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 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 testing
General:
Composition, Concentration, Crystalline phase, Purity
Size:
Grain, Particle, Hydrodynamic size, 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
Needs of standard methods for
Nanoparticles
1. Stability, aggregation and dissolution rates of nanomaterials
2. Assessment of Product Degradation and Release of
Nanomaterials from Consumer Products
3. Nanomaterial Product Labelling
4. Toxicological screening, physical and chemical hazard
5. Risk Assessments on exposure and use
6. Safety standards for consumer of products
7. Reporting Toxicity of Nanomaterials in Consumer Products
8. Determining Exposure to Nanomaterials in Food
9. Life Cycle Analysis for Consumer Products Containing
Nanomaterials
Needs of standard methods for
Nanotubes
1. Inhalation testing
2. Toxicology testing
3. food exposure determination
4. cosmetics and other skin contact products
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
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.
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)
Citotoxicity: macrophage & C60
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
Cytotoxicity: human epidermal
keratinocytes of C60 functionalized
with aminoacids.
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: CNT
A
B
CNT interacts with the cytoskeleton (A) and reduce the
adhesivity of the cells to the substrate (B).
(http://www.coltgroup.com/colt-foundation/ )
Cyto- and genotoxicity tests
Test reference/Name
Effect
System
OECD 471
(Ames assay)
genotoxicity
Prokaryotes, bacterial
reverse mutation test
OECD 473
genotoxicity
“in vitro” chromosome
aberration test
OECD 474
genotoxicity
“in vivo” micronuclei test
COMET assay
genotoxicity
Isolated DNA
Dye exclusion,
MTT uptake test
viability
Eukaryotes, cell lines
Apotosis/autophagy programmed cell Eukaryotes, cell lines, model
genes
death
organisms
Genes expression in
embryos
embryotoxocity
Multicellular model
organisms
“in vitro” models.
ORGANISM
EFFECT
TEST NAME
1 Eukaryote, cell
Viability, apoptosis
Trypan blue dye exclusion,
MTT uptake test,
Apotosis genes
2 Eukaryote, cell
Lipid peroxidation
MDA determination assay
3 Eukaryote, cell
Substrate adhesion
4
Gene expression,
altered development
Eukaryote,
“in vitro”
developing
organs
Ex.: micro organ from cultured
nasal epithelium, embryonic
heart
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 (HL60); 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.
The COMET test for genotoxicity
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
Single Walled Carbon
NanoTubes (SWCNT)
induce DNA damage in
renal epithelial cells
(NRK-52E).
The viability of the cells is
reduced, and apoptosisassociated genes are
overexpressed.
Nam et al., 2011. Arch Pharm Res 34: 661-669
“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
nanotoxicology
Mammals: Rodents (mice, rat, Hamster), rabbit, swine
Fish: Danio rerio
Amphibians: Xenopus laevis
Invertebrate: C.elegans
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 ).
A comprehensive approach:
metabonomics.
Venoms
Physical,
chemical,
Physical agents
biologicaL injuries
Chemical agents
Genes
Genomics
Proteins
Proteomics
Metabolites
Metabonomics
Genotype Phenotype
Metabonomics
Modified from: Duarte, 2011. Journal of Controlled Release 153: 34–39
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.
Organism or cells
Sample: tissue, cell, blood
or other biological fluids
Blue: signal in control
Red: signal after exposition
NMR, SPR, GC, HPLC…
Modified from: Duarte, 2011. Journal of Controlled Release 153: 34–39
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
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
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