1

Bice FubiniBice FubiniDip. di Chimica I.F.M. e Centro Dip. di Chimica I.F.M. e Centro InterdipartimentaleInterdipartimentale “G. Scansetti” per lo Studio “G. Scansetti” per lo Studio degli Amianti e degli degli Amianti e degli AltriAltri Particolati Particolati NociviNocivi, , Università di TorinoUniversità di Torino

Workshop MINERALI E SALUTERoma: 14-15 giugno 2007

Dalla silicosi alle nanoparticelle: danno causatoda micro e nanopolveri minerali.

Dalla silicosi alle nanoparticelle: danno causatoda micro e nanopolveri minerali.

2

La silicosi, la più antica malattia professionale, causata dall’esposizione ad alcune forme di silice ancora non vede una spiegazione completa a livello molecolare

Molte altre polveri minerali risultano patogene, ma non è ancora chiaro cosa impartisca tossicità ad un particolato

… intanto cresce l’allarme per un nuovo rischio: le nanoparticelle prodotte dalle nanotecnologie

3

health effects of silica health effects of silica

silicosischronic

acuteone of the most ancient occupational disease

lung cancer International Agency for Research on Cancer (IARC), 1997, still controversial

autoimmune diseasesrheumatoid arthritis, scleroderma, systemic sclerosis, lupus, chronic renal disease

recently reported

4

Only some silica particles, when inhaled, are pathogenic

What makes a silica particle toxic?

How to predict toxicity of different sources of silica dusts?

open questions

Does variability concern in similar ways fibrogenicity, carcinogenicity and abnomal immune responses?

5

silica carcinogenicitysilica carcinogenicity

“ crystalline silica inhaled in the form of quartz or cristobalite from occupational sources is carcinogenic to humans”

International Agency for Research on Cancer (IARC)monograph, 1997

6

silica carcinogenicity - IARCsilica carcinogenicity - IARC

crystalline silica does not act as a carcinogen in all circumstances

“ Carcinogenicity in humans was not detected in all industrial circumstances studied. Carcinogenicity may be dependent on inherent characteristics of the crystalline silica or on external factors affecting its biological activity or distribution of its polymorphs”

...but, quite exceptionally for IARC, the statement was preceded by the following comment:

7

to chemistry the task of finding out which silica sources are pathogenic…

particle dimensions

particle micromorphology

state of the surface: freshly fractured, hydrophilic…

determined by the origin of the silica dusts

inherent characteristics?

external factors? ???metal ions (e.g. Al, Fe) contaminants,

associated minerals (clay)

impurities acquired during processing

deposited macromolecules, polymers, etc

great variability

8

inhaled silicas: accepted mechanisms of action

clearance

damage to target cells

direct action on target cells

release of oxidants,cytokines,growth factors recruitment of AM and PMN

macrophage activation

cell death

clearance

reactions withendogenousmolecules

9

clearance

successful

unsuccessful macrophage activationPMN recruitment:release of cytokines, growth factors and oxidants (ROI, RNI)

cell death

autoimmune diseases

the peculiar role of macrophages

fibroblast stimulation

fibrosis

damage to target cells

lung cancer

1 0

Inhaled silicas: primary vs secondary genotoxicity

clearance

damage to target cells

clearance

PRIMARY GENOTOXICITY

release of oxidants,cytokines,growth factors recruitment of AM and PMN

macrophage activation

cell death SECONDARY GENOTOXICITY

1 1

Outline silicaOutline silica

• the physico –chemical basis of the variability of silica pathogenicity

• from different polymorphs to different sources of the same polymorph

• association of various physico-chemical features to each of the steps of the pathogenic mechanisms proposed

1 2

The physico –chemical basis of the variability of silica pathogenicity

1 3

silica peculiaritiescausing the complexity of the mechanisms of toxicity

covalency and flexibility of the Si-O bond

large variety of SiO2 forms(in a metastable state both crystalline and amorphous)

the origin of the dust determines the state of the surface

poorly soluble solid: “particulate toxicant”

several chemical functionalities at the surface

1 4

silicas in nature: the mineralogical world

α−quartz β2-tridymite β-cristobalite fused silica570ºC 1470ºC 1723ºC

β−quartz

β1-tridymite

α-tridymite

α-cristobalite

silica glass

870ºC

163ºC

117ºC

200-275ºC

3.8x106 kPacoesite

13x106 kPastishoviteα−quartz

effect of pressure

effect of temperature

large variety of SiO2 forms:

1 5

silicas in nature: biogenic materials

large variety of SiO2 forms:

plantssponges

diatoms

Sponge spiculas

1 6

Industrial or laboratory production

large variety of SiO2 forms:

prepared by :

pyrolysis (pyrogenic silicas)precipitation from aqueous solutions (precipitated silicas)

porous structures: nano, micro, macromostly amorphous and biologically inert

It is commonly expected that any amorphous silica is non-toxic

1 7

comminution of crystalse.g. mining, grinding, milling

combustione.g. fly ashes

biogenice.g.mildly heated diatomaceous earth

the origin of the dust determines the state of the surface

Fresh surfaces, sharp edges

Heat cured, smooth particles

Irregular shapes, partially crystallized

1 8

quartz particles obtained by grinding

DQ12

SEM TEM

Electron microscopy

1 9

diatomaceous earths

Cyclotella stelligera

often the heated product retains memory of the original shape

2 0

isolated silanols,- SiOH geminal silanols -Si (OH)2H-bonded silanols

dissociated silanols SiO-

regular siloxane bridges Si-O-Sidistorted siloxane bridges Si---O-Si

peroxide bridges Si-O-O-SiSi . radical (E' center) SiO . radicalSi+ surface charges SiO - surface charges SiO2

. peroxyradical Si+O2

- . superoxide radical

partially hydrated surfaces

aqueous suspensions

heated particles

mechanical grinding

several chemical functionalities at the surface

2 1

role of contaminants

particulate toxicant

covalency of the Si-O bond

surface associated metal ions: a new chemical entity

2+

OSi Si

SiO

O

HOO-

O

-O

OSi

O O

O-Si

OSiO

O

O-O-

Si

FeO-

O

O

SiOOO

Si OO

O

O

Mn+

catalytic reactions

1% in weight, spread at the surface, mayfully cover it

2 2

All theories taking into account just one chemical feature failed…

not a single physico chemical property but a set of features impart pathogenicity to a given dust

several physico-chemical properties involved

multiple cellular responses and signalling pathways activated

intensity of the adverse reaction

2 3

physico-chemical properties which modulate silica pathogenicity

surface coatingsshape and dimension of particlesmechanical origin of the dust, dangling bondparticle derived free radical release silanol patches and hydrophilicity/hydrophobicityH-bondingsurface chargespresence of metal ions as aluminium or iron

2 4

active sites generated on crystalline silica by grinding

homolytic rupture of silica frameworko

sisi si si

o

dangling bonds

surface charges

o

sisi

heterolytic rupture of silica framework

si si

o+

-

2 5

EPR spectrum of surface dangling bonds and ROS

the EPR spectrum is the superposition of lines from surface peroxides and superoxides and dangling bonds

2 6

free radical generation

Active site

H2O2, R-H ROS, Ri

O2i-

H2O2

HOi

superoxide anion

hydrogen peroxide

hydroxyl radical

ROS

2 7

free radicals generating sites on crystalline silica

iron ions trapped at surface charges

2+

OSi Si

SiO

O

HOO-

O

-O

OSi

O O

O-Si

OSiO

O

O-O-

Si

FeO-

O

O

SiOOO

Si OO

O

O

“surface ROS” (Reactive Oxygen Species) from dangling bonds

si si

o

si si

o+ O2

oo oo

2 8

ROS and free radicals generated by silica

HOiO2

-iH2O2

1O2

Ri

Shi et al., J Toxicol Environ Health, 25, 237-245, 1988Shi et al., Appl Occup.Environ Hyg 1138-1144, 1995

Giamello et al. Colloids and Surfaces 45,155-165,1989Fenoglio et al. Chem Res Toxicol 13,971-975, 2000

LipidsDNA

ProteinsROS

OH

1 nm

2 9

hydrophilicity/hydrophobicity

SiSiO

siloxanesilanols

Si

OH

Si

OH

heat

- H2O

Si

OH

Si

OH

Si

OH

Si

OH

Si

OH

Hydrophilic surfaceHydrophilic surfaceSi

OH

SiSiO

SiSiO

SiSiO

Hydrophobic surfaceHydrophobic surfaceMay be evaluated from the adsorption of water by means of calorimetry and/or IR spectroscopy

3 0

From different polymorphs to differentsources of the same polymorph

3 1

different polymorphse.g. quartz vs stishovite or amorphous forms

same polymorph, but different origine.g. cristobalite: ex mineral, ex heated quartz dust, ex diatoms

same polymorph, same origin, but different sourcee.g various industrial quartz dusts

variability among surface modified samples may even exceed variability among different polymorphs

e.g. variously heated, ground in different jars, aged vs freshly ground…same sample but variously modified

3 2

biogenic origin *(diatomaceous earth, rice husks)volcanic origin *(vitreous silica,)

natural

artificialporosils *chemically prepared silicas *(precipitated or pyrogenic)ground silica glass *

quartz *tridymite *cristobalite *coesite *stishovite *

toxicity: * established *debated *unknown * inert

crystalline amorphous

different polymorphs

3 3

three cristobalite dusts generated by different ways:

CRS-m obtained by grinding cristobalite crystals

CRS-q cristobalite obtained by heating a quartz dust at high temperature (> 1300°C)

CRS-d calcined diatomaceous earth, crystallized into cristobalite

same polymorph, but different origin

3 4

heat of interaction with water

Adsorption of water vapour

CRS-m 58 kJ/mol

CRS-q 25 kJ/molCRS-d 45 kJ/mol

remarkable differences in hydrophilicity: CRS-q hydrophobic

same polymorph, but different origin

isotherms

three cristobalite dusts generated by different ways: hydrophilicity/hydrophobicity

3 5

same polymorph, but different origin

CRS-d

CRS-m

CRS-q

CRS-q

CRS-d

CRS-d

CRS-d

CRS-m

colony forming efficiency

transformation frequency %

Elias et al.,Toxicology in vitro, 2000,14, 409-422

remarkable differences in cytotoxity and morphologicaltransformation of cells(SHE): CRS-q inert

three cristobalite dusts generated by differentways: cytotoxicity and transforming potency

3 6

CRS-q cristobalite obtained by heating a quartz dust at high temperature (> 1300°C)

Inert !

Once taken at high temperature crystalline silicaparticles loose their pathogenic potential?

same polymorph, but different origin

3 7

water adsorbed as a functionof the thermal treatment

Si

OH

Si

OH

Si

O

Si + H2O

H2O+Si

OH

Si

OH

Si

OH

HO

HH

O

Si

300 600 900 1200 15000

T/°C40

2

4

6

8

10a

-2n a

/µm

ol m

-2

hydrogen bonding

adsorption of water

decrease in hydrophilicityupon heating Fubini et al. Chem. Res.Toxicol.

12, 737-745, 1999

same sample but variously modified

Effect of heating a cristobalitedust at increasing temperatures

T

3 8

µg/well

% to

tal L

DH

rele

ase

0 25 50 75 100 1250

25

50

75cristobalite 500cristobalite 800

cristobalite 1300**

****

cytotoxicity of variously heatedcristobalite on mouse alveolarmacrophages, measured by the extent of LDH release.

CRIS 500

CRIS 800

CRIS 1300

Fubini et al. Chem.Res.Toxicol.12, 737-745, 1999

A simlar result was obtained on the proliferation of a macrophage-cell line J774 and on the colony forming efficiency of rat alveolar epithelial cells AE6

same sample but variously modified

3 9

thermal inactivation of silica

a cristobalite dust generated or treated at a temperature at which becomes fully hydrophobic is non-cytotoxic

thermal treatments modulate the pathogenicity of silica dusts

open questionswhat is the hazard of quartz in fly ashes? how persistent is the inactivation?

4 0

same sample but variously modified

Effect of grinding: role of the material of the grinding chambers

quartz ground in a ball mill: widia / tungsten carbideagate / amorphous silicasteel / iron based alloycorundum / alumina

Fenoglio, et al.. Material Synthesis and Processing 8, 145-154, (2001).

4 1

mechanochemical activation/depression of radical yield:steel activates, corundum inhibits

kinetics of free radical (OHi) release

0 10 20 30 40 50 600

5

10

15

20

25

30

35

inte

nsity

(A

.U.)

time (minutes)

OHi from H2O2

same sample but variously modifiedEffect of grinding: role of the material of the grinding chambers

steel

agate

tungsten carbidecorundum

4 2

The variability in epidemiological data for lung cancer from different mining settings (IARC, 1997) may be related to mechanochemical reactions at the quartz surface, i.e. speciation of metal impurities acquired during mining procedures

outcome

Each mine should be tested for the toxicity of the dust

4 3

---+

-++-

+-

++++

++

++++

•ROS•Glucuronidase•TNF•Toxicity/viability

Qz 5/1-cQz 11/1-cQz 3/1-cQz 2/1-c

4 quartz samples selected on the basis of macrophage responses

same polymorph, same origin, but different source

4 4

In vivo testInflammatory, fibrogenic and genotoxic activity examined in a rat lung model.(intratracheal injection)

2.4 mg

0.6 mg

1.2 mg 90 days of incubation

pathogenic inert

--++8-OH-dG--+Mutation (90d)++++++Inflammation (90d)

(total cells, PMN)

+++++Fibrosis (90d)+++Proliferation (Ki-67)

Qz 11/1-cQz 3/1-cQz 2/1-c

+--++

Qz 5/1-c

The most active on macrophages the most pathogenic in vivo

4 5

Adsorption of water vapour on quartz dusts

Qz 2/1-c

Qz 3/1-c

Qz 5/1-c

Qz 11/1-c

Adsorption isotherms Interaction energy

two non-toxic dusts

Strong irreversible adsorptionon other than silica siteson other than silica sites

4 6

98.928396017.2103.049.768.1167.9600.22509Qz 11/1-c

99.0834307.626.034.980.290.0954.72993Qz 5/1-c

99.681135923.0133.087.639.2100.772.21110Qz 3/1-c

99.6793806.321.832.641.068.6112.91049Qz 2/1-c

SiO2%weigh

t

Cppm

Feppm

Mnppm

Nippm

Nappm

Mgppm

Cappm

Kppm

Alppm

quartz

Level of contaminants of the 4 samples

These amounts are sufficient, if dispersed, to cover all the surface

Aluminium is renown to inhibit quartz pathogenicity, quartz from clay orcoal mine is less pathogenic…

two non-toxic dusts

4 7

Even if a complex interplay exist between cell responses and physicochemical features, one single surface modification may inhibit several biochemical reactions generated by different surface sites

outcome

4 8

association of various physico-chemical features to each of the steps of the pathogenic mechanisms proposed

4 9

HO• radical generation and morphologicaltransformation in cells

depletion of the antioxidant defences in the lung lining layer

surface properties and cellular responses

5 0

B. Fubini et al. J. EnvironmentalPathology, Toxicologyand Oncology, 20 87-100, 2001

10

0,1

1

tras

form

atio

n fre

quen

cy (%

)

amount of radicals released (A .U.)

quartz

diatomaceous earth

Quartz dfx

quartz-HF

free radical release parallels cell transformation!

HO• radical generation and morphologicaltransformation in cells

5 1

surfactantsproteinsascorbate glutathion antioxidant defenses

Depletion of the antioxidant defences in the lung lining layer

Lung lining layer wheredeposition of the particlestakes place

5 2

selective depletion of ascorbate by quartz (comparison crystalline and amorphous)

ascorbate in solution is progressively consumed in the presence of quartz, much more than on amorphous silica

0 20 40 60 80 100 120 140 1600

1

2

3

4

5

6

amou

nt c

onsu

med

( µ

mol

/m2 )

time(hours)

quartz

amorphous silica

ascorbyl radical concentration decreases in the presence of quartz

H2O, 10’ H2O, 6 days

quartz suspension, 10’ quartz suspension, 6 days

Fenoglio I. et al.Chem. Res. Toxicol., 13, 971, (2000).

detection of ascorbyl radical in solution by EPR spectroscopy

5 3

control A 50 QRZs QRZg-a QRZg-s0

100

200

300

400

500

600

700Am

ount

of g

luta

thio

ne c

onsu

med

(nm

ol/g

)

amorphous silica

agedquartz

quartz groundsteel jar

control

GSH oxidation of by surface modified quartz

freshly ground quartz oxidizes more GSH

quartz groundagate jar

HO NHNH

OH

O

NH2

OSH

O

O

GSH

Fenoglio et al. Free Rad. Biol. Med 35 (7); 752-762, (2003).

5 4

ROI, RNI from AM, PMN + particle derived free radicalsSurfactant and protein adsorption;

depletion of antioxidants (ascorbic acid, glutathione) in the lung lining layer: surface radicals and Fe(III)

hydrophobicity, surface coating, aluminium ions

damage to target cells

clearanceclearance

surface properties and cellular responses

HO• generation and morphological transformation in cells

5 5

…a general alarm within the scientific community, on the possible health damage caused by the exposure to nano-particles, arising from the new nanotechnologies

… has rapidly attained the media

5 6

Nature, 424, 246, 2003

It started in 2003…

5 7

5 8

A new term/discipline has been established

Nanotoxicology

Many conferences and workshops, only devoted to thistopic, have been organized in different countries aimingto deal with the subject on sound chemical and biological basis

5 9

A new journal is launched

6 0

The number of published papers appears to increaseexponentially!!!

G.Oberdörster et al. 'Toxicology of nanoparticles: A historical perspective', Nanotoxicology, 1:1, 2 - 25

6 1

But the alarm is still going on…

Nano TiO2 in sunscreens

Nanoparticles in sun creams can stress brain cells

…tiny particles used in some sun creams have the potential to cause neurological damage, researchers in the United States have found

Nature, June 2006

6 2

Toxic to brain cells in culture

6 3

inhalednanoparticles may attain the brain!!!

reported, so far, for carbon, gold and manganese oxide particles

6 4

Titania powders have been used since more than 15 years…

Nano TiO2, however, may constitute a health hazard

…producers, aware of the photocatalyticproperties of titania, have proposed various coating procedures

6 5

The hazard is perceived as if any nanoparticle - irrespective of its crystallographic and chemical composition - will threaten our health

This is not the case

6 6

natural in the environment, e.g. gas-to-particle conversions, forest fires, volcanoes, biogenic magnetite

in most finely divided inorganic materials employed asheterogeneous catalysts since more than 50 years

e.g. Aerosil (silica); Alon C (alumina); P25 (titania) etc.

e.g. silica asbestos, urban PM

also as an ill-defined small fraction of several mineral dusts

we have been in contact with various kinds of nanoparticles in our daily life

6 7

quartz particles (DQ12)obtained by grinding

TEM

Several particlesbelow 100 nm

6 8

nanoparticles cover a large variety of chemical compositions

With very different outcomesone from the other !

therefore they cannot be consideredtoxic just because of their nano size

Few have been tested, so far, in cellular tests o in vivo

Carbon based new forms:

Oxides:

Metals:

fullerene, nanotubes

TiO2 , ZnO, Mn2O3

Au

6 9

NANOTOXICOLOGY 2007: International conference – VeniceNanoparticles investigated

ZnO

environmental pollution

SiO2

polystyrene

TiO2

carbon

gold

other < 5

A picture of the kind of materials under investigation

quantum dots

diesel

NiO

ZnONi

Co

Zr

PVC

CeO2Fe2O3

Cu

MgO

CoFe2O4

MnO

LiposomeCo3O4 WC WC-Co

Other < 5%

7 0

urgent requirement for multidisciplinary approach

Which physico-chemical features impart a peculiar toxicity to nano-sized particles

Which chemical compounds areor become toxic at the nanolevel

7 1

outlineoutline

What makes nanoparticles different from their largercounterparts ?

7 2

What makes nanoparticles so different from their largercounterparts ?

nano-size

high ratio of surface to bulk atoms

strong interparticle forces

specific properties at the nanolevel, hence reactivity

7 3

nano-size

7 4

nano-size

cellular responses differentfrom larger particles

1

7 5

cellular responses to micron sized inhaled particlesclearance clearance

adsorption of endogenousmolecules

cell death

macrophage activation

release of oxidants,cytokines,growth factors recruitment of AM and PMN

damage to target cells

direct action on target cells

7 6

alveolar macrophages epithelial/interstitial retention in the lung

(Oberdoster et al. Envir. Health Persp., 2005, 113, 823)

Smaller particlesretained in lung

Selective uptake of larger particles bymacrophages

Animal studies: deposition into the lung and retention

7 7

inhaled nanoparticles : different cellular responses?

damage to target cells

direct action on target cells

macrophage activation

clearanceclearance

release of oxidants,cytokines,growth factors recruitment of AM and PMN

cell death

endogenousmolecules are of similarsize:selectiveeffects on cellmembrane receptors

endocytosis and tissue penetration

7 8

nano-size

migration through differentbiological compartments

several target organs have to be considered

2

various penetration routes,beside inhalation e.g skin, blood stream

7 9

high ratio of surface to bulk atoms

8 0

high ratio of surface to bulk atoms

extremely high specific surfaces

1

8 1

Nel et al. Science, 2006, 311, 622-627

With NP huge increase in SSA and number of particles per unit weight or concentration upon decreasing particle size

Specific surface inversely related to diameterSSA ∝ 1/∅

Number of particles inversely related to … diameterN ∝ 1/∅3

N ∝ SSA3

8 2

Can we easily move from one to the other metric?

Relationships between weight, particle diameter, number of particles, surface area

500 nm

Nanosilica spheres prepared from water ammonia/alchoolsolution of TEOS (tetraethossiysilane) byhydrolysis and condensation

Renato Mortera e Barbara Onida, Dipartimento di scienza dei materiali e d’ingegneria chimica, Politecnico di Torino.

8 3

Pyrogenic nanosilica Aerosil ®

SSA close to what would be for individual particles

Number of particles not !

Aggregated particles…

0.1 µm

8 4

Oberdoster et al. Envir. Health Persp. 2005, 113, 823

TiO2 Inflammatory cells in lung lavage

rats

mice

20 nm vs 250 nm

8 5

Oberdoster et al. Envir. Health Persp. 2005, 113, 823

TiO2

At equal mass the nanosized specimen was much more toxic, howevereffects were comparable when expressed at equal surface exposed

Inflammatory cells in lung lavage

rats

mice

20 nm vs 250 nm

8 6

**

**

0

50

100

150

200

250

F MF UF MUF DQ12

IL-8

(% c

ontro

l)

3 16 80 400 µg/cm2

TiO2Nano (UF)

(F) quartz

Effect of size and composition on inflammation as measured by IL-8 in human lung epithelial cells (A549)

Sing et al. TAAP, 2006 in press

(F)

F-TiO2 < UF TiO2 < Quartz

as a function of weighted dose

8 7

0

50

100

150

200

250

0 1 10 100 1000surface area/cell surface (cm2/cm2)

IL-8

(% c

ontr

ol)

FMFUFMUFDQ12nativemethyl

as a function of exposed surface

Quartz >> TiO2 TiO2 all same effect

quartz

TiO2

Effect of size and composition on inflammation as measured by IL-8 in human lung epithelial cells (A549)

Sing et al. TAAP, 2006 in press

8 8

high ratio of surface to bulk atoms

enhanced surface reactivity

some nano particles are much more reactivethan larger conterparts

2

8 9

Es. kinks and steps in a ionic oxide es. MgO: ions with 5-, 4- and 3-coordination i.e. highly uncoordinated

these are propersites of adsorptionand reaction

Mn+

free radical

active surface site

e.g.

in small particles, a larger fraction of atoms or ions are stronglycoordinatively unsaturated, hence their reactivity.

9 0

Fe2O3 fine vs ultrafine

micron-size (average 0.1 µm) nano-size average 3 nm)

Sustained radical yield fromnanoparticles

3 3 2 0 3 3 4 0 3 3 6 0 3 3 8 0 3 4 0 0 3 4 2 0 3 4 4 0

C a m p o ( G )

EPR spectra of the CO2- radicals released

3 3 0 0 3 3 2 0 3 3 4 0 3 3 6 0 3 3 8 0 3 4 0 0 3 4 2 0 3 4 4 0

C a m p o ( G )

No radicals from micron -size

Ceschino et al unpublished results

9 1

strong interparticle forces

9 2

strong interparticle forces

Agglomeration, aggregation, clumping

close to intermolecular forces ?

9 3

Carbon nanotubes in aqueous environment

Pulskamp et al. Toxicol. Letters 2007, 168, 58-74

Uptake CNTs (alveolar macrophages)

9 4

G.Oberdörster et al. 'Toxicology of nanoparticles: A historical perspective', Nanotoxicology, 1:1, 2 - 25

agglomeration & aggregation mechanisms

9 5

Does a gap exists between intermolecular forces of largemolecules in solids and interpaticle forces in nano-agglomerates ?

Graphite vs carbon nanotubes

9 6

300 nm

Chrysotile bundles and fibrils

9 7

Open questions related to interparticle forces

Do really nanoparticles act as single entities ? If yes, under which circumstances

Do agglomerate NPs disperse in body fluids

Does aggregation following agglomeration favor clearance?

How should cellular and animal testsbe conducted in order to mimic real human exposures?

How do we measure interparticle forces ?

How to be assured that anaggregate will stay as such in any biological compartment ?

How may we influence agglomeration and aggregation ? Coatings, hydrophobicity

9 8

is the DLVO theory applicable ?

9 9

specific properties at the nanolevel, hence reactivity

1 0 0

specific properties at the nanolevel, hence reactivity

nanoscale: a lenghtscale whereproperties diverge from the bulk

J.F.Banfield & A.Navrotsky, “Nanoparticles and the Environment” Reviews in Mineralogyand Geochemistry, vol 44, 2001 Mineral Soc

thus nanoparticles may exhibit differentphysico-chemicalnature from theirlarger conterparts

1 0 1

Energy distribution of 3s and 3p electronic states in an isolated Mg atom and larger and larger Mg clusters (adapted from Thomas et al. 2002).

M.Hochella and A.S.Madden, Elements, 1,199-203, 2005

1 0 2

Possible chemical approaches to the production of safe nano TiO2,

…producers, aware of the photocatalytic properties of titania, have proposed various coating procedures and research is still going on

Photocatalytic effects of nano-TiO2

1 0 3

Crystal features also appear to play a role

Sayes et al. Toxicological Sciences 92(1), 174–185, 2006

Cytotoxicity of various polymorphs of nano - TiO2

Pure anatase Pure rutile

1 0 4

Carbon based materials

Very different entities

Diesel exaust

Carbon nanotubes

Carbon wires and fibers

Carbon particles

Fullerenes

Different biological activitiesand health hazard

Nanodiamonds

1 0 5

Figure 2. The diverse formats of engineered nanomaterials.(a) C60 dried onto filter paper is a black powder (inset: molecular structure of C60). (b) Fullerenes are easily dissolved in nonpolar solvents, such as toluene, and form a purple solution (top layer). (c) With relatively mild chemical treatments, such as evaporation of the nonpolar phase, some C60 becomeswater stable in this yellow solution. Although chemical analysis shows the presence of C60, light scattering and electron microscopy confirm thatthe material is present as colloidal aggregates that contain between 100 and 1,000 fullerene molecules.

Fullerenes

1 0 6

Variously oxidized to increase hydrophilicity

Carbon frame + metal

Carbon nanotubes, a typical product of the new nanotechnologies

unpurifiedthe metal is still in the carbon frame

purifiedmetals present only in traces

fully purified

complete elimination of metals

great variabilityin toxicity

SWNT

MWNT

Single Wall Carbon Nanotubes

Multi Wall Carbon Nanotubes

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They share relevant properties with asbestos

Fibrous habit, needle like shape

nano + fiber effects?

High biopersistence

long term effects

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Toxicity to cells and to experimental animals is still controversial

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do carbon nanotubes generate free radicals ?

•MWCN do not generate oxygen or carbon centered free radicals detectable with the spin-trapping technique, not even after grinding.

3320 3340 3360 3380

Field (G)

generation of HO•

3320 3340 3360 3380

Field (G)

generation of CO2•

MWCN

MWCN ground

Positive control (quartz)

Fenoglio et al. Free Rad. Biol. Med, 2006, 40, 1227-1233

1 1 0

carbon nanotubes: potential to quench free radicals

MWCN show a scavenger activity towards oxygen-centred free radicals

effect is specific for MWNC (powders with similar surface area are not able to quench radicals)

Effect of MWCN compared to nanosized silica on HO•

3280 3300 3320 3340 3360 3380 3400 3420

after addition of MWCN

after addition of nanosized silica

starting solution

Field (G)

H2O2 HO•hv

Effect of MWCN, compared to SDA alone, on O2

•-

3320 3340 3360 3380

after addition of MWCN

after addition of SDS

starting solution

Field (G)

riboflavine O2-•hv

Fenoglio et al. Free Rad. Biol. Med, 2006, 40, 1227-1233

1 1 1

Thus, as far as surface reactivity is concerned, carbon nanotubes are very different from asbestos

1 1 2

ConclusionsConclusions