Directional K+ channel insertion in a single phospholipid bilayer: Neutron reflectometry and electrophysiology in the joint exploration of a model membrane functional platform.

We investigated the insertion of small potassium (K+) channel proteins (KcvMA-1D and KcvNTS) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment.

Water response to ganglioside GM1 surface remodelling.

BACKGROUND: Gangliosides are biological glycolipids participating in rafts, structural and functional domains of cell membranes. Their headgroups are able to assume different conformations when packed on the surface of an aggregate, more lying or standing. Switching between different conformations is possible, and is a collective event. Switching can be induced, in model systems, by concentration or temperature increase, then possibly involving ganglioside-water interaction. In the present paper, the effect of GM1 ganglioside headgroup conformation on the water structuring and interactions is addressed. METHODS: Depolarized Rayleigh Scattering, Raman Scattering, Quasielastic Neutron Scattering and NMR measurements were performed on GM1 ganglioside solutions, focusing on solvent properties. RESULTS: All used techniques agree in evidencing differences in the structure and dynamics of solvent water on different time-and-length scales in the presence of either GM1 headgroup conformations. CONCLUSIONS: In general, all results indicate that both the structural properties of solvent water and its interactions with the sugar headgroups of GM1 respond to surface remodelling. The extent of this modification is much higher than expected and, interestingly, ganglioside headgroups seem to turn from cosmotropes to chaotropes upon collective rearrangement from the standing- to the lying-conformation. SIGNIFICANCE: In a biological perspective, water structure modulation could be one of the physico-chemical elements contributing to the raft strategy, both for rafts formation and persistence and for their functional aspects. In particular, the interaction with approaching bodies could be favoured or inhibited or triggered by complex-sugar-sequence conformational switch. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Ganglioside GM1 forces the redistribution of cholesterol in a biomimetic membrane.

Neutron reflectivity has been applied to investigate different mixed asymmetric lipid systems, in the form of single "supported+floating" bilayers, made of phospholipids, cholesterol and GM1 ganglioside (Neu5Acα2-3(Galß1-3GalNAcß1-4)Galß1-4Glcß1Cer)) in bio-similar mole ratios. Bilayer preparation was carried out layer-by-layer with the Langmuir-Blodgett Langmuir-Schaefer techniques, allowing for compositional asymmetry in the system buildup. It is the first time that such a complex model membrane system is reported. Two important conclusions are drawn. First, it is experimentally shown that the presence of GM1 enforces an asymmetry in cholesterol distribution, opposite to what happens for a GM1-free membrane that, submitted to a similar procedure, results in a full symmetrization of cholesterol distribution. We underline that natural cholesterol has been used. Second, and most interesting, our results suggest that a preferential asymmetric distribution of GM1 and cholesterol is attained in a model membrane with biomimetic composition, revealing that a true coupling between the two molecular species occurs.

Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems.

We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Neutrons for rafts, rafts for neutrons.

The determination of the structure of membrane rafts is a challenging issue in biology. The selection of membrane components both in the longitudinal and transverse directions plays a major role as it determines the creation of stable or tunable platforms that host interactions with components of the outer environment. We focus here on the possibility to apply neutron scattering to the study of raft mimics. With this aim, we realized two extreme experimental models for the same complex membrane system (phospholipid : cholesterol : ganglioside GM1), involving two of the characteristic components of glycolipid-enriched rafts. One consists of a thick stack of tightly packed membranes, mixed and symmetric in composition, deposited on a silicon wafer and analyzed by neutron diffraction. The other consists of a free floating individual membrane, mixed and asymmetric in composition in the two layers, studied by neutron reflection. We present here results on the ganglioside-cholesterol coupling. Ganglioside GM1 is found to force the redistribution of cholesterol between the two layers of the model membranes. This causes cholesterol exclusion from compositionally symmetric ganglioside-containing membranes, or, alternatively, asymmetric cholesterol enrichment in raft-mimics, where gangliosides reside into the opposite layer.

DNA sperm damage correlates with nuclear ultrastructural sperm defects in teratozoospermic men.

Sperm morphology has consistently been the best indicator of male fertility. Transmission electron microscopy currently provides the most information on the subcellular details of sperm structure. Recently, assessment of sperm DNA damage has been employed to assess fertility potential. The purpose of this work was to link sperm DNA damage, evaluated by an intercalated fluorescent dye, with the structural characteristics of sperm. Conventional semen analysis was performed on samples from men undergoing fertility evaluation. Thirty men were evaluated and assigned to three subgroups based on strict criteria for sperm morphology: normal morphology (>14% normal forms), intermediate morphology (5-14% normal forms), and poor morphology (<5% normal forms). By quantifying acridine orange-positive cells and ultrastructural sperm defects, we found that the poor morphology pattern group showed a positive association between sperm carrying damaged DNA and the percentage of sperm nucleus with vacuoles (P = 0.01). No statistically significant correlations were established in other ultrastructural characteristics of sperm, including immature chromatin, lytic changes, or abnormal sperm tails. These results suggest that zones without chromatin in the sperm nucleus reflect underlying chromosomal or DNA defects in severe teratozoospermic men. This association should be considered in the evaluation of male fertility.

A multiscale optimisation method for bone growth scaffolds based on triply periodic minimal surfaces.

Tissue engineered bone scaffolds are potential alternatives to bone allografts and autografts. Porous scaffolds based on triply periodic minimal surfaces (TPMS) are good candidates for tissue growth because they offer high surface-to-volume ratio, have tailorable stiffness, and can be easily fabricated by additive manufacturing. However, the range of TPMS scaffold types is extensive, and it is not yet clear which type provides the fastest cell or tissue growth while being sufficiently stiff to act as a bone graft. Nor is there currently an established methodology for TPMS bone scaffold design which can be quickly adopted by medical designers or biologists designing implants. In this study, we examine six TPMS scaffold types for use as tissue growth scaffolds and propose a general methodology to optimise their geometry. At the macro-scale, the optimisation routine ensures a scaffold stiffness within suitable limits for bone, while at the micro-scale it maximises the cell growth rate. The optimisation procedure also ensures the scaffold pores are of sufficient diameter to allow oxygen and nutrient delivery via capillaries. Of the examined TPMS structures, the Lidinoid and Split P cell types provide the greatest cell growth rates and are therefore the best candidates for bone scaffolds.

Polymeric micelles in drug delivery: An insight of the techniques for their characterization and assessment in biorelevant conditions.

Polymeric micelles, i.e. aggregation colloids formed in solution by self-assembling of amphiphilic polymers, represent an innovative tool to overcome several issues related to drug administration, from the low water-solubility to the poor drug permeability across biological barriers. With respect to other nanocarriers, polymeric micelles generally display smaller size, easier preparation and sterilization processes, and good solubilization properties, unfortunately associated with a lower stability in biological fluids and a more complicated characterization. Particularly challenging is the study of their interaction with the biological environment, essential to predict the real in vivo behavior after administration. In this review, after a general presentation on micelles features and properties, different characterization techniques are discussed, from the ones used for the determination of micelles basic characteristics (critical micellar concentration, size, surface charge, morphology) to the more complex approaches used to figure out micelles kinetic stability, drug release and behavior in the presence of biological substrates (fluids, cells and tissues). The techniques presented (such as dynamic light scattering, AFM, cryo-TEM, X-ray scattering, FRET, symmetrical flow field-flow fractionation (AF4) and density ultracentrifugation), each one with their own advantages and limitations, can be combined to achieve a deeper comprehension of polymeric micelles in vivo behavior. The set-up and validation of adequate methods for micelles description represent the essential starting point for their development and clinical success.

inPentasomes: An innovative nose-to-brain pentamidine delivery blunts MPTP parkinsonism in mice.

Preclinical and clinical evidences have demonstrated that astroglial-derived S100B protein is a key element in neuroinflammation underlying the pathogenesis of Parkinson's disease (PD), so much as that S100B inhibitors have been proposed as promising candidates for PD targeted therapy. Pentamidine, an old-developed antiprotozoal drug, currently used for pneumocystis carinii is one of the most potent inhibitors of S100B activity, but despite this effect, is limited by its low capability to cross blood brain barrier (BBB). To overcome this problem, we developed a non-invasive intranasal delivery system, chitosan coated niosomes with entrapped pentamidine (inPentasomes), in the attempt to provide a novel pharmacological approach to ameliorate parkinsonism induced by subchronic MPTP administration in C57BL-6 J mice. inPentasomes, prepared by evaporation method was administered daily by intranasal route in subchronic MPTP-intoxicated rodents and resulted in a dose-dependent manner (0.001-0.004 mg/kg) capable for a significant Tyrosine Hydroxylase (TH) positive neuronal density rescue in both striatum and substantia nigra of parkinsonian mice. In parallel, inPentasomes significantly decreased the extent of glial-related neuroinflammation through the reduction of specific gliotic markers (Iba-1, GFAP, COX-2, iNOS) with consequent PGE2 and NO2- release reduction, in nigrostriatal system. inPentasomes-mediated S100B inhibition resulted in a RAGE/NF-κB pathway downstream inhibition in the nigrostriatal circuit, causing a marked amelioration of motor performances in intoxicated mice. On the basis of our results, chitosan coated niosomes loaded with pentamidine, the inPentasome system, self-candidates as a promising new intranasal approach to mitigate parkinsonism in humans and possibly paves the way for a possible clinical repositioning of pentamidine as anti-PD drug.

DC(13)PC bilayers from anomalous swelling to main transition: an X-ray scattering investigation.

We have performed small-angle (SAXS) and wide-angle X-ray scattering (WAXS) measurements on the lamellar phase and on large unilamellar vesicles (LUVs) of DC(13)PC in the temperature range corresponding to the anomalous swelling regime of multibilayer systems, adjacent to the chain melting transition, and across the transition. Our SAXS measurements indicate that on cooling from the L(alpha) phase, a uniform progressive swelling of the lamellar system to anomalous distances, starting approximately 2 degrees C above the main transition, is followed by a region of coexistence, covering the width of the transition ( approximately 0.6 degrees C). Across the transition region, a progressively increasing volume fraction of gel phase with a constant P (beta') interlamellar distance coexists with a decreasing amount of nongel phase that keeps on swelling to longer distances. Along both the swelling and the transition regions, anomalies in the specific heat are observed revealing a two-step process. Simultaneous WAXS experiments show a progressive "density" increase along the swelling region, constituting a direct spectroscopic evidence of an "evolving membrane" approaching the transition in a bulk real system. Calorimetric and densitometric measurements on LUVs are also presented, together with WAXS results, that show the existence of a double step main transition in a single component nanosized closed bilayer.

Endotoxin tolerance is associated with reduced secretion of tumor necrosis factor.

Bacterial endotoxin effects are partially mediated by tumor necrosis factor (TNF). It is known that sublethal doses of endotoxin induce transient refractoriness (tolerance) to some of its effects. We studied the role of TNF in endotoxin tolerance in rats. Weight loss, lethality, and TNF production were measured after an initial dose of endotoxin and after subsequent doses. Weight loss reached its peak 72 hours after the initial endotoxin challenge, followed by recovery even under continued administration of endotoxin. While tolerant, rats could survive a dose of endotoxin that was lethal for 100% of naive rats. The high serum levels of TNF, observed 90 minutes after the first dose of endotoxin, markedly diminished when rechallenged during tolerance. Recovery of responsiveness to these effects followed the refractory phase by 3 weeks. We concluded that endotoxin tolerance is associated with a reduced secretion of TNF.

Tumor necrosis factor alone does not explain the lethal effect of lipopolysaccharide.

Lethality and tumor necrosis factor production induced by different types of lipopolysaccharide were studied in naive (non-primed) rats during the late phase of endotoxin tolerance. The correlation with antilipopolysaccharide antibodies was also analyzed. No correlation was found between tumor necrosis factor levels and lipopolysaccharide-induced mortality in naive animals. Low-toxicity lipopolysaccharide preparations induced levels of tumor necrosis factor similar to those induced with more toxic types of lipopolysaccharide. Late tolerance was associated with progressively lower levels of lipopolysaccharide-induced tumor necrosis factor and increasing titers of antilipopolysaccharide antibodies after repeated injections of homologous lipopolysaccharide. During late endotonxin tolerance, a direct correlation between the lipopolysaccharide dose and peak tumor necrosis factor serum levels was found. We conclude that since tumor necrosis factor serum levels do not correlate with mortality, tumor necrosis factor alone cannot explain the lethal effect of lipopolysaccharide.

Application of scattering techniques in the domain of amphiphiles.

Light and neutron scattering techniques are discussed in connection with experiments on micellar solutions of gangliosides--amphiphilic molecules of biological origin. Micellar properties, like molecular weight, hydrodynamic radius and shape, are obtained with precise scattering measurements in conditions of negligible intermicellar interactions. It has also been verified that the GM1 ganglioside micelle does not change molecular weight and shape on addition of salt. Scattering techniques are also shown to be useful in studying intermicellar interactions, like the Coulomb repulsion between GM1 micelles, in the regime fo the long-range interactions obtainable at very low ionic strength.

Aggregation properties of semisynthetic GD1a ganglioside (IV3Neu5AcII3Neu5AcGgOse4Cer) containing an acetyl group as acyl moiety.

GD1a ganglioside containing an acetyl group as acyl moiety, GD1a(acetyl), was synthesized from natural GD1a. The aggregative properties in aqueous solution of GD1a(acetyl) have been studied by static and dynamic laser light-scattering measurements. GD1a(acetyl) spontaneously aggregates as small micelles showing a hydrodynamic radius and molecular mass of 33 A and 96 kDa, respectively. Vibrio cholerae sialidase showed a very high activity on the micelles of GD1a(acetyl), compared to GD1a. This has been explained as a consequence of the high surface curvature of the the small micelles. High resolution proton NMR spectra were recorded from micelles of GD1a(acetyl) in deuterated water. The low overall correlation time of the GD1a(acetyl) micelles was calculated to be about 2 x 10(-8)s, a value one order of magnitude lower than that determined for natural GD1a.

Physicochemical behavior of the ganglioside GM3 in dilute aqueous solution.

The aggregative properties of the ganglioside GM3 have been studied with small-angle X-ray scattering and dynamic light scattering in dilute aqueous solutions. Dynamic light scattering experiments show that GM3 solutions are very polydisperse containing a large amount of small aggregates (hydrodynamic radius of 7-9 nm) in addition to a quite broad distribution of aggregates with an average hydrodynamic radius of 50-60 nm and a small fraction of very large aggregates (> 200 nm). This together with small-angle X-ray scattering scattering experiments and model calculations suggests the coexistence of a lamellar phase (vesicles or extended lamellae) with small aggregates (modelled as lamellar platelets). The latter can also be viewed as lamellar fragments coming from GM3 vesicles which constantly break and reform.

Evidence for spontaneous segregation phenomena in mixed micelles of gangliosides.

A light scattering study of the effect of mixing in aqueous solution two gangliosides, GM2 and GT1b, having different hydrophilic headgroups and similar lipid moieties is presented. Mixed micelle formation with spatial segregation of one ganglioside with respect to the other was observed. It is also shown that segregation is a spontaneous phenomenon which is explainable only in terms of simple geometrical arguments, that is by the fact that the large headgroup of GT1b provides the lipidic core of the aggregate with a better shielding from water in the highly curved regions than the smaller headgroup of GM2 can do. This finding may be of help in understanding the behaviour of gangliosides in artificial and natural membranes.

Light scattering measurements on gangliosides: dependence of micellar properties on molecular structure and temperature.

Static and dynamic laser light scattering measurements on micellar aqueous solutions of gangliosides GM2, GM1, GD1a are reported. The aggregation number, the hydrodynamic radius and the micellar shape depend on the type of ganglioside and the unsaturation degree of the hydrocarbon chains. At a temperature of 25 degrees C the molecular weights of GM2, GM1 and GD1a are 740,000, 470,000 and 418,000 DA respectively. A significant decrease of micellar size with temperature has been found for saturated GM1 in the region 25 degrees-40 degrees C. The strong sensitivity of the micellar parameters to the ganglioside structure is explained by making reference to some simple model which takes into account geometrical packing considerations. By measuring the scattered light intensity at low ionic strength of the solution (0.1-30 mE) it was possible to evaluate the ganglioside micellar charge, 100 electronic units for GM2, 48 for GM1 and 60 for GD1a.

Aggregation properties of GM3 ganglioside (II3Neu5AcLacCer) in aqueous solutions.

The aggregative properties of highly pure GM3 ganglioside in aqueous solution have been studied by static and dynamic laser light scattering measurements and fluorescence spectroscopy experiments performed by the use of a GM3 derivative containing the pyrene group at the end of the fatty acid moiety. GM3 ganglioside spontaneously aggregates as unilamellar vesicles, down to a concentration of 1.25 x 10(-8) M, showing molecular weight and hydrodynamic radius ranging from 15,000 to 30,000 kDa and from 350 to 470 A, respectively. GM3 vesicles are stable with dilution and can be stored at room temperature for some weeks without appreciable change.

Aggregation properties of GD1b, II3Neu5Ac2GgOse4Cer, and of GD1b-lactone, II3[alpha-Neu5Ac-(2----8, 1----9)-alpha-Neu5Ac]GgOse4Cer, in aqueous solution.

The relevance of the presence of an inner ester in the oligosaccharide chain on the aggregative properties of gangliosides is investigated. Micellar molecular weight and hydrodynamic radius of natural GD1b and of semisynthetic GD1b-lactone are measured by the laser light scattering technique. The presence of the lactone ring causes an increase of 36% for the molecular weight and 16% for the hydrodynamic radius. Measurements on mixtures of GD1b and GD1b-lactone show that mixed micelles are formed with microdomain structure. The results are interpreted in terms of the geometrical packing model for the aggregation of amphiphilic molecules and are correlated to membrane processes.

Aggregative properties of gangliosides in solution.

The aggregative properties of gangliosides in diluted aqueous solutions are discussed on the basis of simple and well-established thermodynamic concepts. Theoretical assumptions are compared with experimental data obtained, mainly by scattering techniques, on GM3, GM2, GM1, GD1a, GalNAc-GD1a, GD1b, GD1b lactone and GT1b gangliosides, all containing ceramide portions of similar composition, and on GM1 molecular species containing different well-defined ceramide structures. We also report on mixed aggregates with amphiphilic compounds and on the ganglioside aggregate-soluble protein interaction effects which give rise to very stable lipoproteic complexes of well-defined ganglioside-protein composition.