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FARMACOLOGIA e TOSSICOLOGIA applicate ai nanofarmaci.

FARMACOLOGIA e TOSSICOLOGIA applicate ai nanofarmaci. A.A. 2011-2012. The following Expectations are listed in the presentation of Dr Peter Hatto (Chairman ISO TC 229, Director of Research, IonBond Ltd I), at the

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FARMACOLOGIA e TOSSICOLOGIA applicate ai nanofarmaci.

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  1. FARMACOLOGIA e TOSSICOLOGIAapplicate ai nanofarmaci. A.A. 2011-2012

  2. The following Expectations are listed in the presentation of Dr Peter Hatto (Chairman ISO TC 229, Director of Research, IonBond LtdI), at the International Workshop on Documentary Standards for Measurement and Characterization for Nanotechnologies (Gaithersburg, Maryland, USA, 26 –28 February 2008)

  3. Critical physical-chemical parameters for characterization prior to toxicity testing • General: Composition, Concentration, Crystalline phase, Purity • Size: Grain, Particle, Hydrodynamicsize, Distribution • Shape and surface: Shape, Length, Specific surface area, Surface charge and chemistry, Zeta potential • Interactions: Agglomeration/ aggregation, Catalytic properties, Fat solubility/oleophilicity, Water solubility/hydrophilicity, dustiness

  4. Needs of standard methods for Nanoparticles • Stability, aggregation and dissolution rates of nanomaterials • Assessment of Product Degradation and Release of Nanomaterials from Consumer Products • Nanomaterial Product Labelling • Toxicological screening, physical and chemical hazard • Risk Assessments on exposure and use • Safety standards for consumer of products • Reporting Toxicity of Nanomaterials in Consumer Products • Determining Exposure to Nanomaterials in Food • Life Cycle Analysis for Consumer Products Containing Nanomaterials

  5. Needs of standard methods for Nanotubes • Inhalation testing • Toxicology testing • food exposure determination • cosmetics and other skin contact products

  6. Interactions of nanomaterials with lipid bilayers Nanoparticles enter the biological membranes (A): the process can disrupt the lipid bilayer (B) and can cause lipid peroxidation. As a consequence, the following release of dangerous oxygen radicals is poorly quenched. Image refers to Au55

  7. Interaction of nanomaterials with the components of the cell: oxidative damage Nanomaterials can induce oxidative damage to the structures of the cells through the formation of oxygen radicals. The membrane bilayer undergoes lipoperoxidation. The DNA (plastidic, mitochondrial or nucleic) can be damaged; the genes for the DNA repair inhibited, and the apoptotic proteins induced.

  8. Interactions of nanomaterials with nucleic acids: direct interaction. Highly reactive clusters of nAu55 directly reacts with the DNA double helix. (Liu et al., 2003. Angewandte Chemie International Edition, 42: 2853–2857)

  9. B A Fullerenes (C60, or p) are present inside the macrophage, in the cytoplasm (A), or in lysosomes and nucleus (B). No toxicity recorded. A.E. Porter et al. 2006. Acta Biomaterialia 2: 409–419 Citotoxicity: macrophage & C60

  10. Functionalization of C60 with AA helps the nanoparticles passage through the membrane (A), but enhaces the toxicity (B) A B J.G. Rouse et al. 2006. Toxicology in Vitro, 20: 1313–1320 Cytotoxicity: human epidermal keratinocytes of C60 functionalized with aminoacids.

  11. B A CNT interacts with the cytoskeleton (A) and reduce the adhesivity of the cells to the substrate (B). (http://www.coltgroup.com/colt-foundation/ ) Cytotoxicity: CNT

  12. Cyto- and genotoxicity tests

  13. “in vitro” models.

  14. Cell lines used in nanotoxicology • Healthy cells: • Chinese hamster: Lung, ovary • Human: keratinocytes,fibroblasts, colon cells, respiratory epithelia, • hepatocytes • Mouse: fibroblasts respiratory epithelia, mesothelia, endothelia and • umbilical endothelia. • Tumor or modified cells lines: Immortalized, lymphoblastoid (WIL2-NS), lung epithelial tumor (A549), human small cell lung cancer (NCI-H69), promyelocytic leukemia (HL-60); human hepatoma (BEL-7402), liver carcinoma (HepG2), squamous carcinoma (A431), human fibrosarcoma (HT-1080), human gastric cancer (SGC-7901) • Others: retinal pigment epithelial cells, nasal epithelia, renal epithelia, endothelia, neurons.

  15. Metal oxide NPs induce DNA damage. The persistence of NPs in the head of the “comet” is responsible for an artifact, the persistent fluorescence, after fading of that due to the DNA. Karlsson, 2010. Anal Bioanal Chem 398: 651–666 The COMET test for genotoxicity

  16. Single Walled Carbon NanoTubes (SWCNT) induce DNA damage in renal epithelial cells (NRK-52E). The viability of the cells is reduced, and apoptosis-associated genes are overexpressed. Nam et al., 2011. Arch Pharm Res 34: 661-669 The COMET test for genotoxicity

  17. “in vitro” cell adhesion (Eukaryote) • Quantification of human dermal fibroblast adhesion and viability • on two different polymeric scaffolds (fibers diameter: 800 nm ca). • Green: Viable cells Red: dead cells. • Grafahrend et al. 2011. Nature Materials, 10: 67–73. doi:10.1038/nmat2904

  18. Mammals: Rodents (mice, rat, Hamster), rabbit, swine Fish: Danio rerio Amphibians: Xenopus laevis Invertebrate: C.elegans Model organisms used in nanotoxicology

  19. Nanoparticles and liver toxicity in rats The systemic administration of uncoated USPIO to rats induces liver inflammation and necrosis (B1 and 2). Hepatitis signs do not follow the administration of dextran-coated USPIO (C ).

  20. Venoms Physical agents Chemical agents Genes Proteins Metabolites Genomics Proteomics Metabonomics Phenotype Genotype A comprehensive approach: metabonomics. Physical, chemical, biologicaL injuries Metabonomics Modified from: Duarte, 2011. Journal of Controlled Release 153: 34–39

  21. Why and when use Metabonomics? Metabonomics is recognized as a valuable complement for pharmaco- and toxicologic studies. The FDA includes it in the biomarker development design. Main features: simultaneous and non-selective collection of quantitative data for a large range of metabolites, limited manipulation of the sample. Implementations: metabonomics provides powerful and advanced analytical platforms with high sensitivity.

  22. Sample: tissue, cell, blood or other biological fluids Organism or cells Blue: signal in control Red: signal after exposition NMR, SPR, GC, HPLC… Modified from: Duarte, 2011. Journal of Controlled Release 153: 34–39

  23. Effects of USPIO on rat liver: light microscopy. Rat liver: Control (A); and after treatment with uncoated USPIO (B) or dextran-coated USPIO (C ) Feng et al., 2011. Biomaterials 32: 6558-6569

  24. Effects of USPIO on rat liver: Metabonomics, the 1H-NMR spectra Each peak is due to the signal of a different compuond (metabolite). 2: Isoleucine; 7: Lactate; 8: Alanine; 10: Lysine; 12: Lipid, eCH2eCH ¼ CH; 14: O-Acetyl glycoprotein signal; 15, Glutamate; 21: Lipid: ¼ CHeCH2eCH¼; 23: Malonate; 25: Phosphocholine; 28: Taurine; 29: Trimethylamine N-oxide; 31: myo-Inositol; 32, Glycine; 34: Glyceryl, CH2OCOR. Feng et al., 2011. Biomaterials 32: 6558-6569

  25. Effects of USPIO on rat liver: Metabonomics, the PCA Principal component analysis (PCA) of 1H-NMR spectra (metabonomes) of the liver of control rats compared with those of rats treated with coated and uncoated USPIO, 6h after injection. Feng et al., 2011. Biomaterials 32: 6558-6569

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