives on membranes, characterizing non-ideal mixing, domain formation, bilayer stability, curvature strain, permeability, solubilization, and fusion. Studies of membrane proteins in lipid environments elucidate lipid-protein interactions in membranes. Finally, a plethora of relaxation phenomena toward equilibrium thermodynamic structures can be also investigated. The systems are described in terms of enthalpic and entropic forces, equilibrium constants, heat capacities, partial volume changes, volume and area compressibility, and so on, also shedding light on the stability of the structures and the molecular origin and mechanism of the structural changes.

A semiquantitative theory to describe the adhesion mechanism between an elastic membrane and a solid substrate (or another membrane) was developed. Since the membrane bending deformation requires a relatively small energy cost, thermally excited fluctuations may give rise to a local protrusion connecting the membrane to the substrate. This transient adhesion site is stabilized by short-range adhesion forces and it is destabilized by repulsion and elastic deformation energy. Above a critical radius of the contact site, adhesion forces prevail, enabling the contact site to expand until complete membrane-substrate adhesion is attained. This represents a typical nucleation mechanism involving both growth and dissolution processes. However, here we prove that also in the barrierless region, well beyond the critical radius, the spreading rate of a membrane still remains rather small, even under the favorable assumption of strong, sudden, and irreversible membrane-substrate adhesion. A detailed analysis of the membrane vibrational behavior near the adhesion patch rim suggests a reasonable mechanism for the spreading rate that has been analyzed by nonequilibrium statistical mechanics approaches. In relevant limiting cases, the model yields simple analytical formulas. Approximate relationships between the spreading rate and parameters like membrane elastic bending modulus, membrane-substrate interaction, temperature, and solvent viscosity have been found.

A semiquantitative theory aimed to describe the adhesion kinetics between soft objects, such as living cells or vesicles, has been developed. When rigid bodies are considered, the adhesion kinetics is successfully described by the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) picture, where the energy profile of two approaching bodies is given by a two asymmetrical potential wells separated by a barrier. The transition probability from the long-distance to the short-distance minimum defines the adhesion rate. Conversely, soft bodies might follow a different pathway to reach the short-distance minimum: thermally excited fluctuations give rise to local protrusions connecting the approaching bodies. These transient adhesion sites are stabilized by short-range adhesion forces (e.g., ligand-receptor interactions between membranes brought at contact distance), while they are destabilized both by repulsive forces and by the elastic deformation energy. Above a critical area of the contact site, the adhesion forces prevail: the contact site grows in size until the complete adhesion of the two bodies inside a short-distance minimum is attained. This nucleation mechanism has been developed in the framework of a nonequilibrium Fokker-Planck picture by considering both the adhesive patch growth and dissolution processes. In addition, we also investigated the effect of the ligand-receptor pairing kinetics at the adhesion site in the time course of the patch expansion. The ratio between the ligand-receptor pairing kinetics and the expansion rate of the adhesion site is of paramount relevance in determining the overall nucleation rate. The theory enables one to self-consistently include both thermodynamics (energy barrier height) and dynamic (viscosity) parameters, giving rise in some limiting cases to simple analytical formulas. The model could be employed to rationalize fusion kinetics between vesicles, provided the short-range adhesion transition is the rate-limiting step to the whole adhesion process. Approximate relationships between the experimental fusion rates reported in the literature and parameters such as membrane elastic bending modulus, repulsion strength, temperature, osmotic forces, ligand-receptor binding energy, solvent and membrane viscosities are satisfactory explained by our model. The present results hint a possible role of the initial long-distance-->short-distance transition in determining the whole fusion kinetics.