Giancarlo Ortaggi

Comparison of different commercially available cationic liposome-DNA lipoplexes: Parameters influencing toxicity and transfection efficiency.

Lipid-DNA complexes (lipoplexes) are widely used, since several years, as gene carriers. However, their transfection efficiency, both in vitro and in vivo, depends, in a rather complex way, on different interconnected parameters, ranging from the chemical composition of the lipid components to the size and size distribution of the complexes and, moreover, to the composition of the suspending medium. In this paper, we have investigated the behavior of nine different commercially available transfection agents (liposomal and non-liposomal) and their lipoplexes, at different molar charge ratios and in different experimental conditions. The size and the time stability of the resulting lipoplexes were investigated by means of dynamic light scattering methods and their toxicity and transfection efficiency were assayed in vitro in a model tumor cell line (C6 rat glioma cell line). An attempt to correlate the different parameters governing the complex phenomenology observed has been made. Whereas all the formulations investigated display a low toxicity, that increases with the increase of the lipid-DNA molar charge ratio, the transfection efficiency markedly depends, besides the molar charge ratio, on the lipid composition and on the lipoplex size, in a rather correlated way. The aim of this work is to present, in a wide scenario, an example of the inter-correlation among the different parameters that influence the transfection efficiency of lipoplexes and to suggest the role exerted by the average size of the resulting aggregates in their overall effectiveness as carriers in gene therapy.

A novel near-infrared indocyanine dye-polyethylenimine conjugate allows DNA delivery imaging in vivo

Near-infrared (NIR) fluorescence light has been applied to monitor several biological events in vivo since it penetrates tissues more efficiently than visible light. Dyes exhibiting NIR fluorescence and having large Stokes shift are key elements for this promising optical imaging technology. Here, we report the synthesis of a novel conjugate between a near-infrared indocyanine dye and an organic polyamine polymer (polyethylenimine, PEI) (IR820-PEI) with high chemical stability and good optical properties. IR820-PEI absorbs at 665 nm, emits at 780 nm, and displays a large Stokes shift (115 nm). Moreover, the reported conjugate is able to bind DNA, and the delivery process can be monitored in vivo with noninvasive optical imaging techniques. These characteristics make IR820-PEI one of the most effective and versatile indocyanine dye polymeric-conjugate reported so far.

Chitosan Micro- and Nanospheres: Fabrication and Applications for Drug and DNA Delivery

Polysaccharides and other cationic polymers have been recently used in pharmaceutical research and industry for their properties to control the release of antibiotics, DNA, proteins, peptide drugs or vaccines. They have been also extensively studied as non viral DNA carriers for gene delivery and therapy. Chitosan is one of the most used since it can promote long-term release of incorporated drugs. Here, we reviewed the recent literature on the preparation of chitosan micro- and nanospheres using different manufacturing processes (nanofabrication). Moreover, the preparation of chitosan and chitosan/DNA nanospheres using a novel and simple osmosis-based method has been recently reported. This novel nanofabrication method may be a useful alternative to obtain small DNA-containing nanospheres (38+/-4 nm) for biomedical applications. The reported method has general applicability to various synthetic or natural biopolymers. Solvent, temperature and membrane cut-off are the physicochemical parameters able to control the overall osmotic process leading to obtain several nanostructured systems with different size and shape that may be used in several biotechnological applications.

Common plants as alternative analytical tools to monitor heavy metals in soil.

BackgroundHerbaceous plants are common vegetal species generally exposed, for a limited period of time, to bioavailable environmental pollutants. Heavy metals contamination is the most common form of environmental pollution. Herbaceous plants have never been used as natural bioindicators of environmental pollution, in particular to monitor the amount of heavy metals in soil. In this study, we aimed at assessing the usefulness of using three herbaceous plants (Plantago major L., Taraxacum officinale L. and Urtica dioica L.) and one leguminous (Trifolium pratense L.) as alternative indicators to evaluate soil pollution by heavy metals.ResultsWe employed Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) to assess the concentration of selected heavy metals (Cu, Zn, Mn, Pb, Cr and Pd) in soil and plants and we employed statistical analyses to describe the linear correlation between the accumulation of some heavy metals and selected vegetal species. We found that the leaves of Taraxacum officinale L. and Trifolium pratense L. can accumulate Cu in a linearly dependent manner with Urtica dioica L. representing the vegetal species accumulating the highest fraction of Pb.ConclusionsIn this study we demonstrated that common plants can be used as an alternative analytical tool for monitoring selected heavy metals in soil.

Polyethylenimine In Medicinal Chemistry

Polyethylenimine (PEI), an organic branched or linear polyamine polymer, has been successfully used in the past for DNA complexation and transfection in vitro and in vivo into several cell lines and tissues. PEI was also applied in different fields from gene therapy and several studies have emphasized the importance of this polymer in medicinal chemistry. In this brief critical review the uses and applications of this versatile polymeric molecule will be discussed.

Study of binding affinity and selectivity of perylene and coronene derivatives towards duplex and quadruplex DNA by ESI-MS.

In this paper, we report an extensive electrospray ionization mass spectrometry (ESI-MS) study of the noncovalent interactions between different intermolecular and intramolecular G-quadruplex structures and several perylene and coronene ligands. The selectivity of these compounds toward quadruplex structures with respect to duplex DNA, a fundamental topic for the biological evaluation and the pharmacological application of these ligands as potential chemotherapeutic agents, has also been investigated. After exploring this topic according to the classical approach based on the very simple duplex model of an autocomplementary dodecamer, we extended our analysis reporting for the first time a competition ESI-MS experiment in the presence of genomic DNA fragments. Whereas those ligands showing a high level of selectivity between quadruplex and duplex oligonucleotides, in terms of binding constants and percentage of bound DNA, confirmed their selectivity in the competition experiment, the contrary was not always true: some ligands showing poor selectivity with the autocomplementary dodecamer resulted selective in the presence of genomic DNA fragments. This result suggests that physiologically nonrelevant interactions are possible with a short duplex oligonucleotide. This means that the dodecamer can fail in representing a biologically significant structural model, or, better, that it can be used to quickly screen potentially selective molecules, but bearing in mind the high probability of false negative results.

The behaviour of ferrocene and ruthenocene in weakly to strongly protic media. Implications on the mechanism of substitutions involving proton as the electrophile

Abstract The interaction of ferrocene and ruthenocene with proton donors has been investigated by NMR spectroscopy in a broad range of protic media, including such weakly protic agents as chloroform, moderately acidic systems such as TFA, and more strongly acidic systems such as conc. H2SO4. The results indicate that these metallocenes undergo two kinds of interactions with acidic media, viz., π-hydrogen bonding in which the Cp ring is the electron donor and, in the most strongly protic solvents, metal protonation. The (H0) 1 2 of ruthenocene and a group of ferrocene derivatives have been determined by a combined extractive-spectrophotometric technique. These values show that the metal atom acts as a very weak base. The H/D exchange rate of ruthenocene in conc. D2SO4 decreases on increasing acid concentration, which rules out the metal-protonated species as an intermediate in the reaction.

N-cyclic bay-substituted perylene G-quadruplex ligands have selective antiproliferative effects on cancer cells and induce telomere damage.

A series of bay-substituted perylene derivatives is reported as a new class of G-quadruplex ligands. The synthesized compounds have differing N-cyclic substituents on the bay area and differing side chains on the perylene major axis. ESI-MS and FRET measurements highlighted the strongest quadruplex binders in this series and those showing the highest quadruplex/duplex selectivity. Several biological assays were performed on these compounds, which showed that compound 5 (PPL3C) triggered a DNA damage response in transformed cells with the formation of telomeric foci containing phosphorylated γ-H2AX and 53BP1. This effect mainly occurred in replicating cells and was consistent with Pot1 dissociation. Compound 5 does not induce telomere damage in normal cells, which are unaffected by treatment with the compound, suggesting that this agent preferentially kills cancer cells. These results reinforce the notion that G-quadruplex binding compounds can act as broad inhibitors of telomere-related processes and have potential as selective antineoplastic drugs.

A hydrophilic three side-chained triazatruxene as a new strong and selective G-quadruplex ligand

A new hydrosoluble triazatruxene derivative (Azatrux) is reported to selectively bind to G-quadruplex DNA, as derived by ESI-MS measurements and competition experiments.

Ferrocenyl carbocations. The ionization of ferrocenyl alcohols in aqueous sulfuric acid

A detailed study of the ionization equilibrium between ferrocenyl alcohols and α-ferrocenyl carbocations in aqueous sulfuric acid has been performed by spectral methods. The spectral characterization of the carbocations, the extent of their formation as a function of the proton-donor ability of the solvent, and the determination of the equilibrium constants are reported. The behavior of ferrocenyl alcohols as indicators compared to that of triarylmethanols is discussed. The transmission of the polar effects of the heteroannular substituents, the heteroannular bridging of an α-hydroxytrimethylene chain, and the high charge delocalization power of the carbocation are also examined.