Laura D'Alfonso

Competitive binding of fatty acids and the fluorescent probe 1‐8‐anilinonaphthalene sulfonate to bovine β‐lactoglobulin

The use of spectroscopy in the study of fatty acids binding to bovine β‐lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty‐acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS–BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid‐binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

Luminescent conjugates between dinuclear rhenium(I) complexes and peptide nucleic acids (PNA) for cell imaging and DNA targeting

New luminescent dinuclear rhenium(I) tricarbonyl complex-PNA conjugates have been synthesized through a reliable solid-phase synthetic methodology. Their photophysical properties have been measured. The most luminescent Re-PNA conjugate 7 showed interesting two-photon absorption (TPA) properties, that were exploited for imaging experiments, to demonstrate its easy uptake into living cells.

Synthesis of branched Au nanoparticles with tunable near-infrared LSPR using a zwitterionic surfactant

Asymmetric branched gold nanoparticles are obtained using for the first time in the seed-growth approach a zwitterionic surfactant, laurylsulfobetaine, whose concentration in the growth solution allows to control both the length to base-width ratio of the branches and the LSPR position, that can be tuned in the 700-1100 nm near infrared range.

A new class of luminescent tricarbonyl rhenium(I) complexes containing bridging 1,2-diazine ligands: electrochemical, photophysical, and computational characterization.

A novel class of luminescent tricarbonyl rhenium(I) complexes of general formula [Re2(mu-X)2(CO)6(mu-diaz)] (X=halogen and diaz=1,2-diazine) was prepared by reacting [ReX(CO)5] with 0.5 equiv of diazine (seven different ligands were used). The bridging coordination of the diazine in these dinuclear complexes was confirmed by single-crystal X-ray analysis. Cyclic voltammetry in acetonitrile showed for all the complexes (but the phthalazine derivative) a chemically and electrochemically reversible ligand-centered reduction, as well as a reversible metal-centered bielectronic oxidation. With respect to the prototypical luminescent [ReCl(CO)3(bpy)] complex, the oxidation is more difficult and the reduction easier (about +0.3 V), so that a similar highest occupied molecular orbital-lowest unoccupied molecular orbital gap is observed. All of the complexes exhibit photoluminescence at room temperature in solution, with broad unstructured emission from metal-to-ligand charge-transfer states, at lambda in the range 579-620 nm. Lifetimes (tau=20-2200 ns) and quantum yields (Phi up to 0.12) dramatically change upon varying the bridging ligand X and the diazine substituents: in particular, quantum yields decrease in the series Cl, Br, and I and in the presence of substituents at the alpha positions of the pyridazine ring. A combined density functional and time-dependent density functional study of the geometry, relative stability, electronic structure, and photophysical properties of all the pyridazine derivatives was performed. The nature of the excited states involved in the electronic absorption spectra was ascertained, and trends in the energy of the highest occupied and lowest unoccupied molecular orbitals upon changing the pyridazine substituents and the bridging halogen ligands were discussed. The observed emission properties of these complexes were shown to be related to a combination of steric and electronic factors affecting their ground-state geometry and their stability.

A Luminescent Poly(amidoamine)-Iridium Complex as a New Singlet-Oxygen Sensitizer for Photodynamic Therapy

A polymer complex (1P) was synthesized by binding bis(cyclometalated) Ir(ppy)2(+) fragments (ppy = 2-phenylpyridyl) to phenanthroline (phen) pendants of a poly(amidoamine) copolymer (PhenISA, in which the phen pendants involved ∼6% of the repeating units). The corresponding molecular complex [Ir(ppy)2(bap)](+) (1M, bap = 4-(butyl-4-amino)-1,10-phenanthroline) was also prepared for comparison. In water solution 1P gives nanoaggregates with a hydrodynamic diameter of 30 nm in which the lipophilic metal centers are presumed to be segregated within polymer tasks to reduce their interaction with water. Such confinement, combined with the dilution of triplet emitters along the polymer chains, led to 1P having a photoluminescence quantum yield greater than that of 1M (0.061 vs 0.034, respectively, in an aerated water solution) with a longer lifetime of the (3)MLCT excited states and a blue-shifted emission (595 nm vs 604 nm, respectively). NMR data supported segregation of the metal centers. Photoreaction of O2 with 1,5-dihydroxynaphthalene showed that 1P is able to sensitize (1)O2 generation but with half the quantum yield of 1M. Cellular uptake experiments showed that both 1M and 1P are efficient cell staining agents endowed with two-photon excitation (TPE) imaging capability. TPE microscopy at 840 nm indicated that both complexes penetrate the cellular membrane of HeLa cells, localizing in the perinuclear region. Cellular photodynamic therapy tests showed that both 1M and 1P are able to induce cell apoptosis upon exposure to Xe lamp irradiation. The fraction of apoptotic cells for 1M was higher than that for 1P (74 and 38%, respectively) 6 h after being irradiated for 5 min, but cells incubated with 1P showed much lower levels of necrosis as well as lower toxicity in the absence of irradiation. More generally, the results indicate that cell damage induced by 1M was avoided by binding the iridium sensitizers to the poly(amidoamine).

Micelles as Containers for Self‐Assembled Nanodevices: A Fluorescent Sensor for Lipophilicity

Potentiometric titrations, fluorescence versus pH titrations, dynamic light scattering and fluorescence polarization anisotropy studies demonstrate that inside the nanodimensioned Triton X-100 micelles, 1-pyrenecarboxylic acid, PCOO(-), forms an apical complex with the Zn(2+) cation encircled by a lipophilic cyclen ligand and hugely increasing its fluorescence. The ability of the Zn(2+)-cyclen-PCOO(-) complex plus its micellar container to act as a fluorescent sensor to evaluate the lipophilicity of molecular species is demonstrated on the fatty acid series CH(3)(CH(2))(x)COOH (x=0-16). At pH 7.4 a decrease in fluorescence is observed on the addition of fatty acids that is directly related to their chain length, that is, to their tendency to enter the micellar containers, where they dislocate PCOO(-) from the Zn(2+) centre. The independent determination of fatty acid pK(a) values in the presence of Triton X-100 micelles confirms that our fluorescent micellar device is capable of sensing their lipophilicity.

Porcine beta‐lactoglobulin chemical unfolding: Identification of a non‐native α‐helical intermediate

The chemical unfolding behavior of porcine beta‐lactoglobulin (PLG) has been followed at pH 2 and 6 in the presence of guanidinium hydrochloride. The PLG unfolding transition, monitored by tryptophan fluorescence, far and near UV circular dichroism and 1D‐NMR, can be described by a three‐state transition suggesting the presence of at least one intermediate state that appears to display an excess of non‐native α‐helical structures. The thermodynamic parameters, as determined through a global analysis fitting procedure, give estimates of the free energy differences of the transitions connecting the native, the intermediate and the unfolded state: ΔG  NI0 = 2.8 ± 0.7 kcal mol−1 (pH 2) and 4.2 ± 0.5 kcal mol−1 (pH 6) and ΔG  NU0 = 7.2 ± 0.6 kcal mol−1 (pH 2) and 6.9 ± 0.6 kcal mol−1 (pH 6). CD unfolding data of the bovine species (BLG) have been collected here under the same experimental conditions of PLG to allow a careful comparison of the two beta‐lactoglobulins. Intermediates with different characteristics have been identified for BLG and PLG, and their nature has been discussed on a structural analysis basis. The thermodynamic data reported here for PLG and BLG and the comparative analysis with data reported for equine beta lactoglobulin, show that homologous beta‐barrel proteins, belonging to the same family and displaying high sequence identity (52–64%) populate unfolding intermediates to different extents, even though a common tendency to the formation of non‐native alpha‐helical intermediates, can be envisaged. The present results provide a prerequisite foundation of knowledge for the design and interpretation of future folding kinetic studies. Proteins 2005.

In Vivo Flow Mapping in Complex Vessel Networks by Single Image Correlation

We describe a novel method (FLICS, FLow Image Correlation Spectroscopy) to extract flow speeds in complex vessel networks from a single raster-scanned optical xy-image, acquired in vivo by confocal or two-photon excitation microscopy. Fluorescent flowing objects produce diagonal lines in the raster-scanned image superimposed to static morphological details. The flow velocity is obtained by computing the Cross Correlation Function (CCF) of the intensity fluctuations detected in pairs of columns of the image. The analytical expression of the CCF has been derived by applying scanning fluorescence correlation concepts to drifting optically resolved objects and the theoretical framework has been validated in systems of increasing complexity. The power of the technique is revealed by its application to the intricate murine hepatic microcirculatory system where blood flow speed has been mapped simultaneously in several capillaries from a single xy-image and followed in time at high spatial and temporal resolution.

Gold nanostars coated with neutral and charged polyethylene glycols: A comparative study of in-vitro biocompatibility and of their interaction with SH-SY5Y neuroblastoma cells.

Gold nanostars (GNS) have been coated with four different polyethylene glycols (PEGs) equipped with a -SH function for grafting on the gold surface. These PEGs have different chain lengths with average MW=2000, 3000, 5000 and average number of -O-CH2-CH2 - units 44, 66, and 111, respectively. Two are neutral and two are terminated with -COOH and -NH2 functions, thus bearing negative and positive charges at physiological pH, thanks to the formation of carboxylate and ammonium groups. The negative charge of the GNS coated with PEG carboxylate has also been exploited to further coat the GNS with the PAH (polyallylamine hydrochloride) cationic polymer. Vitality tests have been carried out on SH-SY5Y cells treated with the five differently coated GNS for 4, 24, and 48 h, at Au concentrations ranging from 1.25 to 100 μg/mL. The same tests have been repeated with the pure PEGs and PAH. Excellent biocompatibility was found for all PEGs, independently on charge and chain length, both for coated GNS and for the pure polymers. On the contrary, poor biocompatibility was found for PAH overcoated GNS and for pure PAH, although the latter only at high concentrations. Exploiting the two-photon luminescence of GNS, we have found by confocal laser scanning microscopy that when GNS are coated with PEGs they do not enter SH-SY5Y cells, while when overcoated with PAH they massively penetrate into the cytoplasm. This causes cell death by dramatically changing cell morphology, as demonstrated also by atomic force microscopy.

A biophysical model of intracellular distribution and perinuclear accumulation of particulate matter

We have measured in human alveolar cells the cytoplasmic distribution of the fluorophore coumarin-6 carried by Solid Lipid Nanoparticles (SLNs) and observed a perinuclear accumulation of the fluorescence that can be described by a single exponential growth along an ideal line joining the plasma membrane to the nuclear border and by a sigmoidal relationship as a function of time. Intracellular distribution was affected by lowering the temperature from 37 to 4° C and by the disruption of cytoskeleton by cytochalasin D, but it was minimally perturbed by the inhibition of ATP dependent molecular motors. A biophysical model was developed for an accumulation of loaded particles against a diffusion gradient based on a mean field interaction energy, whose origin we ascribe to the actin structure of the cytoskeleton. The estimated value for the load diffusion coefficient was four and two orders of magnitude less than that of free coumarin-6 and of SLNs in aqueous solutions, respectively, suggesting that the load moves within the cell cytoplasm in a form still reminiscent of the nanocarrier structure.