Chemical synthesis and characterization of silver-protected vasoactive intestinal peptide nanoparticles.

UNLABELLED: We characterized a method to conjugate functional silver nanoparticles with vasoactive intestinal peptide (VIP), which could be used as a working model for further tailor-made applications based on VIP surface functionality. Despite sustained interest in the therapeutic applications of VIP, and the fact that its drugability could be largely improved by the attachament to functionalized metal nanoparticles, no methods have been described so far to obtain them. MATERIALS & METHODS: VIP was conjugated to tiopronin-capped silver nanoparticles of a narrow size distribution, by means of proper linkers, to obtain VIP functionalized silver nanoparticles with two different VIP orientations (Ag-tiopronin-PEG-succinic-[His]VIP and Ag-tiopronin-PEG-VIP[His]). VIP intermediate nanoparticles were characterized by transmission-electron microscopy and Fourier transform infrared spectroscopy. VIP functionalized silver nanoparticles cytotoxicity was determined by lactate dehydrogenase release from mixed glial cultures prepared from cerebral cortices of 1-3 days-old C57/Bl mice. Cells were used for lipopolysaccharide stimulation at day 18-22 of culture. RESULTS: Two different types of VIP-functionalized silver nanoparticles were obtained; both expose the C-terminal part of the neuropeptide, but in the first type VIP is attached to silver nanoparticle through its free amine terminus (Ag-tiopronin-PEG-succinic-[His]VIP), while in the second type, VIP N-terminus remains free (Ag-tiopronin-PEG-VIP[His]). VIP-functionalized silver nanoparticles did not compromise cellular viability and inhibited microglia-induced stimulation under inflammatory conditions. CONCLUSION: The chemical synthesis procedure developed to obtain VIP-functionalized silver nanoparticles rendered functional products, in terms of biological activity. The two alternative orientations designed, reduced the constraints for chemical synthesis that depends on the nanosurface to be functionalized. Our study provides, for the first time, a proof of principle to enhance the therapeutic potential of VIP with the valuable properties of metal nanoparticles for imaging, targeting and drug delivery.

Tiopronin monolayer-protected silver nanoparticles modulate IL-6 secretion mediated by Toll-like receptor ligands.

AIMS: Capped silver nanoparticles that can be coupled to a variety of molecules and biomolecules are of great interest owing to their potential applications in biomedicine. However, there are no data about their toxicity or functional effects on a key innate immune response, such as IL-6 secretion, after the engagement of the main group of pathogen-associated molecular patterns receptors, that is, the Toll-like receptors (TLRs). MATERIALS & METHODS: N-(2-mercaptopropionyl)glycine (tiopronin)-capped silver (Ag@tiopronin) nanoparticles of a narrow sized distribution ( approximately 5 nm) were synthesized and characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, Raman, (1)H-NMR and total correlation spectroscopy. Cytotoxicity was determined by lactate deshidrogenase and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium assays in Raw 264.7 macrophages. IL-6 was measured by ELISA. RESULTS & DISCUSSION: Ag@tiopronin nanoparticles have a narrow size distribution ( approximately 5 nm), high solubility and stability in aqueous environment with no cytotoxicity in terms of mitochondrial function or plasma-membrane integrity at concentrations as high as 200 microg/10(6) cells. Ag@tiopronin nanoparticles were not proinflammatory agents, but remarkably they specifically impaired the IL-6 secretion mediated by TLR2, TLR2/6, TLR3 or TLR9 stimulation in co-treatment experiments. However, in pretreatment experiments, nanoparticles enhanced the susceptibility of macrophages to inflammatory stimulation mediated by TLR2/1 and TLR2/6 specific ligands while severely impairing the IL-6 secretion activated by the TLR3 or TLR9 ligands. CONCLUSIONS: Contrary to what is found for bare silver nanoparticles, Ag@tiopronin nanoparticles are noncytotoxic to macrophages. Ag@tiopronin nanoparticles showed differential effects on TLR signaling of a high degree of specificity, without proinflammatory effects by themselves. These effects have to be borne in mind when using bioconjugates of Ag@tiopronin nanoparticles for future medical applications.

Phylogenetic analysis of chloroplast DNA matK gene and ITS of nrDNA sequences reveals polyphyly of the genus Sonchus and new relationships among the subtribe Sonchinae (Asteraceae: Cichorieae).

Phylogenetic relationships among genera of the subtribes Sonchinae (11 genera and ca. 130 species) and Dendroseridinae (2 genera and 12 species) sensu Bremer were assessed with ITS and matK gene sequences. The Sonchinae is strongly supported as paraphyletic, and subtribal rank of Dendroseridinae is poorly supported. This study re-delimits Sonchinae, which now includes 14 genera and ca. 146 species. Two genera, Reichardia and Launaea, diverge first, followed by a major radiation of the remaining species, including Dendroseris and Thamnoseris. The monotypic Aetheorhiza is more closely related to Sonchus than to Launaea. Sonchus is highly polyphyletic, occurring across nearly all clades of the trees. It is highly probable that several Pacific and Atlantic island endemics evolved within the Sonchus group. Polyploidization processes appear to have played an important role in evolutionary differentiation and speciation in Sonchus, especially in subgenus Sonchus. This study identifies 10 major clades within the Sonchinae, and emphasizes the need to re-delimit and re-circumscribe Sonchus and the Sonchinae.