Atomistic Simulations of Bicelle Mixtures

AbstractMixtures of long- and short-tail phosphatidylcholine lipids are known to self-assemble into a variety of aggregates combining flat bilayerlike and curved micellelike features, commonly called bicelles. Atomistic simulations of bilayer ribbons and perforated bilayers containing dimyristoylphosphatidylcholine (DMPC, di-C14 tails) and dihexanoylphosphatidylcholine (DHPC, di-C6 tails) have been carried out to investigate the partitioning of these components between flat and curved microenvironments and the stabilization of the bilayer edge by DHPC. To approach equilibrium partitioning of lipids on an achievable simulation timescale, configuration-bias Monte Carlo mutation moves were used to allow individual lipids to change tail length within a semigrand-canonical ensemble. Since acceptance probabilities for direct transitions between DMPC and DHPC were negligible, a third component with intermediate tail length (didecanoylphosphatidylcholine, di-C10 tails) was included at a low concentration to serve as an intermediate for transitions between DMPC and DHPC. Strong enrichment of DHPC is seen at ribbon and pore edges, with an excess linear density of ∼3 nm−1. The simulation model yields estimates for the onset of edge stability with increasing bilayer DHPC content between 5% and 15% DHPC at 300 K and between 7% and 17% DHPC at 323 K, higher than experimental estimates. Local structure and composition at points of close contact between pores suggest a possible mechanism for effective attractions between pores, providing a rationalization for the tendency of bicelle mixtures to aggregate into perforated vesicles and perforated sheets

Investigation of Domain Formation in Sphingomyelin/Cholesterol/POPC Mixtures by Fluorescence Resonance Energy Transfer and Monte Carlo Simulations

We have recently proposed a phase diagram for mixtures of porcine brain sphingomyelin (BSM), cholesterol (Chol), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) on the basis of kinetics of carboxyfluorescein efflux induced by the amphipathic peptide δ-lysin. Although that study indicated the existence of domains, phase separations in the micrometer scale have not been observed by fluorescence microscopy in BSM/Chol/POPC mixtures, though they have for some other sphingomyelins (SM). Here we examine the same BSM/Chol/POPC system by a combination of fluorescence resonance energy transfer (FRET) and Monte Carlo simulations. The results clearly demonstrate that domains are formed in this system. Comparison of the FRET experimental data with the computer simulations allows the estimate of lipid-lipid interaction Gibbs energies between SM/Chol, SM/POPC, and Chol/POPC. The latter two interactions are weakly repulsive, but the interaction between SM and Chol is favorable. Furthermore, those three unlike lipid interaction parameters between the three possible lipid pairs are sufficient for the existence of a closed loop in the ternary phase diagram, without the need to involve multibody interactions. The calculations also indicate that the largest POPC domains contain several thousand lipids, corresponding to linear sizes of the order of a few hundred nanometers

The Permeation Behavior of Nanoparticles in Lipid Membranes

The number of engineered nanoparticles for applications in the biomedical arena has grown tremendously over the last years due to advances in the science of synthesis and characterization. For most applications, the crucial step is the transport through a physiological cellular membrane. However, the behavior of nanoparticles in a biological matrix is a very complex problem that depends not only on the type of nanoparticle, but also on its size, shape, phase, surface charge, chemical composition and agglomeration state. In this thesis, I introduce a streamlined theoretical model that predicts the average time of entry of nanoparticles in lipid membranes, using a combination of molecular dynamics simulations and statistical approaches. The uniqueness of the model lies in the ability to identify four parameters that separate the contributions of nanoparticle characteristics (i.e. size, shape, solubility) from the membrane properties (density distribution and dynamics). This factorization allows the inclusion of data obtained from both experimental and computational sources, as well as a rapid estimation of large sets of permutations in membranes. The robustness of the model is supported by experiments carried out in lipid vesicles encapsulating graphene quantum dots as nanoparticles. The model is applied to the study of various nanoparticles, biological membranes (i.e. mammalian cellular organelles, viral envelopes), and environmental conditions. Overall, this work contributes to the understanding and prediction of interactions between nanoparticles and lipid membranes, responding to the high level of interest across multiple areas of study in modulating intracellular targets, and the need to understand and improve the applications of nanoparticles and to assess their effect on human health (i.e. cytotoxicity, bioavailability).PHDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163056/1/desmliu_1.pd

Mobility and Translocation of TAT Peptides in Model Membranes

Cell penetrating peptides (CPPs) like HIV1-TAT have the special property to traverse the cell membrane and to function as vectors for various macromolecular cargoes such as fluorophores, nucleotides, drugs, proteins, DNA, and peptide-nucleic acids, and even liposomes and magnetic nanoparticles. In spite of the fact that TAT peptides were intensively investigated, the exact internalization mechanism is still controversial. Despite the controversy and uncertainty regarding the uptake mechanism, the property of TAT to deliver non-permeable molecules into living cells makes it an attractive tool for biological sciences as well as medicine and biotechnology. It is therefore essential to identify precisely the criteria which can yield an efficient cell penetration with a high degree of drug transfer. To elucidate the non-endocytic entry routes and the transduction mechanism, one possibility is to analyse interaction of TAT peptides with model membrane systems. In this study we use giant unilamellar vesicles (GUVs) as cyto-mimetic model system since the micrometer scale of the GUVs enables microscopic observation of these liposomes. In this study we applied high-speed single-particle tracking (SPT) and confocal laser scanning microscopy to systematically examine factors that affect membrane binding, mobility and penetration of fluorescence labelled TAT peptides in the GUVs with different composition. To focus onto interaction between TAT and lipids the first experiments were performed in sucrose/glucose solution with all ions excluded from the media. As a reference we first examined the mobility of fluorescent lipids within the GUV bilayer. As expected, lipid mobility varied clearly with the phase state of the membranes, whereas peptide mobility was independent on membrane hydrophobic core, but dependent on headgroup of lipids in the bilayer. CLSM experiments revealed that in GUVs formed by phosphatidylcholine (PC) and cholesterol no translocation of TAT peptides but just accumulation on the membrane. The same effect was observed also for anionic GUVs containing 15-30 mol % phosphatidylserine (PS). Additional SPT experiments and evaluation of diffusion coefficients revealed that TAT peptides “float” on neutral membranes and they are partial inserted in the headgroup of anionic bilayers. Introduction of a significant amount of anionic lipids (40 mol %) or lipids inducing locally a negative curvature into the membranes (20 mol %) affected TAT translocation across these membranes. Notably, we discovered that TAT peptides were not only able to directly penetrate such membranes in a passive manner, but they were also capable of forming physical pores, which could be passed by small but not large dye tracer molecules. For the physiological relevance of the study, additional experiments in the presence of salt solutions were performed. CLSM experiments showed that physiological salt solution dramatically changed the TAT interaction with the GUV membrane. Binding of TAT to GUVs of all employed compositions was completely lost, and the peptides now efficiently translocated into the GUV interior. In confocal images no membrane staining was observable and dye release indicated again pore formation. Also the sensitive single molecule microscope did not detect any trace of peptides on the GUV surface. This result was obtained for neutral or anionic, liquid-ordered or liquid-disordered membranes. Also, there was no difference for GUVs without cholesterol or in case of other salt solutions at the same concentration, 100 mM (CaCl2, CaCO3 or PBS)

EXPERIMENTAL AND MOLECULAR DYNAMICS SIMULATION STUDIES OF PARTITIONING AND TRANSPORT ACROSS LIPID BILAYER MEMBRANES

Most drugs undergo passive transport during absorption and distribution in the body. It is desirable to predict passive permeation of future drug candidates in order to increase the productivity of the drug discovery process. Unlike drug-receptor interactions, there is no receptor map for passive permeability because the process of transport across the lipid bilayer involves multiple mechanisms. This work intends to increase the understanding of permeation of drug-like molecules through lipid bilayers. Drug molecules in solution typically form various species due to ionization, complexation, etc. Therefore, species specific properties must be obtained to bridge the experiment and simulations. Due to the volume contrast between intra- and extravesicular compartments of liposomes, minor perturbations in ionic and binding equilibria become significant contributors to transport rates. Using tyramine as a model amine, quantitative numerical models were developed to determine intrinsic permeability coefficients. The microscopic ionization and binding constants needed for this were independently measured. The partition coefficient in 1,9-decadiene was measured for a series of compounds as a quantitative surrogate for the partitioning into the hydrocarbon region of the bilayer. These studies uncovered an apparent long-range interaction between the two polar substituents that caused deviations in the microscopic pKa values and partition coefficient of tyramine from the expected values. Additionally the partition coefficients in the preferred binding region of the bilayer were also measured by equilibrium uptake into liposomes. All-atom molecular dynamics simulations of lipid bilayers containing tyramine, 4- ethylphenol, or phenylethylamine provided free energies of transfer of these solutes from water to various locations on the transport path. The experimentally measured partition coefficients were consistent with the free energy profiles in showing the barrier in the hydrocarbon region and preferred binding region near the interface. The substituent contributions to these free energies were also quantitatively consistent between the experiments and simulations. Specific interactions between solutes and the bilayer suggest that amphiphiles are likely to show preferred binding in the head group region and that the most of hydrogen bonds involving solutes located inside the bilayer are with water molecules. Solute re-orientation inside the bilayer lowers the partitioning barrier by allowing favorable interactions

Interaction of graphene nanoparticles and lipid membranes displaying different liquid ordering: a molecular dynamics study

Understanding the effects of graphene-based nanomaterials on lipid membranes is fundamental to determine their environmental impact and the efficiency of their biomedical use. By means of molecular dynamics simulations of simple model lipid bilayers, we analyse in detail the different interaction modes. We have studied bilayers consisting of lipid species (including cholesterol) which display different internal liquid ordering. Nanometric graphene layers can be transiently adsorbed onto the lipid membrane and/or inserted in its hydrophobic region. Once inserted, graphene nanometric flakes display a diffusive dynamics in the membrane plane, they adopt diverse orientations depending on their size and oxidation degree, and they show a particular aversion to be placed close to cholesterol molecules in the membrane. Addition of graphene to phase-segregated ternary membranes is also investigated in the context of the lipid raft model for the lipid organization of biological membranes. Our simulation results show that graphene layers can be inserted indistinctly in the ordered and disordered regions. Once inserted, nanometric flakes migrate to disordered and cholesterol-poor lipid phases