FARMACOLOGIA e FARMACOLOGIA e TOSSICOLOGIATOSSICOLOGIA

applicate ai nanofarmaci.applicate ai nanofarmaci.

A.A. 2011-2012A.A. 2011-2012

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)

Critical physical-chemical parameters for characterization prior

to toxicity testingGeneral: Composition, Concentration, Crystalline phase, PuritySize: Grain, Particle, Hydrodynamic size, DistributionShape and surface: Shape, Length, Specific surface area, Surface charge and chemistry, Zeta potentialInteractions: Agglomeration/ aggregation, Catalytic properties, Fat solubility/oleophilicity, Water solubility/hydrophilicity, dustiness

Needs of standard methods for Needs of standard methods for NanoparticlesNanoparticles

1. Stability, aggregation and dissolution rates of nanomaterials2. Assessment of Product Degradation and Release of

Nanomaterials from Consumer Products3. Nanomaterial Product Labelling 4. Toxicological screening, physical and chemical hazard 5. Risk Assessments on exposure and use6. Safety standards for consumer of products 7. Reporting Toxicity of Nanomaterials in Consumer Products 8. Determining Exposure to Nanomaterials in Food9. Life Cycle Analysis for Consumer Products Containing

Nanomaterials

1. Inhalation testing2.Toxicology testing3. food exposure determination 4. cosmetics and other skin contact products

Needs of standard methods for Needs of standard methods for NanotubesNanotubes

Interactions of nanomaterials Interactions of nanomaterials with lipid bilayerswith 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

Interaction of nanomaterials with the Interaction of nanomaterials with the components of the cell: oxidative damagecomponents 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.

Interactions of nanomaterials with Interactions of nanomaterials with nucleic acids: direct interaction.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)

Citotoxicity: macrophage & C60Citotoxicity: macrophage & C60

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

A

B

Cytotoxicity: human epidermal Cytotoxicity: human epidermal keratinocytes of C60 functionalized keratinocytes of C60 functionalized

with aminoacids.with aminoacids.

J.G. Rouse et al. 2006. Toxicology in Vitro, 20: 1313–1320

Functionalization of C60 with AA helps the nanoparticles passage through the membrane (A), but enhaces the toxicity (B)

A

B

Cytotoxicity: CNTCytotoxicity: CNT

CNT interacts with the cytoskeleton (A) and reduce the adhesivity of the cells to the substrate (B).

(http://www.coltgroup.com/colt-foundation/ )

A B

Cyto- and genotoxicity testsCyto- and genotoxicity tests

Test reference/NameTest reference/Name EffectEffect SystemSystem

OECD 471OECD 471

(Ames assay)(Ames assay)

genotoxicity Prokaryotes, bacterial reverse mutation test

OECD 473OECD 473 genotoxicity “in vitro” chromosome

aberration test

OECD 474OECD 474 genotoxicity “in vivo” micronuclei test

COMET assayCOMET assay genotoxicity Isolated DNAIsolated DNA

Dye exclusion,Dye exclusion,

MTT uptake testMTT uptake test

viabilityviability Eukaryotes, cell linesEukaryotes, cell lines

Apotosis/autophagy Apotosis/autophagy genesgenes

programmed cell programmed cell deathdeath

Eukaryotes, cell lines, model Eukaryotes, cell lines, model organismsorganisms

Genes expression in Genes expression in embryosembryos

embryotoxocityembryotoxocity Multicellular model Multicellular model organismsorganisms

““in vitro” models.in vitro” models.

ORGANISMORGANISM EFFECTEFFECT TEST NAMETEST NAME

11 Eukaryote, cellEukaryote, cell Viability, apoptosisViability, apoptosis Trypan blue dye exclusion,Trypan blue dye exclusion,

MTT uptake test,MTT uptake test,

Apotosis genesApotosis genes

22 Eukaryote, cellEukaryote, cell Lipid peroxidationLipid peroxidation MDA determination assayMDA determination assay

33 Eukaryote, cellEukaryote, cell Substrate adhesionSubstrate adhesion

44 Eukaryote, Eukaryote,

““in vitro” in vitro” developing developing

organsorgans

Gene expression, Gene expression, altered developmentaltered development

Ex.: micro organ from cultured Ex.: micro organ from cultured nasal epithelium, embryonic nasal epithelium, embryonic

heartheart

Cell lines used in nanotoxicologyCell lines used in nanotoxicology Healthy cells:Healthy cells: Chinese hamster: Lung, ovaryHuman: keratinocytes,fibroblasts, colon cells, respiratory epithelia, hepatocytesMouse: fibroblasts respiratory epithelia, mesothelia, endothelia and umbilical endothelia.

Tumor or modified cells lines: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:Others:retinal pigment epithelial cells, nasal epithelia, renal epithelia, endothelia, neurons.

The COMET test for genotoxicityThe COMET test for genotoxicity

Metal oxide NPs induce DNA damage.The persistence of NPs in thehead 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 genotoxicityThe COMET test for genotoxicity

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

““in vitro” cell adhesion (Eukaryote)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

Model organisms used in Model organisms used in nanotoxicologynanotoxicology

Mammals: Rodents (mice, rat, Hamster), rabbit, swineFish: Danio rerioAmphibians: Xenopus laevisInvertebrate: C.elegans

Nanoparticles and liver toxicity Nanoparticles and liver toxicity in ratsin 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 ).

A comprehensive approach: A comprehensive approach: metabonomics. metabonomics.

Modified from: Duarte, 2011. Journal of Controlled Release 153: 34–39

Genes

Proteins

Metabolites

Genomics

Proteomics

Metabonomics

Metabonomics

Genotype Phenotype

Venoms

Physical agents

Chemical agents

Physical, chemical, biologicaL injuries

Why and when use Metabonomics?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.

Blue: signal in controlRed: signal after exposition

Organism or cells Sample: tissue, cell, blood or other biological fluids

NMR, SPR, GC, HPLC…

Modified from: Duarte, 2011. Journal of Controlled Release 153: 34–39

Effects of USPIO on rat liver: light Effects of USPIO on rat liver: light microscopy.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

Effects of USPIO on rat liver: Effects of USPIO on rat liver: Metabonomics, the Metabonomics, the 11H-NMR spectraH-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

Effects of USPIO on rat liver: Effects of USPIO on rat liver: Metabonomics, the PCAMetabonomics, 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