Tommaso Del Rosso

Lipid rafts: integrated platforms for vascular organization offering therapeutic opportunities

Research on the nanoscale membrane structures known as lipid rafts is relevant to the fields of cancer biology, inflammation and ischaemia. Lipid rafts recruit molecules critical to signalling and regulation of the invasion process in malignant cells, the leukocytes that provide immunity in inflammation and the endothelial cells that build blood and lymphatic vessels, as well as the patterning of neural networks. As angiogenesis is a common denominator, regulation of receptors and signalling molecules critical to angiogenesis is central to the design of new approaches aimed at reducing, promoting or normalizing the angiogenic process. The goal of this review is to highlight some of the key issues that indicate the involvement of endothelial cell lipid rafts at each step of so-called ‘sprouting angiogenesis’, from stimulation of the vascular endothelial growth factor to the choice of tip cells, activation of migratory and invasion pathways, recruitment of molecules that guide axons in vascular patterning and maturation of blood vessels. Finally, the review addresses opportunities for future studies to define how these lipid domains (and their constituents) may be manipulated to stimulate the so-called ‘normalization’ of vascular networks within tumors, and be identified as the main target, enabling the development of more efficient chemotherapeutics and cancer immunotherapies.

Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid‐rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar‐lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro‐angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid‐supported mobile bilayer lipid membranes with raft‐like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR‐GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1‐enriched biomimetic membranes, were validated by identifying a pro‐angiogenic activity of GM1‐enriched EPCs, based on GM1‐dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti‐angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar‐raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar‐raft partitioning of uPAR, as opposed to control and GM3‐challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.

Surface-enhanced fluorescence and surface-enhanced Raman scattering of push-pull molecules: sulfur-functionalized 4-amino-7-nitrobenzofurazan adsorbed on Ag and Au nanostructured substrates.

AbstractWe investigated the chemisorption of self-assembled monolayers of sulfur-functionalized 4-amino-7-nitrobenzofurazan on gold and silver nanoisland films (NIFs) by means of surface-enhanced fluorescence (SEF) and surface-enhanced Raman scattering (SERS). The ligand is a push–pull molecule, where an intramolecular charge transfer occurs between an electron-donor and an electron-acceptor group, thus exhibiting nonlinear optical properties that are related to both SERS and SEF effects. The presence of different heteroatoms in the molecule ensures the possibility of chemical interaction with both silver and gold substrates. The SERS spectra suggest that furazan is bound to silver via lone pairs of the nitrogen atoms, whereas the ligand is linked to gold via a sulfur atom. Silver NIFs provide more efficient enhancement of both fluorescence and Raman scattering in comparison with gold NIFs. The present SEF and SERS investigation could provide useful information for foreseeing changes in the nonlinear responses of this push–pull molecule. FigureSurface-enhanced fluorescence on Ag and Au substrates

The Binding of EGFR to GM1(3) Hosted in Lipid Raft-Like Biomembranes Insighted by Plasmonic Resonance Techniques

We exploit Au/SiO2 plasmonic structures to check the effective binding activity of GM1(3) gangliosides hosted in physiological-like biomembranes, in presence of the Epidermal Growth Factor Receptor (EGFR). To this aim, we used bilayers that support the propagation of optical surface plasmon modes (plasmonic transducers, PTs) or guided modes (Plasmon Waveguide Resonators, PWRs). First, we measured the binding of EGFR to GM1(3) by using PTs. Indeed, effective interactions were evidenced, but with faint signals that prevented resolving dissociation kinetics. In order to enhance the optical responses, we turned our attention to PWRs. We first refined the design of a previously adopted Au/SiO2 PWR, finding that the nominal sensitivity is independent on SiO2 thickness but strongly dependent on its residual losses, due typically to a nonoptimal deposition process. We fabricated an improved Au/SiO2 resonator and tested the predicted signal enhancement by monitoring the binding of EGFR to GM3-enriched biomembranes. The measured signal was ~12-fold higher than that one measured using a PT, close to the maximum theoretical enhancement. The higher PWR response enabled us to detect the dissociation of EGFR from GM3, and the value of the apparent dissociation constant of the GM3-EGFR complex could be obtained.

Metal nanoisland films for the enhancement of the chemico-physical properties of molecular adsorbates

This contribution is aimed at describing the most recent activity carried out in the labs of ISC-CNR Florence concerning preparation and spectroscopic investigation of two dimensional silver and gold metal nanostructures (metal nanoisland films - MeNIFs), with particular focus on the role played by the morphology of the metal dielectric interfaces on the control and enhancement of the properties of different molecular adsorbates, i.e. fluorescence emission and Raman response. In particular, it shows how the fabrication procedure of Au nanoisland films permits fine tuning of the extinction spectrum of the nanostructures and controlled enhancement of the Raman response of a novel thiol-functionalized fluorophore.

Noble metal nanoparticles functionalized with novel organic luminophores

Design and synthesis of new organic luminophores with desired structure and photophysical properties for application as fluorescent markers for noble metal nanoparticles is performed. The novel luminophores are 4methoxy- and 4-pyrollidine derivatives of 1,8-naphtalimide bearing at the imide N atom long methylene chain with SH end groups, which enables their covalent binding to noble metals. The steady state and dynamic photophysical characteristics of the lowest lying singlet and triplet electronically excited states of naphthalimides that specify the luminescence properties of organic molecules on the basis of UV-VIS absorption and fluorescence spectroscopy in solution are determined. The 4-methoxy substituted naphtalimide has strong fluorescence in solution in the blue region of the spectrum at 420 – 450 nm, while the 4-pyrollidine-derivative is characterized by intensive green emission around 500 – 540 nm. The corresponding relative fluorescence quantum yields are extremely high - up to 0.9. The measured natural life time of the first singlet electronically state of 4-methoxy- and 4-pyrrolodine-naphthalimide is between 6 and 10 ns. The novel organic luminophores are applied for fluorescence labeling of both synthetically prepared monolayer protected clusters of Au nanoparticles, and of Au and Ag nanoparticles obtained by picosecond laser ablation.

Degenerate parametric down-conversion at 1570 nm by periodically poled lithium niobate waveguide: a route to single photon source and photon entanglement

In this paper we measure the efficiency of photon pair production from parametric downconversion at the photon counting level, generated in a laboratory prototype of PPLN waveguide at 1572 nm. The aim is to enhance the brightness of an heralded single photon source and to improve compactness for quantum communication applications, namely quantum key distribution. We observed a maximum of the conversion efficiency corresponding to 7.9 106 photon pairs/s for a pump power of 28 μW. This value is compared with the measured pairs production in a classical experiment of difference frequency generation, realized with the same sample by analogue classical detection. We highlight the advantages of using the waveguide versus a bulk PPLN for such applications and we foresee further possible improvements by adopting waveguide in a single photon level experimental setup.