Luca Pasotti

Antibacterial Activity of Glutathione-Coated Silver Nanoparticles against Gram Positive and Gram Negative Bacteria

In the present paper, we study the mechanism of antibacterial activity of glutathione (GSH) coated silver nanoparticles (Ag NPs) on model Gram negative and Gram positive bacterial strains. Interference in bacterial cell replication is observed for both cellular strains when exposed to GSH stabilized colloidal silver in solution, and microbicidal activity was studied when GSH coated Ag NPs are (i) dispersed in colloidal suspensions or (ii) grafted on thiol-functionalized glass surfaces. The obtained results confirm that the effect of dispersed GSH capped Ag NPs (GSH Ag NPs) on Escherichia coli is more intense because it can be associated with the penetration of the colloid into the cytoplasm, with the subsequent local interaction of silver with cell components causing damages to the cells. Conversely, for Staphylococcus aureus, since the thick peptidoglycan layer of the cell wall prevents the penetration of the NPs inside the cytoplasm, the antimicrobial effect is limited and seems related to the interaction with the bacterial surfaces. Experiments on GSH Ag NPs grafted on glass allowed us to elucidate more precisely the antibacterial mechanism, showing that the action is reduced because of GSH coating and the limitation of the translational freedom of NPs.

Synthesis, Characterization and Antibacterial Activity against Gram Positive and Gram Negative Bacteria of Biomimetically Coated Silver Nanoparticles

In the present work, we describe a simple procedure to produce biomimetically coated silver nanoparticles (Ag NPs), based on the postfunctionalization and purification of colloidal silver stabilized by citrate. Two biological capping agents have been used (cysteine Cys and glutathione GSH). The composition of the capped colloids has been ascertained by different techniques and antibacterial tests on GSH-capped Ag NPs have been conducted under physiological conditions, obtaining values of Minimum Inhibitory Concentration (MIC) of 180 and 15 μg/mL for Staphylococcus aureus and Escherichia coli, respectively. The antibacterial activity of these GSH capped NPs can be ascribed to the direct action of metallic silver NPs, rather than to the bulk release of Ag(+).

Water soluble extended naphthalene diimides as pH fluorescent sensors and G-quadruplex ligands

Extended naphthalene diimides (NDIs) fused to 1,4-dihydropyrazine-2,3-dione, containing two solubilizing moieties, have been synthesized. Fluorescence spectra of the new NDIs were remarkably affected by pH, as the second deprotonation of the dihydropyrazinedione moiety (pK(a) 6.9) switched off the emission. Binding to a G-quadruplex folded oligonucleotide and stoichiometry were evaluated by FRET melting assay and CD analysis. G-quadruplex binding was strongly enhanced shifting from pH 7.4 to pH 6.0 as a consequence of the dihydropyrazinedione moiety protonation. Cytotoxicity studies using two human telomerase-positive cell lines (HT29 and A549) revealed that the best G-quadruplex ligand was very active against the colon cell line, with an EC(50) of 300 nM.

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.

Protective Effects of Novel Metal-nonoates on the Cellular Components of the Vascular System

At the cardiovascular level, nitric oxide (NO) controls smooth muscle functions, maintains vascular integrity, and exerts an antihypertensive effect. Metal-nonoates are a recently discovered class of NO donors, with NO release modulated through the complexation of the N-aminoethylpiperazine N-diazeniumdiolate ligand to metal ions, and thus representing a significant innovation with respect to the drugs traditionally used. In this study, we characterized the vascular protective effects of the most effective compound of this class, Ni(PipNONO)Cl, compared with the commercial N-diazeniumdiolate group derivate, diethylenetriamine/nitric oxide (DETA/NO). Ni(PipNONO)Cl induced a concentration-dependent relaxation of precontracted rat aortic rings. The ED50 was 0.67 µM, compared with 4.3 µM obtained with DETA/NO. When tested on cultured microvascular endothelial cells, Ni(PipNONO)Cl exerted a protective effect on the endothelium, promoting cell proliferation and survival in the picomolar range. The administration of Ni(PipNONO)Cl to vascular smooth muscle cells reduced the cell number, promoting their apoptosis at a high concentration (10 µM). Inhibition of smooth muscle cell migration, a hallmark of atherosclerosis, was accompanied by cytoskeletal rearrangement and loss of lamellipodia. When added to isolated platelets, Ni(PipNONO)Cl significantly reduced ADP-induced aggregation. Since atherosclerosis is accompanied by an inflammatory environment, cultured endothelial cells were exposed to interleukin (IL)-1β. In the presence of IL-1β, Ni(PipNONO)Cl inhibited cyclooxygenase-2 and inducible nitric oxide synthase upregulation, and reduced endothelial permeability and the platelet and monocyte adhesion markers CD31 and CD40 at the plasma membrane. Overall, these data indicate that Ni(PipNONO)Cl exerts vascular protective effects relevant for vascular dysfunction and prevention of atherosclerosis and thrombosis.

Optical Method for Predicting the Composition of Self-Assembled Monolayers of Mixed Thiols on Surfaces Coated with Silver Nanoparticles

With a simple optical method, based on UV-vis absorption spectra on glass slides, it is possible to predict the composition of self-assembled monolayers of mixed thiols, grafted on monolayers of silver nanoparticles. Glass slides are modified with the layer-by-layer technique, first forming a monolayer of mercaptopropyltrimethoxysilane, then grafting a monolayer of silver nanoparticles on it. These surfaces are further coated by single or mixed thiol monolayers, by dipping the slides in toluene solutions of the chosen thiols. Exchange constants are calculated for the competitive deposition between the colorless 1-dodecanethiol or PEG5000 thiol and BDP-SH, with the latter being a thiol-bearing molecule containing the strongly absorbing BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) moiety, synthesized on purpose. The constants are calculated by determining the fraction of BDP-SH deposited on the surface from a solution with a given molar fraction, directly measuring the absorption spectra of BDP-SH on the slides. Then, the exchange constant for the competitive deposition between 1-dodecanethiol and PEG5000 thiol is calculated by combining their exchange constants with BDP-SH. This allows to predict the fraction of the two colorless thiols coating the silver nanoparticles slides obtained from a toluene solution with a given molar fraction, for example, of PEG5000 thiol. The correctness of the calculated surface fraction is verified by studying the coating competition between 1-dodecanethiol and a PEG5000 thiol remotely modified with a strongly absorbing fluorescein fragment.

The Cu(II) complex of a C-lipophilized 13aneN4 macrocycle with an additional protonable amino group as micellar anion receptor

Three 13aneN4 macrocyclic ligands have been prepared bearing a -CH(2)NHR side arm (R = H, n-C(5)H(11), n-C(10)H(21)) on a carbon atom. When Cu(2+) is complexed in the macrocyclic ring, the amino group of the side arm undergoes an acid-base protonation equilibrium but it is not able to coordinate apically the metal cation even when it is deprotonated. The Cu(2+) complex with the ligand bearing the longest appended aliphatic chain is fully confined inside Triton X-100 micelles, and its ability to bind and sequestrate a series of anions inside micelles has been studied at two different pH values, i.e. both with protonated and neutral side-arm amino group. The favourable role played by the protonated amino group in the side arm has been demonstrated.

A Fluorescent Molecular Sensor for pH Windows in Traditional and Polymeric Biocompatible Micelles: Comicellization of Anionic Species To Shift and Reshape the ON Window

A new approach is presented to obtain fluorescent sensors for pH windows that work in water and under biomimetic conditions. A single molecule that features all-covalently linked components is used, thus making it capable of working as a fluorescent sensor with an OFF/ON/OFF response to pH value. The components are a tertiary amine, a pyridine, and a fluorophore (pyrene). The forms with both protonated bases or both neutral bases quench the pyrene fluorescence, whereas the form with the neutral pyridine and protonated amine groups is fluorescent. The molecular sensor is also equipped with a long alkyl chain to make it highly hydrophobic in all its protonated and unprotonated forms, that is, either when neutral or charged. Accordingly, it can be confined at any pH value either in traditional (i.e., low-molecular-weight) nonionic surfactant micelles or inside polymeric, biocompatible micellar containers. Relevant for future applications in vivo, thanks to its strong hydrophobicity, no leakage of the molecular sensor is observed from the polymeric biocompatible micelles. Due to the proximity of the pyridine and amine functions in the molecular structure and the poor hydration inside the micelles, the observed pK(a) values are low so that the ON window is positioned at very low pH values. However, the window can be shifted to biologically relevant values by comicellization of anionic species. In particular, in the micelles of the nonionic surfactant TritonX-100, a shift of the ON window to pH 4-6 is obtained by addition of the anionic sodium dodecyl sulphate surfactant, whose negative charge promotes the stability of the protonated forms of the pyridine and amine fragments. In the case of the polymeric micelles, we introduce the use of the amphiphilic polystyrene sulfonate anionic polyelectrolyte, the comicellization of which induces a shift and sharpening of the ON window that is centered at pH 4.

Residual and exploitable fluorescence in micellar self-assembled ON–OFF sensors for copper(II)

A set of new ligands, L2H(2)-L5H(2), containing the 1,4,8,11-tetraaza-5,7-dione framework has been prepared, The ligands feature lipophilic substituents either on the carbon atom in the 6 position or on the amino groups, or on both. The solution behaviour of the ligands when included in TritonX-100 micelles has been investigated by means of potentiometric titrations and protonation and complexation constants for the Cu(2+) cation have been determined in micellar medium. Micellar assemblies containing the ligands and pyrene have been prepared, and coupled pH-metric and fluorimetric titrations allowed the determination of the response of the systems as ON-OFF fluorescent sensors for Cu(2+). A correlation between the effective lipophilicity of the ligand and the residual fluorescence (i.e. the fluorescence of the OFF state) was observed, and with the more lipophilic ligand, L3H(2), we obtained a residual fluorescence as low as 8%, with a significant improvement with respect to other published systems. On the other hand, introduction of functionalities on the amino groups of 1,4,8,11-tetraaza-5,7-dione brings the drawback of a small but significant decrease of the exploitable fluorescence, i.e. the fluorescence of the system in the absence of added Cu(2+), at the pH value suitable for full metal complexation.

Exploiting micelle-driven coordination to evaluate the lipophilicity of molecules.

We present a systematic study based on the calculation of complexation constants between a Zn-complex solubilized in Triton X-100 micellar solutions and a series of linear mono- and dicarboxylic acids, under physiological pH conditions, that allowed the evaluation of the lipophilicity of these molecules. This empirical lipophilicity parameter describes conveniently the partition of organic molecules between hydrophobic microdomains and water. The results can be used to predict the lipophilicity of molecules with similar structure and allows the distinction of intrinsic contributions of the carboxylates and of the methylene groups to the lipophilicity of the molecule.