Compartmentalized drug localization studies in extracellular vesicles for anticancer therapy.

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.

GxxxG Motif Stabilize Ion-Channel like Pores through Cα-H···O Interaction in Aß (1-40).

Aß (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.

A Coarse-Grained Molecular Dynamics Description of Docetaxel-Conjugate Release from PLGA Matrices.

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 ΔGt* 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 ΔGt* 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.

Management of osteoarthritis: From drug molecules to nano/micromedicines.

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)-1ß, 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.

A unifying framework for amyloid-mediated membrane damage: The lipid-chaperone hypothesis.

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 Aß 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 Aß, 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.

Towards design of drugs and delivery systems with the Martini coarse-grained model.

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.

Beta-amyloid pore linked to controlled calcium influx into the cell: A new paradigm for Alzheimer's Disease.

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 (Aß) 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 Aß-mediated synaptic plasticity and Aß trafficking is central for understanding AD pathogenesis and remains a perplexing relationship. The crossover between Aß pathological and physiological roles is subtle and remains controversial. Based on existing literature, as a signaling molecule, Aß 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 Aß pores exert a protective function will unavoidably revolutionize the entire field of anti-amyloid drug development.

Cytosolic delivery of nucleic acids: The case of ionizable lipid nanoparticles.

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.

Predicting the Miscibility and Rigidity of Poly(lactic-co-glycolic acid)/Polyethylene Glycol Blends via Molecular Dynamics Simulations.

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 νACN = 0.63 ± 0.01 and νW = 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.

Overcoming Nanoparticle-Mediated Complement Activation by Surface PEG Pairing.

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 carboxyPEG2000-stabilized poly(lactic-co-glycolic acid) nanoparticles, surface camouflaging with appropriate combinations and proportions of carboxyPEG2000 and methoxyPEG550 can largely suppress nanoparticle-mediated complement activation through the lectin pathway. This is attributed to the ability of the short, rigid methoxyPEG550 chains to laterally compress carboxyPEG2000 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.

Symmetry-breaking transitions in the early steps of protein self-assembly.

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 ß-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 Aß(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.

Engineering shape-defined PLGA microPlates for the sustained release of anti-inflammatory molecules.

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-1ß, 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.

Protein Adsorption at the Air-Water Interface by a Charge Sensing Interferometric Technique.

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.

Helical Inclusions in Phospholipid Membranes: Lipid Adaptation and Chiral Order.

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 role of scaffold reshaping and disassembly in dynamin driven membrane fission.

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.

Responsive behavior of a branched-chain polymer network: a molecular dynamics study.

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.

Zoledronate derivatives as potential inhibitors of uridine diphosphate-galactose ceramide galactosyltransferase 8: A combined molecular docking and dynamic study.

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-ß-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.

Oscillations of Bubble Shape Cause Anomalous Surfactant Diffusion: Experiments, Theory, and Simulations.

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.

Lipid-assisted protein transport: A diffusion-reaction model supported by kinetic experiments and molecular dynamics simulations.

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 Aß (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

On the physiological/pathological link between Aß peptide, cholesterol, calcium ions and membrane deformation: A molecular dynamics study.

The dynamic interplay between cholesterol, asymmetrically (at physiological condition) or symmetrically (hallmark of aging) distributed in membrane, and ß amyloid peptides is investigated by a computational approach. The drawn overall picture, starting from the very appearance of ß 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 ß 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.