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Delivery of large therapeutic payloads to the central nervous system is challenging owing to the presence of the blood brain barrier (BBB) that inhibits uptake of virtually any macromolecule. Targeted nanostructures could overcome this issue, allowing delivery of otherwise impermeant payloads without suffering serum protein adsorption during circulation in the bloodstream. Here we present our results on the realization of a rationally designed targeted nanostructure tailored to in vivo use. Our strategy relies on the development of a stealth shell that inhibits serum protein adsorption without using immunogenic PEG derivatives. A zwitterionic peptide (EKEKEKE) with known stealth properties was conjugated with lipid tails and used to prepare liposome-like self-assembled structures that outperform PEG in terms of serum protein adhesion, being stable up to 8 hours in pure serum.1 These structures were decorated with a targeting sequence that exploits transferrin receptor pathway. Transferrin (Tfn) is a promising target for delivery to the central nervous system (CNS) due to the overexpression of its receptor on the BBB. We designed and validated experimentally a new peptide aptamer (Tf2) able to recognize with good affinity transferrin without altering binding properties of the latter to its receptor.2 We shall show that the presence of Tf2 on the surface of a nanoparticle induces extensive modulation of the protein corona composition, with significant overrepresentation of Tfn. We will also show that Tf2- decorated nanoparticles are efficiently assimilated by the cells in a transferrin-dependent fashion.2 Finally, we shall present the applicability of our structure to enzyme replacement therapy for the treatment of Batten disease. Our nanostructures can be loaded with recombinant protein palmitoyl thiotransferase 1 fully retaining its activity. We shall discuss the effect of this administration on enzymatic activity recovery and on the phenotypic rescue in primary healthy and patient-derived fibroblasts.

Composite-fermion metals occur in the quantum Hall bilayers at total Landau level filling fraction v(T) = 1 as the tunneling gap Delta(SAS) collapses by application of in-plane magnetic fields. Experimental evidence is obtained from the observation of a spin continuum below the Zeeman energy by resonant inelastic light scattering. These low-lying spin modes are assigned to quasi-particle excitations, where spin and composite-fermion Landau level index change simultaneously. (C) 2007 Elsevier B.V. All rights reserved.

We observe resonant Rayleigh scattering of light from quantum Hall bilayers at Landau level filling factor nu=1. The effect arises below 1 Kelvin when electrons are in the incompressible quantum Hall phase with strong interlayer correlations. Marked changes in the Rayleigh scattering signal in response to application of an in-plane magnetic field indicate that the unexpected temperature dependence is linked to formation of a nonuniform electron fluid close to the phase transition towards the compressible state. These results demonstrate a new realm of study in which resonant Rayleigh scattering methods probe quantum phases of electrons in semiconductor heterostructures.

We report inelastic light scattering measurements of spin excitations on coupled electron bilayers with relatively large tunneling gaps at total filling factor nu(T) = 1. We show that the pseudospin polarization order parameter, where the pseudospin labels the occupation of symmetric and antisymmetric levels, can be determined from the energy of long wavelength spin excitations. Our experiments indicate that the order parameter in the quantum Hall ground state collapses at the incompressible-compressible phase transition. The latter is driven by decreasing the tunneling gap through the application of an in-plane magnetic field. (c) 2006 Elsevier B.V. All rights reserved.

Resonant Rayleigh scattering of light from electrons confined in gallium arsenide double quantum wells displays significant changes at temperatures that are below one degree Kelvin. The Rayleigh resonance occurs for photon energies that overlap a quantum well exciton and when electron bilayers condense into a quantum-Hall state. Marked changes in Rayleigh scattering intensities that occur in response to application of an in-plane magnetic field indicate that the unexpected temperature dependence is linked to formation of non-uniform electron fluids in a disordered quantum-Hall phase. These results demonstrate a new realm of study in which resonant Rayleigh scattering methods probe quantum phases of cold electrons in semiconductor heterostructures.

Recent findings have proved the benefits of Pioglitazone (PGZ) against atherosclerosis and type 2 diabetes. Since the systematic and controllable release of this drug is of significant importance, encapsulation of this drug in nanoparticles (NPs) can minimize uncontrolled issues. In this context, drug delivery approaches based on several poly(lactic-co-glycolic acid) (PLGA) nanoparticles have been rising in popularity due to their promising capabilities. However, a fully reliable and reproducible synthetic methodology is still lacking. In this work, we present a rational optimization of the most critical formulation parameters for the production of PGZ-loaded PLGA NPs by the single emulsification-solvent evaporation or nanoprecipitation methods. We examined the influence of several variables (e.g., component concentrations, phases ratio, injection flux rate) on the synthesis of the PGZ-NPs. In addition, a comparison of these synthetic methodologies in terms of nanoparticle size, polydispersity index (PDI), zeta potential (ζp), drug loading (DL%), entrapment efficiency (EE%), and stability is offered. According to the higher entrapment efficiency content, enhanced storage time and suitable particle size, the nanoprecipitation approach appears to be the simplest, most rapid and most reliable synthetic pathway for these drug nanocarriers, and we demonstrated a very slow drug release in PBS for the best formulation obtained by this synthesis. Recent findings have proved the benefits of Pioglitazone (PGZ) against atherosclerosis and type 2 diabetes. Since the systematic and controllable release of this drug is of significant importance, encapsulation of this drug in nanoparticles (NPs) can minimize uncontrolled issues. In this context, drug delivery approaches based on several poly(lactic-co-glycolic acid) (PLGA) nanoparticles have been rising in popularity due to their promising capabilities. However, a fully reliable and reproducible synthetic methodology is still lacking. In this work, we present a rational optimization of the most critical formulation parameters for the production of PGZ-loaded PLGA NPs by the single emulsification-solvent evaporation or nanoprecipitation methods. We examined the influence of several variables (e.g., component concentrations, phases ratio, injection flux rate) on the synthesis of the PGZ-NPs. In addition, a comparison of these synthetic methodologies in terms of nanoparticle size, polydispersity index (PDI), zeta potential (ζp), drug loading (DL%), entrapment efficiency (EE%), and stability is offered. According to the higher entrapment efficiency content, enhanced storage time and suitable particle size, the nanoprecipitation approach appears to be the simplest, most rapid and most reliable synthetic pathway for these drug nanocarriers, and we demonstrated a very slow drug release in PBS for the best formulation obtained by this synthesis.

When live imaging is not feasible, sample fixation allows preserving the ultrastructure of biological samples for subsequent microscopy analysis. This process could be performed with various methods, each one affecting differently the biological structure of the sample. While these alterations were well-characterized using traditional microscopy, little information is available about the effects of the fixatives on the spatial molecular orientation of the biological tissue. We tackled this issue by employing Rotating-Polarization Coherent Anti-Stokes Raman Scattering (RP-CARS) microscopy to study the effects of different fixatives on the myelin sub-micrometric molecular order and micrometric morphology. RP-CARS is a novel technique derived from CARS microscopy that allows probing spatial orientation of molecular bonds while maintaining the intrinsic chemical selectivity of CARS microscopy. By characterizing the effects of the fixation procedures, the present work represents a useful guide for the choice of the best fixation technique(s), in particular for polarisation-resolved CARS microscopy. Finally, we show that the combination of paraformaldehyde and glutaraldehyde can be effectively employed as a fixative for RP-CARS microscopy, as long as the effects on the molecular spatial distribution, here characterized, are taken into account., 11 pages, 4 figures. This is the peer reviewed version of an article that has been published in final form by Applied Optics. Please note that the copyright statement on the front page of the article allows posting of this manuscript by authors on arXiv, as specified here: https://www.osapublishing.org/submit/review/copyright_permissions.cfm

We address the contribution of kinase domain structure and catalytic activity to membrane trafficking of TrkA receptor tyrosine kinase. We conduct a systematic comparison between TrkA-wt, an ATP-binding defective mutant (TrkA-K544N) and other mutants displaying separate functional impairments of phosphorylation, ubiquitination, or recruitment of intracellular partners. We find that only K544N mutation endows TrkA with restricted membrane mobility and a substantial increase of cell surface pool already in the absence of ligand stimulation. This mutation is predicted to drive a structural destabilization of the αC helix in the N-lobe by molecular dynamics simulations, and enhances interactions with elements of the actin cytoskeleton. On the other hand, a different TrkA membrane immobilization is selectively observed after NGF stimulation, requires both phosphorylation and ubiquitination to occur, and is most probably related to the signaling abilities displayed by the wt but not mutated receptors. In conclusion, our results allow to distinguish two different TrkA membrane immobilization modes and demonstrate that not all kinase-inactive mutants display identical membrane trafficking. © 2019 Elsevier B.V.

Developing safe and high efficiency contrast tools is an urgent need to allow in vivo applications of photoacoustics (PA), an emerging biomolecular imaging methodology, with poor invasiveness, deep penetration, high spatial resolution and excellent endogenous contrast. Eumelanins hold huge promise as biocompatible, endogenous photoacoustic contrast agents. However, their huge potential is still unexplored due to the difficulty to achieve at the same time poor aggregation in physiologic environment and high PA contrast. This study addresses both issues through the design of a biocompatible photoacoustic nanoprobe, named MelaSil_Ag-NPs, relying on silica-templated eumelanin formation as well as eumelanins redox and metal chelating properties to reduce Ag + ions and control the growth of generated metal nanoparticles. This strategy allowed self-structuring of the system into a core-shell architecture, where the Ag core was found to boost PA signal, despite the poor eumelanin content. Obtained hybrid nanoplatforms, showed stable photoacoustic properties even under long irradiation. Furthermore, conjugation with rhodamine isothiocyanate allowed particles detection through fluorescent imaging proving their multifunctional potentialities. In addition, they were stable towards aggregation and efficiently endocytosed by human pancreatic cancer cells (BxPC3 and Panc-1)displaying no significant cytotoxicity. Such numerous features prove huge potential of those nanoparticles as a multifunctional platform for biomedical applications.

We currently lack a satisfactory understanding of the membrane complexes and internalization routes underpinning the pleiotropic biological outcomes of neurotrophins (NTs), which exert their functions via interlaced binding of three different families of neurotrophin receptors (NRs). We are working to answer several open questions in this field: are NRs membrane movements linked to ligand-specific activation processes? Are different NRs functions linked to different movements at the cell membrane? How does p75NTR enhance NGF-TrkA signalling? Are NGF and its precursor proNGF different signalling molecules as far as NRs binding and internalization is concerned? To address these issues, we developed non-invasive means to covalently fluorolabel with 1:1 stoichiometry both NTs and their receptors. This toolbox was exploited to perform single molecule imaging and tracking (SMIT) at the plasma membrane and inside axons of living neuronal cells using wide-field and TIRF microscopy. We report here results in two different directions. First, we analysed by SMIT the lateral mobility of wt TrkA in comparison to a dead-kinase TrkA and to three other mutants having i) kinase activity, ii) recruitment of intracellular effectors, iii) ubiquitination (and further degradation) separately impaired. Obtained data point to kinase activity as a master regulator of TrkA membrane dynamics and hint at possible mechanisms by which the cell handles the trafficking of kinase-inactive TrkA receptors. Second, we undertook a comparative study about the axonal transport displayed by “homologue” fluorescent proNGF and NGF in compartmented DRG neurons. We demonstrate that proNGF is internalized and retrogradely transported across axons like mature NGF, but the two NTs display remarkable differences both in terms of NTs flux and number of molecules carried per vesicle. Furthermore, we unveiled a competition mechanism favoring NGF transport upon coadministration of the two NTs.

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community. Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community. © 2008 Springer-Verlag.