Cydonia oblonga Mill. Pulp Callus Inhibits Oxidative Stress and Inflammation in Injured Cells.

The pharmacological activity of a callus extract from the pulp of Cydonia oblonga Mill., also known as quince, was investigated in murine macrophage (RAW 264.7) and human keratinocyte (HaCaT) cell lines. In particular, the anti-inflammatory activity of C. oblonga Mill. pulp callus extract was assessed in lipopolysaccharides (LPS)-treated RAW 264.7 by the Griess test and in LPS-treated HaCaT human keratinocytes by examining the expression of genes involved in the inflammatory process, including nitric oxide synthase (iNOS), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), nuclear factor-kappa-B inhibitor alfa (ikBα), and intercellular adhesion molecule (ICAM). The antioxidant activity was evaluated by quantizing the reactive oxygen species (ROS) production in the hydrogen peroxide and tert-butyl hydroperoxide-injured HaCaT cell line. The obtained results indicate that C. oblonga callus from fruit pulp extract has anti-inflammatory and antioxidant activities, suggesting its possible application in delaying and preventing acute or chronic diseases associated with aging or in the treatment of wound dressing.

Targeting SARS-CoV-2 by synthetic dual-acting thiol compounds that inhibit Spike/ACE2 interaction and viral protein production.

The SARS-CoV-2 life cycle is strictly dependent on the environmental redox state that influences both virus entry and replication. A reducing environment impairs the binding of the spike protein (S) to the angiotensin-converting enzyme 2 receptor (ACE2), while a highly oxidizing environment is thought to favor S interaction with ACE2. Moreover, SARS-CoV-2 interferes with redox homeostasis in infected cells to promote the oxidative folding of its own proteins. Here we demonstrate that synthetic low molecular weight (LMW) monothiol and dithiol compounds induce a redox switch in the S protein receptor binding domain (RBD) toward a more reduced state. Reactive cysteine residue profiling revealed that all the disulfides present in RBD are targets of the thiol compounds. The reduction of disulfides in RBD decreases the binding to ACE2 in a cell-free system as demonstrated by enzyme-linked immunosorbent and surface plasmon resonance (SPR) assays. Moreover, LMW thiols interfere with protein oxidative folding and the production of newly synthesized polypeptides in HEK293 cells expressing the S1 and RBD domain, respectively. Based on these results, we hypothesize that these thiol compounds impair both the binding of S protein to its cellular receptor during the early stage of viral infection, as well as viral protein folding/maturation and thus the formation of new viral mature particles. Indeed, all the tested molecules, although at different concentrations, efficiently inhibit both SARS-CoV-2 entry and replication in Vero E6 cells. LMW thiols may represent innovative anti-SARS-CoV-2 therapeutics acting directly on viral targets and indirectly by inhibiting cellular functions mandatory for viral replication.

Particle-Bound Mercury Characterization in the Central Italian Herbarium of the Natural History Museum of the University of Florence (Italy).

Museums air quality can be negatively affected by treatments with heavy metals compounds employed to prevent pest infestations. Among these, the past use of mercury dichloride (HgCl2) on herbaria artifacts currently produces high levels of indoor atmospheric gaseous mercury (Hg0) and possibly of particulate bound Hg (PBM), i.e., the particulate matter containing Hg. This study evaluates the PBM pollution in the Central Italian Herbarium (Natural History Museum of the University of Florence, Italy), characterizing the size range and chemical speciation with SEM-EDS microanalysis. The analysis of the total Hg concentration in the samples allowed to calculate the workers exposure risk to this pollutant. PBM is almost totally classifiable as fine particulate with a significant dimensional increase in a period of scarce attendance of the Herbarium rooms. The microanalysis indicates that Hg is essentially bound to S, highlighting the change of Hg speciation from the original association with Cl. The average Hg concentration reveals a potential health risk for workers as result of multiple Hg exposure pathways, mainly by ingestion. The study provides information for characterizing PBM pollution that could affect a workplace atmosphere and a useful basis to evaluate and correctly design solution strategies to reduce the contamination levels and protect workers' health.

Monitoring of Airborne Mercury: Comparison of Different Techniques in the Monte Amiata District, Southern Tuscany, Italy.

In the present study, mercury (Hg) concentrations were investigated in lichens (Flavoparmelia caperata (L.) Hale, Parmelia saxatilis (L.) Ach., and Xanthoria parietina (L.) Th.Fr.) collected in the surrounding of the dismissed Abbadia San Salvatore Hg mine (Monte Amiata district, Italy). Results were integrated with Hg concentrations in tree barks and literature data of gaseous Hg levels determined by passive air samplers (PASs) in the same area. The ultimate goal was to compare results obtained by the three monitoring techniques to evaluate potential mismatches. Lichens displayed 180-3600 ng/g Hg, and Hg concentrations decreased exponentially with distance from the mine. Mercury concentration was lower than in Pinus nigra barks at the same site. There was a moderate correlation between Hg in lichen and Hg in bark, suggesting similar mechanisms of Hg uptake and residence times. However, correlation with published gaseous Hg concentrations (PASs) was moderate at best (Kendall Tau = 0.4-0.5, p > 0.05). The differences occurred because a) PASs collected gaseous Hg, whereas lichens and barks also picked up particulate Hg, and b) lichens and bark had a dynamic exchange with the atmosphere. Lichen, bark, and PAS outline different and complementary aspects of airborne Hg content and efficient monitoring programs in contaminated areas would benefit from the integration of data from different techniques.

Effects of biogenerated ferric hydroxides nanoparticles on truffle mycorrhized plants.

Truffles are highly valuable ectomycorrhizal fungi that grow naturally in alkaline, calcareous soils. Iron deficiency chlorosis is a common problem in truffle (Tuber spp.) cultivation due to the high quantity of lime added to increase the pH of acidic soils. In this work, the effects of ferric hydroxide nanoparticles embedded in an exopolysaccharide (Fe-EPS NPs), extracted from cultures of Klebsiella oxytoca DSM 29614, were investigated on Quercus robur seedlings under greenhouse conditions. The plants were inoculated with Tuber borchii (the bianchetto truffle) and were cultivated with and without iron nanoparticle additions and compared with non-inoculated control plants. The seedlings were grown in limed soil in order to induce iron deficiency. Low doses of Fe-EPS NPs had a beneficial effect on the growth of the plants inoculated with T. borchii, increasing their height and reducing their leaf chlorosis 5 months after the first Fe-EPS NP treatment. Moreover, Fe-EPS NP treatments significantly increased the level of T. borchii mycorrhizal colonization and the ectomycorrhizal mantle thickness. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) applied to cross sections of mycorrhizas showed that Fe accumulated in the fungal mantle and apparently was slowly released serving as a resilient reservoir of iron for the plant. The results suggest that the application of Fe-EPS NPs is a promising technique in the production of Tuber mycorrhized plants in the nursery and could have future applications in the field.

In vitro effects on calcium oxalate crystallization kinetics and crystal morphology of an aqueous extract from Ceterach officinarum: Analysis of a potential antilithiatic mechanism.

Ceterach officinarum Willd is a plant widespread throughout Europe and used in southern Italy as a diuretic. Beliefs in the benefits of C. officinarum aqueous extract in the treatment of calcium oxalate kidney stones are widely held. Little is known, however, about the actual mechanism of its antilithiatic action. Our results in this in vitro study corroborate C. officinarum aqueous extract as a good source of antioxidants with a high antioxidant effects. Our results also demonstrate a major impact of C. officinarum aqueous extract on in vitro induced calcium oxalate crystallization kinetics and crystal morphology, showing its critical role in kidney stone formation and/or elimination. We show that progressively increasing doses of C. officinarum aqueous extract cause a sequence of effects. A powerful inhibitory action on calcium oxalate monohydrate (COM) growth and aggregation is first observed. C. officinarum aqueous extract also appears highly effective in stimulating nucleation increasing the number and reducing the size of COM crystals, which become progressively thinner, rounded and concave in a dose-dependent manner. These shape-modified COM crystals are known to be less adherent to renal tubular cells and more easily excreted through the urinary tract preventing kidney stone formation. Further, C. officinarum aqueous extract promotes the formation of calcium oxalate dihydrate (COD) rather than the monohydrate so that, at the highest concentrations used, only COD crystals are observed, in significant greater numbers with a clear reduction in their size, in a dose-dependent manner. Furthermore, AFM analyses allowed us to reveal the presence of C. officinarum component(s) on the surfaces of COD and modified COM crystals. The crystal surface adsorbed component(s) are shown to be similarly active as the total aqueous extract, suggesting a trigger factor which may direct crystal modification towards COD forms. In urolithiasis pathogenesis COD crystals are less dangerous than the COM forms due to their lower affinity for renal tubular cells. Our results are important in understanding the mechanisms which guide the modification induced by C. officinarum on the crystallization process. Based on these data, together with no adverse toxic effect being observed on the in vitro model of human intestinal enterocytes, C. officinarum aqueous extract could represent an attractive natural therapy for the treatment of urolithiasis.

A multi-analytical approach for the study of red stains on heritage marble.

Red stains are a common discolouration on stone cultural heritage all over the world. These are very difficult to remove and little is known about the reddish pigmentation. Numerous red stains were mapped on the Baptistery of San Giovanni in Florence, one of the most important monuments in Italy. This paper is focused on red stains on marble stone and the results of a detailed multidisciplinary approach are presented. Several analytical and investigation techniques (such as optical microscopy, X-ray fluorescence mapping, X-ray micro-tomography, micro-Raman spectroscopy, scanning electron microscopy with energy dispersive spectroscopy, microbial isolation, colorimetric measurements and isotopic analyses) were used to better understand the origin and processes involved in this kind of alteration. Analyses of the red stains led us to believe the presence of minium (lead tetroxide) and Pb are usually concentrated in the spaces between calcite grains. Red stains of Pb isotopic composition also overlap with data from Sardinian mines. These preliminary data seem to reinforce the suggestion of a source of lead from some metallic items (during restoration campaigns between 1938 and 1944, damaged parts were removed and replaced, and the new marble cladding was fixed with iron brackets treated with minium).

Design, Synthesis, and Biological Activity of Hydrogen Peroxide Responsive Arylboronate Melatonin Hybrids.

Stimulus-responsive cleavage reactions have found broad use to direct drug release at a particular target disease area. Increased levels of reactive oxygen species (ROS) have been associated with the development and progression of cancer and several other disease states, motivating the development of drug conjugates that can undergo a chemoselective ROS-triggered release. Melatonin (MLT) and the reactive electrophile p-benzoquinone methide ( p-QM) have evidenced either cytoprotective or cytotoxic effects in biological systems, depending on the dose, cellular targets, and time of exposure. In this study, we report the synthesis and biological activity of two MLT derivatives linked to ROS-responsive arylboronate triggers (P1 and P2), which can be activated by endogenously generated hydrogen peroxide (H2O2) to release MLT, or 5-methoxytryptamine (5-MeOT), and p-QM-intermediates. Their H2O2-induced activation mechanism was studied by HPLC-DAD-MS. P1, which rapidly releases MLT and p-QM, was able to strongly induce the Nrf2 antioxidant signaling pathway, but was ineffective to provide protection against H2O2-mediated oxidative damage. By contrast, P1 exhibited strong toxic effects in HeLa cancer cells, without causing significant toxicity to normal NCTC-2544 cells. Similar, although more limited, effects were exerted by P2. In both cases, cytotoxicity was accompanied by depletion of cellular glutathione (GSH), probably as a consequence of p-QM release, and increased ROS levels. A role for MLT in toxicity was also observed, suggesting that the P1 released products, MLT and p-QM, contributed additively to promote cell death.

Bacteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles (Tuber borchii).

Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their effects on Tuber borchii ("bianchetto" truffle) mycelium growth and morphology, they were tested on human K562 cell line and Lentinula edodes pure culture and shown to be non-toxic. Using these nanoparticles as iron supplement, the truffles showed extremely efficient iron uptake of over 300 times that of a commercial product. This avoided morphological changes in T. borchii due to lack of iron during growth and, with optimum nanoparticle dosage, increased growth without cell wall disruption or alteration of protoplasmatic hyphal content, the nuclei, mitochondria, and rough endoplasmic reticula being preserved. No significant modifications in gene expression were observed. These advantages derive from the completely different mechanism of iron delivery to mycelia compared to commercial iron supplements. The present data, in fact, show the nanoparticles attached to the cell wall, then penetrating it non-destructively without damage to cell membrane, mitochondria, chromatin, or ribosome. Low dosage significantly improved mycelium growth, without affecting hyphal morphology. Increases in hyphal diameter and septal distance indicated a healthier state of the mycelia compared to those grown in the absence of iron or with a commercial iron supplement. These positive effects were confirmed by measuring fungal biomass as mycelium dry weight, total protein, and ergosterol content. This "green" method for biogenerating iron exopolysaccharide nanoparticles offers many advantages, including significant economic savings, without toxic effects on the ectomycorrhizal fungus, opening the possibility of using them as iron supplements in truffle plantations.

Mercury speciation in Pinus nigra barks from Monte Amiata (Italy): An X-ray absorption spectroscopy study.

This study determined, by means of X-ray absorption near-edge structure (XANES) spectroscopy, the speciation of mercury (Hg) in black pine (Pinus nigra) barks from Monte Amiata, that were previously shown to contain exceptionally high (up to some mg kg-1) Hg contents because of the proximity to the former Hg mines and roasting plants. Linear fit combination (LCF) analysis of the experimental spectra compared to a large set of reference compounds showed that all spectra can be fitted by only four species: ß-HgS (metacinnabar), Hg-cysteine, Hg bound to tannic acid, and Hg0. The first two are more widespread, whereas the last two occur in one sample only; the contribution of organic species is higher in deeper layers of barks than in the outermost ones. We interpret these results to suggest that, during interaction of barks with airborne Hg, the metal is initially mechanically captured at the bark surface as particulate, or physically adsorbed as gaseous species, but eventually a stable chemical bond is established with organic ligands of the substrate. As a consequence, we suggest that deep bark Hg may be a good proxy for long term time-integrated exposure, while surface bark Hg is more important for recording short term events near Hg point sources.

Black pine (Pinus nigra) barks as biomonitors of airborne mercury pollution.

Tree barks are relevant interfaces between plants and the external environment, and can effectively retain airborne particles and elements at their surface. In this paper we have studied the distribution of mercury (Hg) in soils and in black pine (Pinus nigra) barks from the Mt. Amiata Hg district in southern Tuscany (Italy), where past Hg mining and present-day geothermal power plants affect local atmospheric Hg concentration, posing serious environmental concerns. Barks collected in heavily Hg-polluted areas of the district display the highest Hg concentration ever reported in literature (8.6mg/kg). In comparison, barks of the same species collected in local reference areas and near geothermal power plants show much lower (range 19-803µg/kg) concentrations; even lower concentrations are observed at a "blank" site near the city of Florence (5-98µg/kg). Results show a general decrease of Hg concentration from bark surface inwards, in accordance with a deposition of airborne Hg, with minor contribution from systemic uptake from soils. Preliminary results indicate that bark Hg concentrations are comparable with values reported for lichens in the same areas, suggesting that tree barks may represent an additional useful tool for biomonitoring of airborne Hg.

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics.

UNLABELLED: Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152), an N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice. IMPORTANCE: The first report of an association between Th2 polarization and alteration of the redox state in LP-BM5 infection is presented. Moreover, it provides evidence that LP-BM5 infection causes a decrease in the thiol content of peritoneal macrophages, which can influence IL-12 production. The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection, as it reestablishes the Th1/Th2 balance. Immunotherapy based on the use of pro-GSH molecules would permit LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and a Th1/Th2 imbalance to be more effectively combated.

Development of a multilevel approach for the evaluation of nanomaterials' toxicity.

AIM: To develop a multilevel approach that includes different toxicity tests and gene-expression studies for toxicity evaluation of engineered nanomaterials developed for biomedical applications. MATERIALS & METHODS: K-562, MCF-7 and U-937 human-derived cell lines were used as models for in vitro toxicity tests. These tests included viability assays (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium [MTS] assay); evaluation of apoptosis/necrosis by propidium iodide staining and DNA laddering assay; evaluation of mitochondrial toxicity (5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethyl-benzimidazolcarbocyanine iodide [JC-1] assay); transmission electron microscopy analysis and gene expression analysis by DNA microarray. For in vivo toxicity evaluation, Swiss mice were used for monitoring acute or chronic effects. Two superparamagnetic contrast agents approved for human use (Resovist and Primovist) and two new lanthanide-based luminescent nanoparticles were tested. RESULTS & DISCUSSION: The nanomaterials approved for human use did not show significant toxicities in our assays. Toxicity studies performed on lanthanide-based nanoparticles (EDTA120 and EDTA120D) complexed with the chelating agent EDTA revealed that these nanomaterials induced necrosis in U-937 and K-562 cells while no toxicity was observed in MCF-7 cells. Moreover, no in vivo effects have been observed. The comparative analysis of the nanomaterials and their separated components showed that the toxicity in U-937 and K-562 cells was mainly due to the presence of EDTA. CONCLUSION: The multilevel approach proved to be useful for nanomaterial toxicity characterization. In particular, for the lanthanide-based nanoparticles tested in this work, the EDTA was identified as the main cause of the toxicity in vitro, suggesting a possible applicability of these nanoparticle suspensions for in vivo optical imaging.

The use of Eudragit RS 100/cyclodextrin nanoparticles for the transmucosal administration of glutathione.

The aim of this work was to develop and characterize new nanoparticle systems based on Eudragit RS 100 and cyclodextrins (CDs) for the transmucosal administration of glutathione (GSH). For this purpose, nanoparticles (NPs) with the mucoadhesive properties of Eudragit RS 100 and the penetration enhancing and peptide protective properties of CDs were prepared and evaluated. The quasi-emulsion solvent diffusion technique was used to prepare the NPs with natural and chemically modified (HP-beta-CD and Me-beta-CD) CDs. The NPs prepared showed homogeneous size distribution, mean diameters between 99 and 156nm, a positive net charge and spherical morphology. Solid state FT-IR, thermal analysis (DSC), and X-ray diffraction studies suggest that the nanoencapsulation process produces a marked decrease in crystallinity of GSH. The encapsulation efficiency of the peptide was found to be between 14.8% and 24%. The results indicate that mean diameters, surface charges and drug-loaded NPs were not markedly affected by the CD, whereas the presence of the latter influences drug release and to some extent peptide stability and absorption. Finally, it has been shown that CD/Eudragit RS 100 NPs may be used for transmucosal absorption of GSH without any cytotoxicity using the epithelial human HaCaT and murine monocyte macrophage RAW264.7 cell lines.

Enhanced antisense effect of modified PNAs delivered through functional PMMA microspheres.

Peptide nucleic acids (PNA) are very promising antisense agents, but their in vivo application is often hampered by their low bioavailability, mainly due to their limited uptake through cellular and nuclear membranes. However, PNA chemical synthesis easily allows modification with functional structures able to improve the intrinsically low permeability and great interest is arising in finding specific and efficient delivery protocols. Polymeric core-shell microspheres with anionic functional groups on the surface were tested for their ability to reversibly bind lysine modified PNA sequences, whose antisense activity against COX-2 mRNA was already demonstrated in murine macrophages.

Comparison of novel delivery systems for antisense peptide nucleic acids.

Peptide nucleic acids (PNAs) provide a powerful tool to study the mechanism of transcription and translation, an innovative strategy to regulate target gene expression. They have been successfully used in antisense technology, for their ability to specifically bind to messenger RNA (mRNA) targets and to inhibit translation of the target genes. However, unlike most of the DNA and RNA oligonucleotides, PNAs are poorly penetrated through the cell membrane, partially due to their uncharged property. To enhance the efficiency in PNA delivery, many strategies have been explored. We here compare the efficacy of three different delivery strategies for antisense PNA: 1) conjugation to hydrophobic peptides, 2) adsorption onto polymeric microspheres and 3) encapsulation in autologous erythrocytes. To this purpose, we designed and prepared PNA sequences able to inhibit the expression of macrophage enzymes involved in inflammatory process, i.e. nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) and tested their antisense activity in a murine macrophage cellular model. Both delivery through polymeric microspheres and encapsulation into erythrocytes allowed the antisense activity of unmodified PNAs at nanomolar concentration.

Erythrocyte-based drug delivery.

The use of a physiological carrier to deliver therapeutics throughout the body to both improve their efficacy while minimising inevitable adverse side effects, is an extremely fascinating perspective. The behaviour of erythrocytes as a delivery system for several classes of molecules (i.e., proteins, including enzymes and peptides, therapeutic agents in the form of nucleotide analogues, glucocorticoid analogues) has been studied extensively as they possess several properties, which make them unique and useful carriers. Furthermore, the possibility of using carrier erythrocytes for selective drug targeting to differentiated macrophages increases the opportunities to treat intracellular pathogens and to develop new drugs. Finally, the availability of an apparatus that permits the encapsulation of drugs into autologous erythrocytes has made this technology available in many clinical settings and competitive with other drug delivery systems.

Novel biocompatible anionic polymeric microspheres for the delivery of the HIV-1 Tat protein for vaccine application.

Two novel classes of biocompatible core-shell anionic microspheres, composed of an inner hard insoluble core, either made of poly(styrene) (PS) or poly(methyl methacrylate) (PMMA), and a soft outer tentacular shell made of long soluble negatively charged arms derived from the steric stabilizer, hemisuccinated poly(vinyl alcohol) or Eudragit L100/55, respectively, were prepared by dispersion polymerization and characterized. Five types of these novel microspheres, two made of poly(styrene) and hemisuccinated poly(vinyl alcohol) (A4 and A7), and three made of poly(methyl methacrylate) and Eudragit L100/55 (1D, 1E, H1D), differing for chemical composition, size, and surface charge density were analyzed for the delivery of the HIV-1 Tat protein for vaccine applications. All microspheres reversibly adsorbed the native biologically active HIV-1 Tat protein preventing Tat from oxidation and maintaining its biological activity, therefore increasing the shelf-life of the Tat protein vaccine. The microspheres efficiently delivered Tat intracellularly, and were not toxic in vitro nor in mice, even after multiple administrations. These results indicate that these novel microparticles are safe and represent a promising delivery system for vaccination with Tat, as well as for other subunit vaccines, particularly when a native protein conformation is required.

Dinuclear copper(II) complex as nitric oxide scavenger in a stimulated murine macrophage model.

Nitric oxide is a gaseous, short-living free radical which behaves as an important signaling molecule with pleiotropic capacities including vasodilatation, neurotransmission, and microbial and tumor cell killing, as well as in tissue damage and organ-specific autoimmune disorders. Here, a synthesized, dinuclear copper complex system in vitro obtained by the simple aza-phenolic ligand 2,6-bis[[bis-(2-aminoethyl)amino]methyl]phenol (L) and Cu(II) ion has been used. The stability constants of ligand L with Cu(II) ion were determined through potentiometric measurements in aqueous solution (37.1 +/- 0.1 degrees C, I = 0.15 M of NaCl) to mimic the biological medium. The measurements demonstrated that [Cu(2)H(-1)L(OH)](2+) (DCu) is the predominant species present in solution at pH 7.4. The molecular structure of the ligand in this species permits the cooperation of the two copper ions in assembling the substrate, thus the complex can be used as a receptor for small molecules such as NO. As a biological model, we chose the production of NO catalyzed by inducible nitric oxide synthase obtained from RAW 264.7 murine macrophage cell line stimulated with LPS, which enabled us to prove that NO is coordinated by the DCu complex, modifying its EPR spectra. The coordination of NO with DCu reduces the level of nitrite in the culture medium of stimulated RAW 264.7 macrophages without any inhibition in the expression of iNOS.

Core-shell nanospheres for oligonucleotide delivery. V: adsorption/release behavior of 'stealth' nanospheres.

The adsorption/release behavior of oligodeoxynucleotides (ODNs) on new PEGylated core-shell polymethylmethacrylate nanospheres is described. The outer shell consists of alkyl chains containing quaternary ammonium groups and of poly(ethylene glycol) chains, both covalently bound to the inner core. Ion pair formation between negatively charged ODN phosphate groups and positively charged groups on the nanosphere surface is the main interaction mechanism. No cellular toxicity in HL60 cells is observed at nanosphere concentrations required for biologically active ODN delivery. These results indicate that these novel cationic polymeric nanoparticles are safe and represent promising vectors for oligonucleotide delivery.