Paola Brocca

Conformation of the Oligosaccharide Chain of GM1 Ganglioside in a Carbohydrate-Enriched Surface

The solution structure of ganglioside G(M1) carbohydrate moiety at the surface of a 102-kDa lipid-modified-G(M1) micelle is investigated by high-resolution 1H-NMR in H2O. The micellar surface can be considered a cluster-like lateral distribution of the gangliosides, each single monomer being anchored in a carbohydrate-enriched model membrane matrix. 1H NOESY measurements at short mixing times reveal a rigid trisaccharide core -beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal- and a more flexible beta-Gal-(1-3)-beta-GalNAc- terminal glycosidic bond. In the lipid-modified G(M1) ganglioside micellar system, there is no evidence that intermolecular side-by-side carbohydrate interactions modulate, or alter in any way, the head-group spatial arrangement. Possible intermonomer interactions at the level of the branched trisaccharide portion were further investigated on mixed micelles of natural N-glycolyl- and N-acetylneuraminic acid containing G(M1) in D2O, taking advantage of the different NMR features of N-glycolyl- and N-acetylneuraminic acids, which allow discrimination between sialic acid ring proton signals. Measurements of the water/ganglioside-OH proton chemical exchange rates suggest hydroxyl group involvement at position 8 of sialic acid in strong intramolecular interaction processes.

Structure of self-organized multilayer nanoparticles for drug delivery.

The combined use of cryo-TEM, dynamic light scattering, and small-angle X-ray and neutron scattering techniques allows a detailed structural model of complex pharmaceutical preparations of soybean lecithin/chitosan nanoparticles used as drug vectors to be worked out. Charge-driven self-organization of the lipid(-)/polysaccharide(+) vesicles occurs during rapid injection, under mechanical stirring, of an ethanol solution of soybean lecithin into a chitosan aqueous solution. We conclude that beyond the charge inversion region of the phase diagram, i.e., entering the redissolution region, the initial stages of particle formation are likely to be affected by a re-entrant condensation effect at the nanoscale. This behavior resembles that at the mesoscale which is well-known for polyion/amphiphile systems. Close to the boundary of the charge inversion region, nanoparticle formation occurs under a maximum condensation condition at the nanoscale and the complexation-aggregation process is driven toward a maximum multilamellarity. Interestingly, the formulation that maximizes vesicle multilamellarity corresponds to that displaying the highest drug loading efficiency.

Modeling ganglioside headgroups by conformational analysis and molecular dynamics

The conformations and dynamics of gangliosides GM1, GM2, 6′-GM2 and GM4 have been studied by computational means, and the results compared to NMR data. Unconstrained conformational searches were run using the AMBER* force field augmented by MNDO derived parameters for the Neu5Ac anomeric torsion, the GB/SA water solvation model, and the MC/EM alogorithm; extended (10–12[emsp4 ]ns) dynamic simulations in GB/SA water were performed with the MC/SD protocol, and the stored structures were minimized. The overall mobility of the Neu5Acα2,3Gal linkage and the position of its minimum energy conformation have been shown to depend mainly on the presence or the absence of a GalNAc residue at the adjacent position. The best quantitative agreement with the available NOE data was achieved after minimization of the structures stored during the MC/SD dynamic runs. The latter protocol appears to reproduce satisfactorily the available experimental data, and can be used with confidence to build three-dimensional models of ganglioside headgroups.

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 3β-[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.

Structural Basis for the Resistance of Tay-Sachs Ganglioside GM2 to Enzymatic Degradation

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glcβ1–1′Cer) are refractory to β-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6′GM2 (GalNAcβ1→6(NeuAcα2→3)Galβ1→4Glcβ1–1′Cer). While GM2 has GalNAcβ1→4Gal linkage, 6′-GM2 has GalNAcβ1→6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223–227). We have studied the enzymatic susceptibilities of GM2 and 6′GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcβ1→4Galβ1→ 4Glcβ1–1′Cer) and 6′GM2. In addition, the conformational properties of both GM2 and 6′GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6′GM2 were readily hydrolyzed by β-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6′GM2, only the oligosaccharide from GM2 was resistant to β-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6′GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.

Structural aspects of ganglioside-containing membranes

The demand for understanding the physical role of gangliosides in membranes is pressing, due to the high number of diverse and crucial biological functions in which they are involved, needing a unifying thread. To this purpose, model systems including gangliosides have been subject of extensive structural studies. Although showing different levels of complication, all models share the need for simplicity, in order to allow for physico-chemical clarity, so they keep far from the extreme complexity of the true biological systems. Nonetheless, as widely agreed, they provide a basic hint on the structural contribution specific molecules can pay to the complex aggregate. This topic we address in the present review. Gangliosides are likely to play their physical role through metamorphism, cooperativity and demixing, that is, they tend to segregate and identify regions where they can dictate and modulate the geometry and the topology of the structure, and its mechanical properties. Strong three-dimensional organisation and cooperativity are exploited to scale up the local arrangement hierarchically from the nano- to the mesoscale, influencing the overall morphology of the structure.

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, β-Gal-(1-3)-β-GalNAc-(1-4)-[α-Neu5Gc-(2-3)]-β-Gal-(1-4)-β-Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core-β-GalNAc-(1-4)-[α-Neu5Gc-(2-3)]-β-Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal β-Gal-was found to be quite mobile as the β-Gal-(1-3)-β-GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].

1H-NMR study on ganglioside amide protons: evidence that the deuterium exchange kinetics are affected by the preparation of samples

The kinetics of H/2H chemical exchange of the amide proton has been suggested as one of the tools available for investigating hydrogenbond stabilizing interactions in gangliosides.The amide proton/deuterium (NH/2H) exchange rates in GM2 ganglioside were studied by1H-NMR spectroscopy on 12 samples prepared following different procedures. In samples passed through a sodium salt Chelex-100 cation exchange resin column prior to being analysed theN-acetylneuraminic acid NH exchange occurred in less than 10 min and that of ceramide NH in 30 min. TheN-acetylgalactosamine acetamido NH exchange was slower, the half-life of the signal ranging from 15 min to 3.5 h. Contact of the Chelex-treated GM2 samples with water, through a dialysis process, modified the NH/2H exchange rate values, theN-acetylgalactosamine acetamido NH exchange becoming faster than that of ceramide NH and similar to that ofN-acetylneuraminic acid NH. Our results indicate that the deuterium/proton exchange rate strongly depends on sample preparation (ion content and minor contaminants present in water). The three-dimensional model involving theN-acetylgalactosamine acetamido NH and theN-acetylneuraminic acid carboxyl group hydrogen-bonding, which is supported by experimental evidence, cannot be confirmed by NH-exchange measurement.

Niosomes as Drug Nanovectors: Multiscale pH-Dependent Structural Response

The use of nanocarriers, which respond to different stimuli controlling their physicochemical properties and biological responsivness, shows a growing interest in pharmaceutical science. The stimuli are activated by targeting tissues and biological compartments, e.g., pH modification, temperature, redox condition, enzymatic activity, or can be physically applied, e.g., a magnetic field and ultrasound. pH modification represents the easiest method of passive targeting, which is actually used to accumulate nanocarriers in cells and tissues. The aim of this paper was to physicochemically characterize pH-sensitive niosomes using different experimental conditions and demonstrate the effect of surfactant composition on the supramolecular structure of niosomes. In this attempt, niosomes, made from commercial (Tween21) and synthetic surfactants (Tween20 derivatives), were physicochemically characterized by using different techniques, e.g., transmission electron microscopy, Raman spectroscopy, and small-angle X-ray scattering. The changes of niosome structure at different pHs depend on surfactants, which can affect the supramolecular structure of colloidal nanocarriers and their potential use both in vitro and in vivo. At pH 7.4, the shape and structure of niosomes have been maintained; however, niosomes show some differences in terms of bilayer thicknesses, water penetration, membrane coupling, and cholesterol dispersion. The acid pH (5.5) can increase the bilayer fluidity, and affect the cholesterol depletion. In fact, Tween21 niosomes form large vesicles with lower curvature radius at acid pH; while Tween20-derivative niosomes increase the intrachain mobility within a more interchain correlated membrane. These results demonstrate that the use of multiple physicochemical procedures provides more information about supramolecular structures of niosomes and improves the opportunity to deeply investigate the effect of stimuli responsiveness on the niosome structure.

Nanoscale structural response of ganglioside-containing aggregates to the interaction with sialidase.

J. Neurochem. (2011) 116, 833–839.