Simona Motta

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.

Different mutations at V363 MAPT codon are associated with atypical clinical phenotypes and show unusual structural and functional features.

Microtubule-associated protein tau gene (MAPT) is one of the major genes linked to frontotemporal lobar degeneration, a group of neurodegenerative diseases clinically, pathologically, and genetically heterogeneous. In particular, MAPT mutations give rise to the subgroup of tauopathies. The pathogenetic mechanisms underlying the MAPT mutations so far described are the decreased ability of tau protein to promote microtubule polymerization (missense mutations) or the altered ratio of tau isoforms (splicing mutations), both leading to accumulation of hyperphosphorylated filamentous tau protein. Following a genetic screening of patients affected by frontotemporal lobar degeneration, we identified 2 MAPT mutations, V363I and V363A, leading to atypical clinical phenotypes, such as posterior cortical atrophy. We investigated in vitro features of the recombinant mutated tau isoforms and revealed unusual functional and structural characteristics such as an increased ability to promote microtubule polymerization and a tendency to form oligomeric instead of filamentous aggregates. Thus, we disclosed a greater than expected complexity of abnormal features of mutated tau isoforms. Overall our findings suggest a high probability that these mutations are pathogenic.

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

J. Neurochem. (2011) 116, 833–839.

Amyloidβ Peptides in interaction with raft-mime model membranes: a neutron reflectivity insight

The role of first-stage β–amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between β–amyloid (Aβ) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of Aβ(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aβ(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aβ(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aβ(1-6) fragment, the N-terminal portion of Aβ, was also used. Aβ N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment.

Nanoscale structure of protamine/DNA complexes for gene delivery

Understanding the internal packing of gene carriers is a key-factor to realize both gene protection during transport and de-complexation at the delivery site. Here, we investigate the structure of complexes formed by DNA fragments and protamine, applied in gene delivery. We found that complexes are charge- and size-tunable aggregates, depending on the protamine/DNA ratio, hundred nanometers in size. Their compactness and fractal structure depend on the length of the DNA fragments. Accordingly, on the local scale, the sites of protamine/DNA complexation assume different morphologies, seemingly displaying clumping ability for the DNA network only for shorter DNA fragments.

Chitosan-coupled solid lipid nanoparticles: Tuning nanostructure and mucoadhesion.

&NA; Solid Lipid Nanoparticles (SLNs) composed of biodegradable physiological lipids have been widely proposed as efficient drug delivery systems, also for ophthalmic administration. Recently, chitosan‐associated‐SLNs have been developed to further improve the residence time of these colloidal systems in the precorneal area by means of mucoadhesive interaction. In the present study, a one‐step preparation protocol was used aiming both at scale‐up ease and at stronger coupling between chitosan and SLNs. The resulting particles were chitosan associated‐SLNs (CS‐SLNs). These nanoparticles were characterized, as compared to both the chitosan‐free and the usual chitosan‐coated ones, by applying a multi‐technique approach: light, neutron and X‐ray scattering, Zeta‐potential, AFM, calorimetry. It was assessed that, while keeping the features of nano‐size and surface‐charge required for an efficient vector, these new nanoparticles display a strong and intimate interaction between chitosan and SLNs, far more settled than the usual simple coverage. Moreover, this one‐step preparation method allows to obtain a strong and intimate interaction between chitosan and SLNs, firmer than the usual simple coating. This confers to the CS‐SLNs an improved mucoadhesion, opening the way for a high‐performing ophthalmic formulation. Graphical abstract Figure. No caption available.

Transient step-like kinetics of enzyme reaction on fragmented-condensed substrates.

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.

Optimizing the crowding strategy: sugar-based ionic micelles in the dilute-to-condensed regime.

In the present study, we explore the effect of concentration on micelles made by different gangliosides, which are ionic biological glycolipids bearing multisugar headgroups with huge steric hindrance. Moreover, strong preferential interactions exist among like-conformer headgroups that can keep the ganglioside micelles in a trapped configuration. We extend the well-known ionic-amphiphiles paradigm, where local condensation and micelle crowding are matched by forming larger aggregates at increasing concentration. In fact, we force the balance between interparticle and intraparticle interactions while allowing for like conformers to modulate rebalancing. In the vast experimental framework, obtained by Small Angle X-ray scattering (SAXS) experiments, a theoretical model, accounting for a collective conformational transition of the bulky headgroups, is developed and successfully tested. It allows us to shed some light on the nature and coupling of the intermolecular forces involved in the interactions among glycolipid micelles. Energy minimization leads to complex behavior of the aggregation number on increasing concentration, fully consistent with the experimental landscape. From a biological perspective, this result could be reflected in the properties of ganglioside-enriched rafts on cell membranes, with a nonlinear structural response to approaching bodies such as charged proteins.

What the cell surface does not see: The gene vector under the protein corona

The fate of lipid-based nanovectors, used in genetic targeting inside cells, depends on their behavior in biological media. In fact, during both in vitro and in vivo transfection, nanovectors come in contact with proteins that compete for their surface and build the protein corona, their true biological identity while engaging the cell membrane. Nonetheless, after cell internalization, the efficacy of transfection may depend also on structural modifications that occurred under the protein cover, following interaction with biological fluids. Here, based on previous in vivo experiments, two widely used lipid mixtures, namely DOTAP/DOPC and DC-Chol/DOPE, were identified as paradigms to investigate the impact of the inner structure of nanovectors on the transfection efficiency, all being proficiently internalized. The evolution of the inner structure of cationic lipoplexes and nanoparticles based on such lipid mixtures, following interaction with human plasma, could be unraveled. Particles were investigated in high dilution, approaching the biosimilar conditions. Data have demonstrated that the modulation of their inner structure depends on their lipid composition and the plasma concentration, still preserving the genetic payload. Interestingly, protein contact induces a variety of inner structures with different perviousness, including reshaping into cubic phases of different porosity, sometimes observed upon interaction between carrier-lipids and cell-lipids. Cubic reshaping is of biological relevance, as lipid cubic phases have been recently associated to both fusogenicity and to the readiness in releasing the payload to the final target via endosomal escape.

Hierarchical Ordering of Sugar Based Amphiphiles

Colloidal aggregates of amphiphilic molecules provide an example of self-confined liquid crystal, being finite both in extension, nanometric scale, and in dimension, nearly-two dimensional. In fact, although disperse on the macroscopic scale, amphiphiles demix in dilute aqueous solution on the microscopic scale due to their bifunctional nature and form condensed assemblies with a complex internal structure, dictated by the compromise between hydrophobic and hydrophilic requirements. On raising the concentration, these amphiphile-aggregate containing solutions start to develop organised structures on the mesoscale, evidencing a long-range crystalline order, but still preserving the ability to rearrange on the nanoscale giving rise to rich phase diagrams. We present here some peculiar and unexpected effects observed for aggregates of gangliosides, glycosidic amphiphiles with both an extended hydrophobic portion, a double-tailed ceramide, and a bulky saccharidic headgroup, displaying large conformational adaptability and showing relevant preferential interactions. While giving rise to a highly cooperative structural unit, with a strong conformational coupling between surface and core of the colloid, ganglioside aggregates respond to crowding in a counterintuitive fashion, exploiting their structural metamorphism.