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Nanomaterials and Neurodegeneration.Lucia Migliore, Chiara Uboldi, Sebastiano Di Bucchianico, Fabio Coppedè

To cite this version:Lucia Migliore, Chiara Uboldi, Sebastiano Di Bucchianico, Fabio Coppedè. Nanomaterialsand Neurodegeneration.. Environmental and Molecular Mutagenesis, Wiley-Blackwell, 2015,�10.1002/em.21931�. �hal-02353800�

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Review

Nanomaterials andNeurodegeneration

Lucia Migliore,* Chiara Uboldi, Sebastiano Di Bucchianico, andFabio Coppede'

Medical Genetics Unit, Department of Translational Research andNew Technologies in Medicine and Surgery, University of Pisa, Via Roma,

55 - 56126 Pisa, Italy

The increasing application of nanotechnology invarious industrial, environmental, and humansettings raises questions surrounding the poten-tial adverse effects induced by nanosized mate-rials to human health, including the possibleneurotoxic and neuroinflammatory properties ofthose substances and their capability to induceneurodegeneration. In this review, a panel ofmetal oxide nanoparticles (NPs), namely tita-nium dioxide, silicon dioxide, zinc oxide, cop-per oxide, iron NPs, and carbon nanotubeshave been focused. An overview has been pro-vided of the in vitro and in vivo evidence ofadverse effects to the central nervous system.Research indicated that these nanomaterials(NMs) not only reach the brain, but also can

cause a certain degree of brain tissue damage,including cytotoxicity, genotoxicity, induction ofoxidative stress, and inflammation, all potentiallyinvolved in the onset and progression of neuro-degeneration. Surface chemistry of the NMsmay play an important role in their localizationand subsequent effects on the brain of rodents.In addition, NM shape differences may inducevarying degrees of neurotoxicity. However, oneof the potential biomedical applications of NMsis nanodevices for early diagnostic and noveltherapeutic approaches to counteract agerelated diseases. In this context, engineeredNMs were promising vehicles to carry diagnos-tic and therapeutic compounds across theblood–brain barrier, thereby representing very

Abbreviations: AChE, acetilcholinesterase; AD, Alzheimer’s disease; Ag

NPs, silver nanoparticles; AgNO3, silver nitrate; ALS, amyotrophic lat-

eral sclerosis; AMNPs, amorphous silica nanoparticles; APP, ß-amyloid

precursor protein; Aß1–42, amyloid-ß1–42 peptide; BACE1, beta-secre-

tase 1 gene; bax, BCL2-associated X protein; BBB, blood–brain barrier;

bcl-2, B-cell lymphoma 2 protein; casp8, caspase-8 gene; casp9, cas-

pase-9 gene; CBF, cerebral-blood flow; CNS, central nervous system;

CNT, carbon nanotubes; Comt, catechol-O-methyltransferase gene;

COX-2, Cyclooxygenase-2 gene; CuO NPs, copper oxide nanoparticles;

DA, dopamine; DAPI, 4’,6-diamidino-2-phenylindole; DMSA, dimercap-

tosuccinic acid; DNA, deoxyribonucleic acid; DOPAC, 3,4-dihydroxy-

phenylacetic acid; ERK, extracellular-signal-regulated kinase; Fe2O3

NPs, maghemite nanoparticles; Fe3O4 NPs, magnetite nanoparticles;

FITC, fluorescein isothiocyanate; GADD45, growth arrest and DNA

damage-inducible 45; GFAP, glial fibrillary acidic protein; Gpr37, G

protein-coupled receptor 37 gene; Gpx1, glutathione peroxidase 1 gene;

GSH, glutathione; GSH-PX, glutathione peroxidase; 5-HT, 5-hydroxy-

tryptamine; H2O2, hydrogen peroxide; HD, Huntington’s disease;

hGDNF, human glial cell-derived neurotrophic factor gene; HO-1, heme

oxygenase 1; HVA, homovanillic acid; IL-10, interleukin-10; IL-1ß,

interleukin-1 beta; IL-6, interleukin-6; iNOS, inducible nitric oxide syn-

thase; IONPs, iron oxide nanoparticles; LDH, lactate dehydrogenase;

JNK, c-Jun N-terminal kinases; Jun, transcriptional factor AP-1; Maoa,

monoamine oxidase A gene; MCP-1, monocyte chemoattractant protein-

1; MDA, malondialdehyde; MIP-1a, macrophage inflammatory protein 1alpha; MMP-9, matrix metallopeptidase 9; MNPs, magnetic nanopar-

ticles; MRI, magnetic resonance imaging; mRNA, messenger RNA;

MWCNT, multi-walled carbon nanotubes; MTT, 3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide; NE, norepinephrine; NF-jB,nuclear factor kappa-light-chain-enhancer of activated B cells; NMDAR,

N-methyl-d-aspartate receptors; NO, nitric oxide; NOS, nitric oxide spe-

cies; NPs, nanoparticles; Nrf-2, NF-E2-related factor 2; NSC, neural

stem cells; p21, cyclin-dependent kinase inhibitor 1; p53, tumor protein

p53; Park2, Parkinson’s disease (autosomal recessive, juvenile) 2 gene;

PD, Parkinson’s disease; PEG, polyethylene glycol; PGE2, prostaglandin

E2; PI, propidium iodide; PLGA, polylactic-co-glycolic acid; RNA, ribo-

nucleic acid; ROS, reactive oxigen species; SiO2 NPs, silicon dioxide

nanoparticles; SN, substantia nigra; Snca, synuclein gene; SOD1, super-

oxide dismutase-1 gene; SOD, superoxide dismutase; SPIONs, superpar-

amagnetic iron oxide nanoparticles; SWCNT, single-walled carbon

nanotubes; Th, tyrosine hydroxylase gene; TiO2 NPs, titanium dioxide

nanoparticles; TNF-a, tumor necrosis factor-alpha; TUNEL, terminaldeoxynucleotidyl transferase dUTP nick end labeling; Txnrd1, thiore-

doxin reductase 1 gene; USPIONs, ultrasmall superparamagnetic iron

oxide nanoparticles; ZnO NPs, zinc oxide nanoparticles.

The authors declare no conflicts of interests.

Grant sponsor: European Commission’s Seventh Framework Programme

(FP7/2007–2013); Grant number: No 280716 (Sanowork [www.sanowork.eu]).

*Correspondence to: Lucia Migliore; Department of Translational

Research and New Technologies in Medicine and Surgery, University of

Pisa, Medical School, Via Roma 55, 56126 Pisa, Italy.

E-mail: [email protected]

Received 14 August 2014; provisionally accepted 7 November 2014;

and in final form 13 November 2014

DOI 10.1002/em.21931

Published online 00 Month 2014 in

Wiley Online Library (wileyonlinelibrary.com).

VC 2014Wiley Periodicals, Inc.

Environmental andMolecular Mutagenesis 00:00^00 (2014)

timely and attractive theranostic tools in neuro-degenerative diseases. Therefore, a carefulassessment of the risk–benefit ratio must be

taken into consideration in using nanosizedmaterials. Environ. Mol. Mutagen. 00:000–000,2014. VC 2014 Wiley Periodicals, Inc.

Key words: nanomaterials; nanoparticles; neurotoxicity; oxidative stress; neurodegenerative diseases;nanodevices

INTRODUCTION

Nanomaterials are small molecules with distinct biolog-

ical activity that have been progressively and increasingly

applied in various industrial and medical settings over the

last 30 years [Robertson et al., 2010; Schr€ofel et al.,2014; ShannahAn et al., 2012]. However, despite great

progress in nanotechnologies, comparatively little is

known to date on the negative effects that exposure to

NMs may have on the human brain, including the poten-

tial induction of pathways leading to neurodegeration

[Cupaioli et al., 2014]. Although many NMs exhibit

potential benefits for diagnostic and therapeutic purposes,

some of these molecules can exert unfavorable effects,

suggesting that the beneficial and harmful effects should

be compared prior to their application to humans [Iqbal

et al., 2013]. Indeed, NMs can enter the human body

through several routes, including absorption through the

skin or the digestive tract, inhalation, and blood injection.

NMs may cross the blood–brain barrier (BBB) to reach

the central nervous system (CNS), where they have been

suspected to impair several molecular pathways and con-

tribute to neurodegeration [Iqbal et al., 2013; Cupaioli

et al., 2014].

Neurodegenerative diseases are a heterogeneous

group of either hereditary or sporadic conditions all

characterized by progressive nervous system dysfunc-

tion resulting from the degeneration of selected neurons

in the CNS. Some of the most well known neurodege-

nerative diseases are Alzheimer’s disease (AD) and

other dementias, Parkinson’s disease (PD), amyotrophic

lateral sclerosis (ALS), and Huntington’s disease (HD)

[Migliore and Coppedè, 2009]. Despite the heterogene-

ous nature of neurodegenerative diseases, the applica-

tion of recent genome-wide and -omics approaches has

provided novel insights into the critical molecular path-

ways of those disorders, revealing that aggregation and

accumulation of misfolded proteins, mitochondrial dys-

function, oxidative stress, oxidatively damaged DNA

and impaired DNA repair, apoptosis, impaired

autophagy-lysosomal activities, inflammation and micro-

glia activation, perturbation of vesicle trafficking and

synapse dysfunction, RNA processing and protein deg-

radation pathways, as well as epigenetic deregulation of

gene expression, are common pathways in neurodege-

nerative diseases [Coppedè and Migliore, 2010; Gior-

dano et al., 2013; Golde et al., 2013; Ramanan and

Saykin, 2013; Vanderweyde et al., 2013; Amor et al.,

2014; B€aumer et al., 2014].

Recent evidence indicates that NPs can impair dopami-

nergic and serotoninergic systems, the former being rele-

vant for PD and the latter for AD pathogenesis, and can

cause changes of neuronal morphology and cell death. In

addition, NPs can also contribute to neurodegeneration by

inducing mitochondrial dysfunction, redox imbalance and

apoptosis, autophagy and impaired lysosomal activity,

cytoskeletal damage and vesicle trafficking perturbations,

neuroinflammation, and microglia activation [Iqbal et al.,

2013; Cupaioli et al., 2014]. Furthermore, in vitro evi-

dence suggests that engineered NMs are able to induce

changes in the expression of genes involved in DNA

methylation pathways, as well as global changes in epige-

netic marks such as DNA methylation and histone tail

modifications, all potentially involved in human complex

disorders, including neurodegeneration [Stoccoro et al.,

2013].

Conversely, since biodegradable NMs can be engi-

neered to load drugs, contrast agents, and cellular or

intracellular component targeting moieties, they have

emerged as potential alternatives for tracking and treating

human diseases, including neurodegenerative disorders

[Marrache et al., 2013]. Nanoparticulate drug carriers are

able to cross the BBB by virtue of their size, surface

potential, or surface coatings, and are currently under

investigation for effective delivery of pharmaceuticals or

contrast agents active in the treatment and detection of

AD and other neurodegenerative diseases [Garbayo et al.,

2013; Oesterling et al., 2014]. Indeed, during the past

decade, nanotechnology has been widely considered as a

promising tool for theranosis (diagnosis and therapy) of

neurodegenerative diseases [Amiri et al., 2013]. The aim

of this review is to critically discuss available in vitro

and in vivo data on the potential neurotoxic effects of

NMs in the context of neurodegeneration, with a focus on

the induction of cytotoxic and genotoxic effects, oxidative

stress, and inflammatory pathways. Moreover, we also

provide some examples of the potential beneficial uses of

NMs in the context of diagnosis and treatment of neuro-

degenerative diseases.

CYTOTOXIC,GENOTOXIC,OXIDATIVE, ANDINFLAMMATORY POTENTIALOFNMS: IMPLICATIONS FORNEURODEGENERATION

Due to their unique physico-chemical properties (i.e.,

small size, large surface area, composition, and function-

alization) several types of metallic NPs are able to cross

Environmental and Molecular Mutagenesis. DOI 10.1002/em

2 Migliore et al.

the BBB and interact with the CNS components. How-

ever, despite the large number of both in vitro and in

vivo investigations performed so far, the interactions

between NMs and the CNS are still not completely under-

stood and their toxic potential is still unclear. The major-

ity of the data available in the literature report that

metallic NPs induce toxic effects to the target cells or to

the exposed animals, and the toxicity is mainly triggered

via oxidative stress. The evidence that NMs induce cyto-

toxicity and genotoxicity, as well as oxidative stress and

inflammation in various cell lines representative of body

compartments such as the respiratory system, the intes-

tine, and the immune system, amplifies the need for com-

prehensive studies on the neurotoxicity and the

neurodegeneration induced by NMs engineered for the

screening, diagnosis, and therapy of CNS diseases. More-

over, the evidence that the CNS is a potential susceptible

target for nanosized materials and that NMs can penetrate

there through the olfactory bulb and deposit in the hippo-

campus [Oberd€orster et al., 2004; Wang et al., 2008] fur-ther emphasizes the need for studies on the potential

neuronal effects of NMs. Retention of particles in the

CNS, neurotoxicity, apoptosis, and oxidative stress, as

well as changes in gene expression and neuropathological

lesions are the most investigated parameters. The next

sections discuss the evidence available on the most

widely used NMs in industrial or biomedical applications,

and provide a summary of in vitro (Table I) and in vivo

(Table II) studies.

Titanium Dioxide Nanoparticles

Titanium dioxide nanoparticles (TiO2 NPs) are one of

the most frequently used NPs in industrial applications,

ranging from paints to ceramics and from food to cosmet-

ics. Therefore, investigation of risks associated with occu-

pational exposure to TiO2 NPs is of pivotal interest.

However, the neurotoxic potential of these NPs has only

recently been examined.

Among the first studies to demonstrate the cytotoxic

effects of TiO2 micro- and nanoparticles on human neural

cells (U87 astrocytoma cells) as well as in human fibro-

blasts (HFF-1 cells), were Lai et al. [2008]. Both TiO2microparticles (1–1.3 l particle size) and nanoparticles(

TABLEI.

Neu

roto

xic

Eff

ects

ofE

ngi

nee

red

Nan

opar

ticl

esIn

Vit

ro

Nan

opar

ticl

eC

ell

syst

emN

Pch

arac

teri

stic

sE

ffec

tsR

efer

ence

s

Tit

aniu

mdio

xid

eB

V-2

muri

ne

mic

rogli

alce

lls

Deg

uss

aP

25

(am

ixtu

reof

the

anat

ase

(70%

)an

d

ruti

le(3

0%

)fo

rms

of

TiO

2,<

100

nm

par

ticl

e

size

)

TiO

2N

Ps

induce

dra

pid

(<5

min

)an

dpro

longed

(2hr)

rele

ase

of

RO

S.

Inte

rfer

ence

wit

hth

em

itoch

ondri

alm

achin

ery

Long

etal

.,2006

U87

astr

ocy

tom

ace

lls.

HF

F-1

hum

anfi

bro

bla

sts

Anat

ase

TiO

2m

icro

par

ticl

es(1

–1.3

lpar

ticl

esi

ze)

or

nan

opar

ticl

es(<

25

nm

)

Dose

-rel

ated

cyto

toxic

ity

(apopto

sis,

nec

rosi

s,ap

opto

sis

and

nec

rosi

s-li

ke

cell

dea

th).

Both

TiO

2m

icro

par

ticl

esan

dnan

o-

par

ticl

es,

are

equal

lyef

fect

ive

inhum

anneu

ral

cell

san

d

fibro

bla

sts

Lai

etal

.,2008

PC

12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

Anat

ase

(20

nm

insi

ze)

and

ruti

le(2

0nm

insi

ze)

TiO

2-N

Ps.

and

mic

rom

eter

-TiO

2par

ticl

es(1

mm)

Conce

ntr

atio

n-d

epen

den

tcy

toto

xic

ity

and

mem

bra

ne

dam

age.

RO

S,

GS

H,

SO

D,

and

MD

Ain

crea

sed

pro

duct

ion.

Apopto

sis

and

nec

rosi

s;ce

llcy

cle

arre

sted

inG

2/M

phas

e.A

nat

ase

TiO

2

NP

sm

ore

toxic

than

ruti

le.

Mic

ron-s

ized

TiO

2did

not

exhib

it

any

toxic

resp

onse

Wu

etal

.,2010

C6

muri

ne

gli

alce

lls

and

U373

hum

angli

alce

lls

Anat

ase

(96%

)an

dru

tile

(4%

)fo

rms

of

TiO

2,

4–

200

nm

par

ticl

esi

ze

Dose

-rel

ated

cyto

toxic

ity.

Induct

ion

of

apopto

tic

even

ts.

Inte

rnal

izat

ion

on

TiO

2N

Ps

inm

embra

ne-

bound

ves

icle

s.

Morp

holo

gic

alch

anges

M� ar

quez

-Ram

� ırez

etal

.,2012

Pri

mar

yra

tm

icro

gli

alan

dP

C12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

TiO

2,

20

nm

par

ticl

esi

zeO

xid

ativ

est

ress

via

incr

ease

dN

Ore

leas

e.In

crea

sed

secr

etio

nof

TN

F-a

,IL

-1ß,

and

IL-6

.In

crea

sed

expre

ssio

nof

MC

P-1

and

MIP

-1a

Xue

etal

.,2012

SH

SY

5Y

hum

anneu

robla

stom

a

cell

s

Anat

ase

(100%

)an

da

mix

ture

of

anat

ase

(80%

)

and

ruti

le(2

0%

)fo

rms

(2%

nm

insi

ze)

TiO

2-

NP

s

No

cyto

toxic

ity

nor

morp

holo

gic

alal

tera

tions.

Tim

e-an

ddose

-

dep

enden

tin

tern

aliz

atio

n.

Pure

anat

ase

TiO

2N

Ps

alte

red

cell

cycl

ean

din

duce

dap

opto

sis/

nec

rosi

s.M

icro

nucl

eifo

rmat

ion

and

pri

mar

yoxid

ativ

eD

NA

dam

age

Val

dig

lesi

aset

al.,

2013

C6

muri

ne

gli

alan

dU

373

hum

angli

alce

lls

Anat

ase

(96%

)an

dru

tile

(4%

)fo

rms

of

TiO

2,

�50

nm

par

ticl

esi

ze

Cyto

toxic

ity.

Morp

holo

gic

alch

anges

.In

crea

sed

RO

S,

lipid

per

oxid

ase,

GS

H-P

X,

CA

T,

and

SO

D

Huer

ta-G

arc� ı

aet

al.,

2014

Sil

icon

dio

xid

eP

rim

ary

rat

mic

rogli

alce

lls

Am

orp

hous

SiN

Ps

embed

ded

wit

ha

fluore

scen

t

dan

syla

mid

edye,

spher

ical

insh

ape

and

about

150–200

nm

india

met

er

No

cyto

toxic

ity.

SiO

2N

Ps

inte

rnal

izat

ion

inm

embra

ne-

bound

ves

icle

s.O

xid

ativ

est

ress

Choi

etal

.,2010

PC

12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

SiO

2N

Ps

of

dif

fere

nts

izes

(20

and

50

nm

)C

once

ntr

atio

n-d

epen

den

tcy

toto

xic

ity.

Dose

-rel

ated

GS

Hdep

le-

tion

and

enhan

ced

RO

Spro

duct

ion.

Cyto

pla

smic

accu

mula

tion

of

SiO

2N

Ps

agglo

mer

ates

.M

orp

holo

gic

alch

anges

Wan

get

al.,

2011a

Pri

mar

yra

tm

icro

gli

alce

lls

SiO

2N

Ps

(20

nm

india

met

er)

Enhan

ced

secr

etio

nof

TN

F-a

,IL

-1ß

and

IL-6

pro

-infl

amm

atory

cyto

kin

es

Xue

etal

.,2012

SK

-N-S

Hhum

anneu

robla

stom

a

and

N2a

mouse

neu

robla

stom

ace

lls

SiO

2N

Ps,

12.1

nm

mea

npar

ticl

essi

zeC

once

ntr

atio

n-d

epen

den

tcy

toto

xic

ity,

apopto

sis

and

RO

Sre

leas

e.

Morp

holo

gic

alch

anges

.S

iO2

NP

sdis

per

sed

inth

ecy

topla

sm

of

SK

-N-S

Hce

lls.

SiO

2N

Ps

store

din

trac

ellu

larl

yin

ves

icle

s

inN

2a

cell

s

Yan

get

al.,

2014


4 Migliore et al.

TABLEI.

(con

tin

ued

).

Nan

opar

ticl

eC

ell

syst

emN

Pch

arac

teri

stic

sE

ffec

tsR

efer

ence

s

Zin

coxid

eN

SC

mouse

Neu

ral

Ste

mC

ells

ZnO

NP

sw

ith

zinci

tecr

yst

alst

ruct

ure

,of

dif

fere

nt

size

(10,

30,

60,

and

200

nm

)

Conce

ntr

atio

n-d

epen

den

t,but

no

size

-dep

enden

tto

xic

effe

cts.

Alt

ered

morp

holo

gy

and

induct

ion

of

apopto

sis.

Sig

nifi

cant

rele

ase

of

Zn-i

ons

Den

get

al.,

2009

PC

12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

ZnO

NP

s,<

50

nm

par

ticl

esi

zeM

itoch

ondri

alim

pai

rmen

tan

dre

duce

dce

llvia

bil

ity.

Inte

rnal

izat

ion

of

ZnO

NP

sin

mem

bra

ne-

bound

ves

icle

s

Kao

etal

.,2012

RC

S96

rat

Sch

wan

nce

lls

ZnO

NP

sw

ith

dif

fere

nt

arch

itec

ture

:sp

her

ical

(35

nm

india

met

er);

mic

rosp

her

e(4

5nm

in

dia

met

er);

hex

ahed

ral,

pri

sm-l

ike

(�2.5

to6.0

lm

india

met

eran

d�

18.0

to60.0

lm

in

length

.);

flow

ers-

like

(�500

to600

nm

india

m-

eter

and

sever

alm

icro

ns

inle

ngth

)

Shap

e-an

dti

me-

dep

enden

tneu

roto

xic

ity.

Apopto

sis

induct

ion

and

G2/M

phas

ece

llcy

cle

arre

st.

Sig

nifi

cant

rele

ase

of

Zn-

ions

Yin

etal

.,2012

SH

SY

5Y

hum

anneu

robla

stom

aZ

nO

NP

s,100

nm

mea

npar

ticl

essi

zeC

yto

toxic

ity:

apopto

sis

and

cell

cycl

eal

tera

tions

and

gen

oto

xic

ity

(mic

ronucl

ei,

H2A

Xphosp

hory

lati

on

and

pri

mar

y

and

oxid

ativ

eD

NA

dam

age)

,in

aco

nce

ntr

atio

n-

and

tim

e-

dep

enden

tm

anner

Val

dig

lesi

aset

al.,

2013a

Copper

oxid

eP

C12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

Cu

NP

s,90

nm

par

ticl

esi

zeR

educe

dle

vel

sof

DA

,D

OP

AC

and

HV

A.

Dow

nre

gula

tion

of

Gpx

1gen

e.U

pre

gula

tion

of

Txn

rd1,

Snca

,an

dM

aoa

gen

es

Wan

get

al.,

2009

rBM

EC

rat

bra

inm

icro

ves

sel

endoth

elia

lce

lls

Cu

NP

s,40

and

60

nm

par

ticl

esi

zeS

ize-

rela

ted

cyto

toxic

ity.

Incr

ease

dle

vel

sof

PG

E2,

TN

F-a

and

IL-1

ß.

Enhan

ced

bar

rier

per

mea

bil

ity

Tri

ckle

ret

al.,

2012

Sil

ver

PC

12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

Ag

NP

s,15

nm

par

ticl

esi

zeU

pre

gula

tion

of

Gpx

1gen

e.N

ovar

iati

ons

of

the

gen

es

asso

ciat

edto

DA

met

aboli

sman

dP

arkin

son’s

pat

hogen

esis

Wan

get

al.,

2009

Rat

bra

inm

icro

ves

sel

endoth

elia

lce

lls

co-c

ult

ure

d

wit

has

trocy

tes

Ag

NP

sA

gN

Ps

cross

edth

eB

BB

and

accu

mula

ted

inen

doth

elia

lce

lls

Tan

get

al.,

2010

Pri

mar

yra

tbra

inm

icro

ves

sel

endoth

elia

lce

lls

Ag

NP

s,25,

40,

or

80

nm

par

ticl

esi

zeD

ose

-an

dsi

ze-d

epen

den

tA

gN

Ps

inte

rnal

izat

ion.

Cyto

toxic

ity

and

morp

holo

gic

alch

anges

wit

hm

onola

yer

per

fora

tions.

Siz

e-

and

tim

e-dep

enden

tin

crea

seof

TN

F-a

and

IL-1

ßle

vel

s.S

ize-

sele

ctiv

eim

pai

rmen

tof

the

bar

rier

per

mea

bil

ity

Tri

ckle

ret

al.,

2010

Rat

cort

ical

cell

sA

gN

Ps,

20

nm

par

ticl

esi

zeL

oss

of

cyto

skel

eton

stru

cture

and

F-a

ctin

and

ß-t

ubuli

ndeg

rada-

tion.

Inhib

itio

nof

neu

ronal

exte

nsi

on

and

cyto

toxic

ity

Xu

etal

.,2013

SH

SY

5Y

hum

anneu

robla

stom

a

and

D384

hum

anas

trocy

tom

a

cell

s

Ag

NP

s,20

nm

par

ticl

esi

zeD

ose

-rel

ated

impai

red

mit

och

ondri

alm

etab

oli

sman

dm

embra

ne

dam

age.

Dose

-rel

ated

inhib

itio

nof

colo

ny

form

ing

effi

cien

cy.

Sig

nifi

cant

rele

ase

of

Ag-i

ons

Cocc

ini

etal

.,2014

Pri

mar

yporc

ine

bra

in

mic

roves

sel

endoth

elia

lce

lls

Ag

NP

s,25,

40,

and

80

nm

par

ticl

esi

zeIn

crea

sed

PG

E2,

TN

F-a

and

IL-1

ßle

vel

s.B

BB

leak

age.

Enhan

ced

bar

rier

per

mea

bil

ity

Tri

ckle

ret

al.,

2014

CG

Cpri

mar

yra

tce

rebel

lar

gra

nule

cell

s

0.2

%poly

vin

ylp

yrr

oli

done

(PV

P)-

coat

edA

gN

Ps

<100

nm

Cal

cium

imbal

ance

.A

ctiv

atio

nof

NM

DA

R.

Oxid

ativ

est

ress

by

RO

Spro

duct

ion.

Impai

red

mit

och

ondri

alm

embra

ne

pote

nti

al

Zie

m� ın

ska

etal

.,

2014


Nanomaterials and Neurodegeneration 5

TABLEI.

(con

tin

ued

).

Nan

opar

ticl

eC

ell

syst

emN

Pch

arac

teri

stic

sE

ffec

tsR

efer

ence

s

Iron

oxid

eP

rim

ary

rat

cere

bel

lar

cort

ex

astr

ocy

tes

Nan

osi

zed

iron

oxid

esu

per

par

amag

net

icpar

ticl

es

(Fe 3

O4

orc

-Fe 2

O3).

Sev

ere

reduct

ion

of

cell

via

bil

ity

and

cell

adhes

ion.

No

dam

age

toth

ece

llm

embra

ne

inte

gri

ty

Au

etal

.,2007

PC

12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

Anio

nic

Fe 2

O3

nan

opar

ticl

esw

ith

surf

ace

coat

ing

(DM

SA

);nan

opar

ticl

edia

met

ers

bet

wee

n5

and

12

nm

.

Impai

red

cell

via

bil

ity.

Morp

holo

gic

alch

anges

Pis

anic

etal

.,2007

Pri

mar

yra

tas

trocy

te-r

ich

cult

ure

s

DM

SA

-coat

edF

eN

Ps,

60

nm

aver

age

dia

met

erT

emper

ature

-dep

enden

tIO

NP

supta

ke

Gep

per

tet

al.,

2009

OL

N-9

3sp

onta

neo

usl

y

tran

sform

edpri

mar

yra

tbra

in

oli

goden

dro

gli

alce

lls

Cit

rate

d-c

oat

edF

eN

Ps

Cel

lula

rF

e-co

nte

nt

signifi

cantl

yin

crea

sed

ina

conce

ntr

atio

n

dep

enden

tm

anner

.IO

NP

sst

ore

din

mem

bra

ne-

bound

per

inu-

clea

rves

icle

s

Hohnholt

etal

.,2010

Pri

mar

yra

tas

trocy

te-r

ich

cult

ure

s

DM

SA

-coat

edF

eN

P,

60

nm

aver

age

dia

met

erT

ime-

and

dose

-dep

enden

tIO

NP

sin

tern

aliz

atio

nG

epper

tet

al.,

2011

Pri

mar

yra

tas

trocy

te-r

ich

cult

ure

s

DM

SA

-coat

edF

eN

P,

60

nm

aver

age

dia

met

erIO

NP

sst

ore

din

mem

bra

ne-

bound

per

inucl

ear

ves

icle

s.F

e-io

ns

seques

tere

dby

pro

tein

sto

pro

tect

cell

sfr

om

cyto

toxic

effe

cts

Gep

per

tet

al.,

2012

Pri

mar

yra

tas

trocy

te-r

ich

cult

ure

s

DM

SA

-coat

edF

e-N

P,

60

nm

aver

age

dia

met

erT

ime-

,co

nce

ntr

atio

n-

and

tem

per

ature

-dep

enden

tupta

ke

of

ION

Ps

by

endocy

toti

cpro

cess

es

Hohnholt

etal

.,2012

Pri

mar

yra

tas

trocy

te-r

ich

cult

ure

s

DM

SA

-coat

edF

eN

Ps,

60

nm

aver

age

dia

met

erT

ime-

,co

nce

ntr

atio

n-

and

tem

per

ature

-dep

enden

tupta

ke

of

ION

Ps.

ION

Ps

accu

mula

tion

enhan

ced

by

the

pre

sence

of

a

mag

net

icfi

eld

Lam

kow

sky

etal

.,

2012

Pri

mar

yra

tce

rebel

lar

cort

ex

astr

ocy

tes

Fe 3

O4

NP

sco

ated

wit

hpoly

ethyle

nei

min

ean

d

tagged

wit

hrh

odam

ine

Bis

oth

iocy

anat

e(F

e 3O

4-

PE

I-R

ITC

),m

ean

core

size

24.3

65.7

nm

No

cyto

toxic

effe

cts.

Rap

idan

dex

tensi

ve

ION

Ps

upta

ke

Yiu

etal

.,2012

Pri

mar

yra

tm

icro

gli

alce

lls

Fe 3

O4,

45

nm

aver

age

Incr

ease

dex

pre

ssio

nle

vel

sof

TN

F-a

,IL

-1ß

and

IL-6

.N

o

chan

ges

inN

O,

MC

P-1

and

NF

-jB

pro

duct

ion

Xue

etal

.,2012

Car

bon

nan

otu

bes

Pri

mar

ych

icken

embry

os

mix

ed

neu

ronal

and

gli

alce

lls

SW

CN

T-a

gglo

mer

ates

(SW

CN

T-a

),w

ith

a

dia

met

erof

appro

xim

atel

y100

nm

SW

CN

T-

bundle

s(S

WC

NT

-b),

wit

ha

dia

met

erof

appro

x-

imat

ely

20

nm

.

Agglo

mer

ated

SW

CN

Tin

duce

dcy

toto

xic

ity

Bel

yan

skay

aet

al.,

2009

PC

12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

Long

single

-wal

led

carb

on

nan

otu

bes

(LS

WC

NT

:

Oute

rD

iam

eter

1–2

nm

,L

ength

20mm

),sh

ort

single

-wal

led

carb

on

nan

otu

bes

(SS

WC

NT

:O

D

1–2

nm

,L

ength

:0.5

–2mm

).

SW

CN

Tin

duce

dti

me-

and

conce

ntr

atio

n-r

elat

edcy

toto

xic

ity.

Dec

reas

edm

itoch

ondri

alm

embra

ne

pote

nti

al.

Oxid

ativ

est

ress

via

RO

San

dli

pid

per

oxid

atio

nin

crea

se.

Tim

e-an

ddose

-

dep

enden

tdec

reas

edS

OD

,G

SH

-Px,

CA

T,

and

GS

H.

Cel

l

cycl

ear

rest

edin

G2/M

phas

e.D

ose

-dep

enden

tap

opto

tic

rate

Wan

get

al.,

2011b

PC

12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

SW

CN

Ts,

OD

1–2

nm

,le

ngth

20mm

Tim

e-an

ddose

-dep

enden

tap

opto

tic

cell

dea

th.

Form

atio

nof

RO

S.

Dec

reas

edle

vel

sof

lipid

per

oxid

e.In

crea

sed

level

sof

GS

H,

SO

D,

GS

H-P

x,

and

CA

T.

Red

uce

dm

itoch

ondri

alm

em-

bra

ne

pote

nti

al.

Act

ivat

ion

of

casp

ase-

3.

Vit

amin

Epro

tect

s

cell

sfr

om

the

toxic

ity

induce

dby

SW

CN

T

Wan

get

al.,

2012

PC

12

rat

embry

onic

pheo

chro

mocy

tom

ace

lls

Fe-

low

and

Fe-

hig

hM

WC

NT

sbas

edon

thei

r

met

alim

puri

ties

,oute

rdia

met

er2–50

nm

,

length

50mm

Cyto

toxic

ity.

Cyto

skel

eton

dis

rupti

on.

Eff

ects

ampli

fied

by

the

pre

sence

of

met

alim

puri

ties

such

asir

on

Men

get

al.,

2013

Co-c

ult

ure

sof

pri

mar

y

embry

onic

rat

hip

poca

mpal

neu

rons

and

gli

alce

lls

Subst

rate

sco

nta

inin

gpoly

pyrr

ole

and/o

rS

WC

NT

sIm

pai

red

cell

via

bil

ity.

Neu

ropro

tect

ive

effe

cts

inth

epre

sence

of

poly

pyrr

ole

Her

n� an

dez

-Fer

rer

etal

.,2014


6 Migliore et al.

TABLEII.

Neu

roto

xic

Eff

ects

ofE

ngi

nee

red

Nan

opar

ticl

esIn

Viv

o

Nan

opar

ticl

eA

nim

alm

odel

Adm

inis

trat

ion

NP

char

acte

rist

ics

Eff

ects

Ref

eren

ces

Tit

aniu

m

dio

xid

e

CD

-1m

ice,

fem

ale

Intr

anas

alF

our

types

ruti

le-p

has

eT

iO2

NP

s(<

5mm

;<100

nm

;

hydro

phil

ican

dw

ith

sili

ca-

coat

edsu

rfac

e)

Hydro

phobic

ruti

leT

iO2

NP

sac

cum

ula

ted

inth

e

cere

bra

lco

rtex

and

stri

atum

and

cause

morp

holo

gic

alch

anges

toth

eneu

rons.

Hydro

phil

ic

ruti

leT

iO2

NP

sw

ere

not

inte

rnal

ized

inth

ebra

in

but

induce

dre

duce

dN

Ele

vel

sin

hip

poca

mpus,

cere

bra

lco

rtex

,ce

rebel

lum

and

stri

atum

Zhan

get

al.,

2011

Sw

iss

albin

om

ice,

mal

eO

ral

Mix

ture

of

ruti

lean

dan

atas

e

TiO

2N

Ps

(<75

nm

)

Enhan

ced

DA

and

NE

level

s.O

xid

ativ

est

ress

wit

h

incr

ease

dR

OS

Shri

vas

tava

etal

.,

2014

Wis

tar

rats

,pre

gnan

tIn

trag

astr

icA

nat

ase

(10

nm

)R

educt

ion

of

cell

pro

life

rati

on

inth

ehip

poca

mpus

and

impai

red

lear

nin

gan

dm

emory

inoff

spri

ng

Moham

mad

ipour

etal

.,2014

Sil

icon

dio

xid

e

SD

rats

Intr

anas

alS

iO2

NP

s(1

5nm

)In

tern

aliz

atio

nof

SiO

2N

Ps

inth

ebra

in.

Oxid

ativ

e

stre

ss.

Infl

amm

atio

n

Wu

etal

.,2011

SD

rats

,m

ale

Intr

aper

ionea

lin

ject

ion

SiO

2N

Ps

(50–60

nm

)B

BB

dis

rupti

on.

Neu

ronal

dam

age.

Beh

avio

ral

impai

rmen

t

Shar

ma

etal

.,2013a

Zeb

rafi

sh(D

anio

reri

o)

Exposu

rein

wat

erS

iO2

NP

s(1

5-

and

50-n

m)

Siz

e-dep

enden

tbeh

avio

ral

chan

ges

.P

arkin

son’s

like

beh

avio

r

Li

etal

.,2014

Bal

b/c

mic

eIn

trap

erit

onea

lin

ject

ion

Mes

oporo

us

holl

ow

sili

ca

nan

opar

ticl

es(M

HS

Ns)

(110

nm

)

Siz

e-dep

enden

tm

igra

tion

thro

ugh

the

BB

Bof

PE

Gyla

ted

sili

ca.

Tim

e-re

late

dN

Ps

accu

mula

tion

in

the

bra

in

Liu

etal

.,2014

Zin

coxid

eS

Dra

ts,

mal

eIn

tran

asal

ZnO

NP

s(<

50

nm

)Z

nO

NP

str

ansl

oca

tion

inth

eolf

acto

rybulb

and

inth

e

bra

in

Kao

etal

.,2012

Sw

iss

mic

e,m

ale

Intr

aper

itonea

lin

ject

ion

ZnO

NP

s(2

0–80

nm

)Im

pai

rmen

tof

synap

tic

resp

onse

s.D

isru

pte

dsp

atia

l

mem

ory

Xie

etal

.,2012

SD

rats

,m

ale

Ora

lZ

nO

NP

s(4

0nm

)Z

nO

NP

str

ansl

oca

tion

inth

ebra

inC

ho

etal

.,2013

Copper

SD

rats

,m

ale

Intr

aper

itonea

lin

ject

ion

Cu

NP

s(5

0–60

nm

)N

euro

nal

alte

rati

ons.

Bra

indysf

unct

ion.

Cognit

ive

impai

rmen

t

Shar

ma

and

Shar

ma,

2007

SD

rats

,m

ale

Intr

aper

itonea

lin

ject

ion

Cu

NP

s(5

0–60

nm

)B

BB

dam

age.

Bra

ined

ema

form

atio

nS

har

ma

etal

.,2009

SD

rats

,m

ale

Intr

aper

itonea

lin

ject

ion

Cu

NP

s(5

0–60

nm

)N

euro

nal

cell

dam

age,

gli

alce

llac

tivat

ion,

loss

of

myel

inat

edfi

ber

s.B

rain

edem

afo

rmat

ion

Shar

ma

etal

.,2010

Wis

tar

rats

Intr

aper

itonea

lin

ject

ion

CuO

NP

s(1

0–70

nm

)O

xid

ativ

edam

age

inhip

poca

mpus.

Alt

ered

cognit

ive

funct

ions

(poor

per

form

ance

of

anim

als

in

beh

avio

ral

test

s)

An

etal

.,2012

Sil

ver

C57B

L/6

Nm

ice,

mal

eIn

trap

erit

onea

lin

ject

ion

Ag

NP

s(2

5nm

)A

lter

edex

pre

ssio

nof

oxid

ativ

est

ress

and

anti

oxid

ant

gen

es.

RO

Sen

han

cem

ent.

DN

Adam

age

Rah

man

etal

.,2009

SD

rats

,m

ale

Intr

aper

itonea

lin

ject

ion

Ag

NP

s(5

0–60

nm

)B

BB

leak

age.

Bra

ined

ema

form

atio

n.

Gli

al

acti

vat

ion.

Red

uce

dce

rebra

lblo

od

flow

.L

oss

of

myel

inat

edfi

ber

s

Shar

ma

etal

.,2009

ICR

mic

e,m

ale

and

fem

ale

Ora

lS

mal

l-si

zed

Ag

NP

s(2

2,

42,

and

71

nm

)an

dla

rge-

size

d

Ag

NP

s(3

23

nm

)

Siz

e-dep

enden

tA

gN

Ps

inte

rnal

izat

ion

(sm

all-

size

d

NP

sw

ere

dis

trib

ute

dto

the

org

ans

incl

udin

gbra

in,

lung,

liver

,kid

ney

,an

dte

stis

).In

crea

seof

TG

F-ß

level

sin

seru

mby

smal

l-si

zed

Ag

NP

s

Par

ket

al.,

2010

Wis

tar

Han

nover

Gal

as

rats

,fe

mal

e

Ora

lA

gN

Ps

(�14

nm

)A

gN

Ps

and

Ag-i

ons

upta

ke

inth

ebra

in.

Sig

nifi

cant

rele

ase

of

Ag-i

ons

Loes

chner

etal

.,

2011



TABLEII.

(con

tin

ued

).

Nan

opar

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eA

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odel

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inis

trat

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NP

char

acte

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Eff

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tar

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and

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ime-

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ted

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NP

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0nm

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NP

sbet

ter

inte

rnal

ized

than

200

nm

Ag

NP

s

Dzi

endzi

kow

ska

etal

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Jm

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mal

eIn

hal

atio

nA

gN

Ps

(25

nm

)T

ransl

oca

tion

of

Ag

NP

sin

toth

eolf

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rybulb

and

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ral

bra

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les

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ter

etal

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tar

rats

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mal

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ral

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NP

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sed

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and

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vel

s.S

ignifi

cant

rele

ase

of

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ons

Had

rup

etal

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SD

rats

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ale

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lA

gN

Ps<

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ated

and

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NP

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ticl

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lim

ited

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dis

solu

tion

van

der

Zan

de

etal

.,

2012

SD

rats

,m

ale

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aper

itonea

lin

ject

ion

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NP

s(5

0–60

nm

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ize-

rela

ted

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rupti

on.

Age-

rela

ted

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-

age.

Neu

ronal

NO

Supre

gula

tion.

Neu

ronal

impai

rmen

t

Shar

ma

etal

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net

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mal

ean

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mal

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trap

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onea

lin

ject

ion

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ized

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tfe

rrit

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ver

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ed.

[MN

Ps@

SiO

2(R

ITC

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ION

Ps

pas

sed

thro

ugh

the

BB

BK

imet

al.,

2006

ICR

mic

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ale

and

fem

ales

Inhal

atio

nF

luore

scen

tm

agnet

ic

nan

opar

ticl

es(5

0nm

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ION

Ps

pas

sed

thro

ugh

the

BB

BK

won

etal

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ICR

mic

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ale

and

fem

ales

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lF

e3O

4M

NP

s(�

20

nm

)IO

NP

spas

sed

thro

ugh

the

BB

BW

ang

etal

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SD

rats

,m

ale

Intr

aneu

ral

inje

ctio

nF

our

ION

Ps

of

dif

fere

nt

surf

ace

and

core

chem

istr

ies:

DM

SA

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O3,

DM

SA

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O4,

PE

G-

Fe 3

O4

and

PE

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u-F

e 3O

4,

roughly

spher

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at8–10

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rophag

es,

monocy

tes

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lym

phocy

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accu

mula

tion

atsi

tes

of

inje

ctio

n.

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ease

dle

vel

s

of

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K,

casp

ase-

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MM

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O-1

and

IL-1

ß

Kim

etal

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SD

rats

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ale

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atio

nF

e 3O

4N

Ps

(30

nm

)IO

NP

sdep

osi

ted

inolf

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riat

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and

hip

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Oxid

ativ

est

ress

via

upre

gula

tion

of

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dM

DA

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etal

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ICR

mic

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ale

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atio

na

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e2O

3N

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2011

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ula

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of

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Enhan

ced

mR

NA

level

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dju

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de

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vei

raet

al.,

2014

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bon

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aven

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ctio

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Huan

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al.,

2014


8 Migliore et al.

V-FITC and PI staining showed that apoptotic and

necrotic PC12 cells increased significantly with anatase

titania, but flow cytometry demonstrated that both crystal-

line forms were able to arrest the cell cycle in G2/M

phase. Western blot analysis confirmed that anatase TiO2NPs were more potent than rutile in activating apoptosis

and cell cycle checkpoint proteins: the expression of

JNK, p53, p21, GADD45, as well as bax and bcl-2 was

higher following exposure to anatase NPs than to the

rutile ones [Wu et al., 2010].

In contrast to the above, crystalline forms of TiO2 NPs

had no impact on cytotoxic effects in the human neuroblas-

toma SHSY5Y cell line [Valdiglesias et al., 2013b]. MTT

test and neutral red uptake showed that up to 24 hr exposure

to 0–150 mg/mL pure anatase and P25 (80:20 anatase:rutile)TiO2 NPs did not impair the viability of SHSY5Y. More-

over, no morphological alterations were observed and elec-

tron microscopy studies showed that TiO2 NPs were

internalized in a time- and concentration-dependent manner,

although pure anatase TiO2 NPs were slightly more effi-

ciently taken up than P25. Additionally, as previously

observed in PC12 cells [Wu et al., 2010], pure anatase TiO2NPs altered the SHSY5Y cell cycle and induced apoptotic

and necrotic events, while no effects were observed in cells

treated with P25. Interestingly, both types of TiO2 NPs

enhanced the formation of micronuclei and increased pri-

mary, but not oxidatively, damaged DNA observed with the

comet assay [Valdiglesias et al., 2013a, b].

Since TiO2 NPs induced in vitro neurodegeneration, in

vivo studies are critically important to further investigate

the toxic potential of these NPs. To this end, Zhang et al.

[2011a, b, c] focused their attention on the neurological

lesions in the brain of female CD-1 mice induced by

intranasally instilled TiO2 NPs of various size and surface

coating. The results indicated that surface properties play

a role in the neurodegenerative mechanisms of TiO2 NPs:

after 30 days exposure, hydrophobic TiO2 NPs accumu-

lated significantly in the cerebral cortex and in the stria-

tum, while microsized and nano-hydrophilic (silica-

coated) titania did not differ from the unexposed animals.

Moreover, hydrophilic TiO2 NPs caused morphological

changes of neurons in the cerebral cortex. There was also

a significant decrease in norepinephrine (NE) levels in the

hippocampus, cerebral cortex, cerebellum, and striatum

after hydrophilic TiO2 NPs instillation, whereas hydro-

phobic titania did not alter the monoamine neurotransmit-

ter levels in sub-brain regions [Zhang et al., 2011a, b, c].

Shrivastava et al. [2014] exposed Swiss albino mice for

21 days to a single oral dose of TiO2 NPs and observed

enhancement of DA and NE levels. They also detected

oxidative stress conditions with increased ROS and

reduced SOD production, suggesting that TiO2 NPs are

neurotoxic. It is, therefore, interesting to note that muta-

tions of superoxide dismutase 1 (SOD1) cause familialforms of ALS [Rosen et al., 1993].

Finally, an interesting study was recently performed in

pregnant Wistar rats that received intragastric TiO2 NPs

(100 mg/kg body weight) daily from gestational days

2–21. Exposure to TiO2-NPs significantly reduced cell

proliferation in the hippocampus, and impaired learning

and memory in offspring [Mohammadipour et al., 2014].

The TiO2 NPs assessed in the above in vitro and

in vivo experiments differed according to their crystalline

form (anatase or rutile), surface characteristics, or dose

used. Similarly, the endpoints measured (cell viability,

inflammation, oxidative stress markers, cytogenetic

effects) also differed greatly, complicating direct compari-

sons between studies. Nevertheless, both forms (anatase

and rutile) have been shown to be able to induce neuro-

toxicity at various levels, with the anatase form generally

more active than the rutile form. Taken together the

above findings indicate that single neurons, microglial

cells, and the whole CNS (including brain regions critical

for the onset of neurodegenerative diseases) are poten-

tially susceptible targets for TiO2 NPs.

Silicon Dioxide Nanoparticles

Another type of metal oxide used in industry and pro-

posed for drug and gene delivery is silicon dioxide nano-

particles (SiO2 NPs). The mechanism of toxicity of SiO2NPs is linked to the overproduction of ROS and to the

activation of pro-inflammatory responses [Liu and Sun,

2010; Park and Park, 2009]. For example, SiO2 NPs

stimulated the secretion of the pro-inflammatory cytokines

TNF-a, IL-1 ß, and IL-6 in freshly isolated rat microglialcells, but they were not able to stimulate the secretion of

NO, MIP-1a and MCP-1, or NF-jB, which are known tobe involved in the induction of inflammation-related

genes and in microglia activation [Xue et al., 2012].

Using primary microglial cells from Sprague-Dawley

pups, Choi and collaborators reported that SiO2 NPs were

stored intracellularly within phagocytic membrane-bound

vesicles, and that silica induced a significant release of

ROS and nitric oxidative species (NOS) accompanied by

an increased COX-2 gene expression, although the cellviability was not affected [Choi et al., 2010]. Moreover,

exposure of PC12 neuronal cells to SiO2 NPs revealed a

concentration-dependent decrease in cell viability, deple-

tion of GSH, and enhanced ROS production. In this

study, silica nanoparticles were internalized as agglomer-

ates in the cytoplasm and induced significant morphologi-

cal changes, resulting in cells that appeared small and

fragmented and that had reduced ability to grow neurites,

therefore impeding the development of intercellular con-

tacts and the formation of mature cells [Wang et al.,

2011a, b]. Exposure to low doses (10 lg/mL) of 15 nmSiO2 NPs similarly led to morphological alterations and

concentration-dependent cytotoxicity in human SK-N-SH

and mouse Neuro2a (N2a), two common neuroblastoma



cell lines. By electron microscopy, SK-N-SH cells were

shown to be able to internalize silica particles throughout

the cytoplasm, while the particles were found to be stored

within vesicles in N2a cultures [Yang et al., 2014]. In

addition, treatment of SK-N-SH and N2a cells caused

ROS release and significant dose-dependent apoptosis, as

shown by nuclear and TUNEL staining. Interestingly,

Yang et al. [2014] reported that SiO2 NPs (mean particles

size 12.1 nm) increased the deposition of intracellular ß-

amyloid peptide (Aß1–42) due to both the upregulation of

ß-amyloid precursor protein (APP) and the downregula-

tion of the amyloid-ß-degrading enzyme neprylysin, sug-

gesting a possible role for SiO2 NPs in the development

of AD. Indeed, according to the amyloid cascade hypoth-

esis of AD, changes in APP and/or Ab1–42 homeostasisfoster the assembly of Ab peptides into progressivelyhigher order structures, from dimers all the way up to the

insoluble plaques, which finally deposit in the brain; these

events are sufficient to initiate the pathological and clini-

cal changes of the disease [Hardy and Selkoe, 2002].

The use of SiO2 NPs in many applications and their

potential use for drug and gene delivery, makes it essen-

tial to conduct further studies on possible biological

effects. This is particularly important given that in vitro

studies have shown that SiO2 NPs are cytotoxic [Eom

and Choi, 2009; Akhtar et al., 2010], induce oxidative

stress [Napierska et al., 2009; Zhang et al., 2011a, b, c;

Ahmad et al., 2012; Ahamed, 2013] and inflammatory

responses [Panas et al., 2013; Kusaka et al., 2014; Men-

doza et al., 2014] in many cell types, including cells rep-

resentative of the CNS [Choi et al., 2010;Wang et al.,

2011a, b; Xue et al., 2012; Yang et al., 2014]. Further-

more, SiO2 NPs have been reported to induce inflamma-

tion [Lee et al., 2011; Morishige et al., 2012; Brown

et al., 2014], as well as pulmonary [Choi et al., 2008;

Zhao et al., 2014] and hepatic [Nishimori et al., 2009;

Liu et al., 2012] toxicity in vivo.

Wu et al. [2011] exposed SD rats to intranasal instilla-

tion of SiO2 NPs and observed significant brain accumu-

lation of nanoparticles, oxidative stress (increased H2O2and MDA and significant decrease in GSH), and

increased TNF-a and IL-1ß levels, indicative of inflam-mation. Interestingly, when a deeper analysis of the con-

tent of silica in the different brain regions was performed,

it was possible to establish a ranking of SiO2 NPs accu-

mulation that corresponded to olfactory bulb> stria-tum> hippocampus> brain stem> cerebellum> frontalcortex [Wu et al., 2011]. In Balb/c mice polyethylene

glycol-coated silica nanoparticles (PEG-SiO2 NPs)

crossed the BBB, showing size-dependent variation in

transport efficiency. At short exposure times (15 min), 50

and 100 nm PEG-SiO2 NPs were poorly able to migrate

through the BBB but their uptake significantly increased

after 60 min. Smaller (25 nm) PEG-SiO2 NPs, in contrast,

were already significantly taken up after a 15 min incuba-

tion, and their migration across the BBB was further

enhanced after 1 hr [Liu et al., 2014]. In addition to BBB

disruption and neuronal damage, SiO2 NPs have also

been associated with behavioral impairment in rats

[Sharma et al., 2013a]. Similarly, silica nanoparticles dis-

turbed the neural behavior of zebrafish Danio rerio in asize-dependent manner, as 15 nm SiO2 NPs significantly

changed the color preference of the animals and caused

PD-like behavior, neither of which were observed for

50 nm particles [Li et al., 2014].

Overall, both in vitro and in vivo data indicate that

SiO2 NPs can pass through the BBB. The increased pro-

duction of ROS and of pro-inflammatory responses, which

seem a common feature of SiO2 NPs, can adversely affect

different cell types. The major emerging finding is that

transport efficiency of SiO2 NPs across BBB was found

to be size-dependent, with increased particle size resulting

in decreased efficiency. Greater concern is therefore justi-

fied when considering neurotoxicity associated with small

sized silica nanoparticles in biomedical applications and

occupational exposure in large-scale production.

Zinc Oxide Nanoparticles

Zinc oxide nanoparticles (ZnO NPs) are of industrial

interest because of their exceptional optoelectronic, piezo-

electric, ferromagnetic and optical properties. Moreover,

ZnO NPs have been used in sunscreens, biosensors, food

additives, and pigments [Ji and Ye, 2008]. Due to their

antiseptic activity, they also have potential applications in

combatting bacteria-related infections and diseases.

Although some data on the toxic potential of ZnO NPs

are available in the current literature, little is known about

their neurotoxic effects. Deng et al. [2009] showed that in

neural stem cells (NSC), ZnO NPs whose nominal mean

size diameter ranged between 10 and 200 nm impaired

cell viability in a dose-dependent manner. In contrast,

size did not play a role in inducing toxic effects, as a

comparison of the differently sized nanoparticles did not

result in any significant difference in terms of cell viabil-

ity. Electron microscopy analysis and nuclear staining

were used to demonstrate that ZnO NPs induced apopto-

sis in NSC cells [Deng et al., 2009]. Nevertheless, the

authors speculated that the cytotoxicity and apoptosis

induced by ZnO NPs in NSC cells might result from the

zinc ions dissolved either in solution or intracellularly.

This hypothesis was supported also by the fact that the

internalized ZnO NPs were not detectable by electron

microscopy [Deng et al., 2009]. ZnO NPs neurotoxicity

was further evaluated in RSC96 rat Schwann cells com-

paring four different hierarchical structures: monodis-

persed spherical ZnO NPs of 35 nm size, hollow ZnO

microspheres (2.7 mm), prism- (ca., 2.5–6.0 mm in diame-ter and ca., 18–60 lm in length) and flower-like (500–600 nm in diameter and several microns in length)


10 Migliore et al.

structures [Yin et al., 2012]. Results demonstrated that

prism- and flower-like ZnO NPs did not induce cytotoxic

effects after 12 hr exposure, while significant impairment

of the viability of RSC96 cells was observed at 48 hr.

Similarly, spherical monodispersed ZnO NPs and zinc

microspheres exerted concentration- and time-dependent

cytotoxicity. Moreover, they significantly enhanced apo-

ptotic events, and G2/M cell cycle arrest was observed

when RSC96 cells were exposed for 12 hr to 80 lg/mLZnO nanoparticles and microspheres [Yin et al., 2012].

Interestingly, analysis of the levels of zinc ions performed

in culture media at increasing time points revealed that

the observed time-related ion levels enhancement was the

result of a leaching process occurring during the incuba-

tion period, which suggested that the cytotoxic effects

observed in RSC96 rat Schwann cells were also due to

the ionic fraction in the culture environment and not

exclusively to the nanoparticulated fraction [Yin et al.,

2012].

By confocal microscopy, Kao et al. [2012] observed

that ZnO NPs were internalized in membrane-bound

vesicles in PC12 neuronal cells and caused the reduction

of cell viability and mitochondrial impairment. An exten-

sive study on Zn NPs was performed in the human neuro-

blastoma SHSY5Y cell line, testing several concentrations

and exposure times and employing a battery of cytotoxic-

ity and genotoxicity assays. The internalization of the Zn

NPs was assessed by flow cytometry but it was not possi-

ble to demonstrate that ZnO NPs enter the neuronal cells.

However, a wide range of cytotoxic effects were induced

including apoptosis and cell cycle alterations, as well as

genotoxic effects (micronuclei, H2AX phosphorylation,

and primary and oxidatively damaged DNA), in a dose-

and time-dependent manner [Valdiglesias et al., 2013a].

In vivo, Kao et al. [2012] showed that ZnO NPs intra-

nasally administered to Sprague-Dawley rats (6 hr expo-

sure) translocated into the olfactory bulb and the

synaptosomes, as clearly shown by electron microscopy

micrographs. The translocation of ZnO NPs across the

BBB and into the CNS was further confirmed by Cho

et al. [2013]; after 13 weeks of repeated oral administra-

tion, enhanced ZnO NPs levels were measured in rats

brain compared with the untreated group, although the

uptake was not dose-related. Additionally, ZnO NPs were

reported to disrupt spatial memory and to significantly

impair the synaptic responses of Swiss male mice with

depressive-like behavior [Xie et al., 2012].

In many experimental systems in which Zn NPs were

able to induce neurotoxic effects, there was often no evi-

dence for NP uptake by the cells, and no effect of NP

size on cytotoxicity. Instead, a time-dependent increase of

Zn21 concentration in the culture media was sometimes

found, most likley due to decomposition of ZnO NPs and

the subsequent release of ions, as already reported. How-

ever, the question of whether the increase of intracellular

ions is due to the NPs being taken up by cells, or to NP

dissolution in medium, still remains to be resolved [Van-

debriel and De Jong, 2012].

Copper Oxide Nanoparticles

Copper, an essential trace element vital for the life and

the development of organisms, is known to be involved

in neurodegenerative disorders such as Menkes [Kodama

et al., 201], Wilson’s [Lorincz, 2010], AD, HD, and PD

[Desai and Kaler, 2008; Rivera-Mancia et al., 2010;

Greenough et al., 2013; Montes et al., 2014], acting

through the induction of oxidative stress [Halliwell and

Gutteridge, 1984] and the activation of microglial cells

and inflammation [Zhang et al., 2011b]. Although it is

important to understand how nanosized copper oxide

(CuO NPs) can induce neurotoxicity, to date few investi-

gations have been performed. Wang et al. [2009] investi-

gated expression changes in genes associated with the

dopaminergic system and their correlation with DA deple-

tion in PC12 cells. Treatment with CuO NPs significantly

reduced the content of DA, 3,4-dihydroxyphenylacetic

acid (DOPAC) and homovanillic acid (HVA) in PC12

cells, and induced downregulation in the expression of

the redox-status gene glutathione peroxidase 1 (Gpx1)and upregulation of thioredoxin reductase 1 gene

(Txnrd1). In addition, CuO NPs upregulated the expres-sion of the monoamine oxidase A (Maoa), which isrelated to DA metabolism, and of the alpha-synuclein

gene (Snca) associated with the pathogenesis of neurode-generation in PD [Wang et al., 2009]. Indeed, PD results

from loss of neuromelanin-containing dopaminergic neu-

rons in the substantia nigra (SN), with the presence ofeosinophilic, intracytoplasmic inclusions (termed Lewy

bodies) and containing aggregates of a-synuclein as wellas other substances. Furthermore, human SNCA mutationscause autosomal dominant PD, and SNCA polymorphismsor epigenetic changes of SNCA gene expression arebelieved to contribute to sporadic forms of the disease

[Thomas and Beal, 2011]. An additional study on rat

brain microvessel endothelial rBMECs cells showed that

low concentrations of 40 and 60 nm CuO NPs increased

the cellular proliferation while 50 mg/mL Cu-NPs werecytotoxic, and the extracellular concentration of the proin-

flammatory mediators Prostaglandin E2 (PGE2), TNF-a,and IL-1b were significantly increased [Trickler et al.,2012]. Moreover, Trickler et al. [2012] reported that the

enhanced permeability of rBMEC upon exposure to CuO

NPs suggests that the NPs can be neurotoxic and damage

the BBB even at low doses.

To better investigate their involvement in the etiology

of neurodegenerative disorders, CuO NPs were studied in

vivo. CuO NPs of approximately 50–60 nm mean diame-

ter were able to induce brain dysfunction in rats which,

after 7 days of exposure, exhibited mild cognitive



impairment and cellular alterations in the brain [Sharma

and Sharma, 2007]. Additionally, intraperitoneal, intrave-

nous, intracarotid or intracerebroventricular administration

of CuO-NPs significantly altered BBB function in several

regions of the brain and spinal cord 24 hr after adminis-

tration, and a marked decrease in local cerebral blood

flow (CBF) and severe brain edema was observed in brain

areas associated with BBB leakage [Sharma et al., 2009].

Moreover, Sharma et al. [2009] observed that the injured

brain areas exhibited neuronal cell damage, glial cell acti-

vation, heat shock protein upregulation and loss of

myelinated fibers, and that these changes were more evi-

dent in mice compared with rats. Furthermore, by means

of Evans blue leakage, it was possible to show that brain

edema formation took place in rats after intravenous,

intraperitoneal and intracerebral administration of CuO

NPs, and that the most severely damaged areas were the

ventral surface of brain and the proximal frontal cortex,

whereas the dorsal surfaces of cerebellum showed mild to

moderate damage [Sharma et al., 2010]. CuO NP treat-

ment also led to detrimental effects on the cognitive func-

tions of Wistar rats, highlighted by poor performance of

animals in behavioral tests. The occurrence of an imbal-

ance in oxidation–antioxidation homeostasis, and of neu-

ronal damage in the hippocampus, suggested the

induction of oxidative damage and neuronal apoptosis

[An et al., 2012]. Despite the scarcity of available studies,

mainly carried out in a few experimental centers, CuO

NPs seem neurotoxic both in vitro and in vivo. Of partic-

ular concern is the finding that nano-CuO can induce

brain dysfunctions and affect the abilities of learning and

memory in rodents.

Silver Nanoparticles

Due to its bactericidal properties and its role as an

imaging contrast agent, silver nanoparticles (Ag NPs) are

promising tools for biomedical applications. It is well

known that the CNS is sensitive to silver [Carpenter,

2001], and that Ag can be retained in the CNS for long

periods of time [Panyala et al., 2008] and induce neuronal

degeneration and BBB malfunction. The ability of Ag

NPs to translocate into the brain by crossing the BBB

was reported in 2010 by Tang et al. Using an in vitro co-

culture model composed of rat brain microvessel endothe-

lial cells and astrocytes, Ag NPs were observed to pass

the BBB by transcytosis and accumulate in endothelial

cells, as shown by electron microscopy [Tang et al.,

2010]. In freshly isolated rat brain microvessel endothelial

rBMEC cells, 25–40–80 nm Ag NPs accumulated in a

dose- and size-dependent manner, and at high concentra-

tions (25–50 mg/cm3) induced an impairment of the cellviability. Furthermore, size-related morphological changes

and formation of perforations in the monolayer were

observed in rBMECs [Trickler et al., 2010]. In a follow-

up study using confluent porcine brain microvessel endo-

thelial cells, Trickler et al. [2014] observed that 25–40–

80 nm Ag NPs induced pro-inflammatory responses by

enhancing the extracellular levels of PGE2, TNF-a andIL1ß, in addition to causing BBB leakage and signifi-

cantly higher permeability.

Loss of cytoskeleton structure with degradation of

beta-tubulin and F-actin was observed in primary rat cort-

ical cells exposed to Ag NPs (20 nm mean size diameter),

and phase contrast images showed that Ag NPs inhibited

neuronal extension, neuritic overlap, and impaired the

viability of the rat cortical cells [Xu et al., 2013]. Size-

and time-dependent TNF-a and IL-1ß secretion weredetected, while PGE2 was not released in the presence of

40 and 80 nm Ag NPs. In addition, Ag NPs selectively

affected the permeability of rBMECs: small Ag NPs

(25 nm) induced an increased permeability of fluorescein

across rBMECs, whilst 40 nm Ag NPs only slightly dam-

aged the integrity of the barrier, and 80 nm particles did

not exert any effect [Trickler et al., 2010]. In PC12 cells,

expression changes in genes associated with the dopami-

nergic system were analyzed following exposure to

15 nm Ag NPs: Gpx1 was the only upregulated gene,whereas there was no change in the expression of genes

related to DA metabolism (Th, Maoa, and Comt) or genes(Gpr37, Snca and Park2) associated with the pathogenesisof neurodegeneration in PD [Wang et al., 2009]. In

human-derived SHSY5Y neuroblastoma and D384 astro-

cytoma cells, exposure to 20 nm Ag NPs revealed that at

short exposure times (4–48 hr), Ag NPs induced dose-

and time-dependent impairment of mitochondrial metabo-

lism and cell membrane damage. Similarly, longer expo-

sures (10 days) of SHSY5Y and D384 cells treated with

increasing concentrations of Ag NPs showed dose-

dependent reduction in colony forming efficiency.

Since Ag NPs are known to release silver ions in solu-

tion, a comparison with AgNO3 was performed. Cytotox-

icity was more severe when SHSY5Y and D384 cells

were incubated in the presence of AgNO3 compared with

Ag NPs at both short (4–48 hr) and at long (10 days)

time points [Coccini et al., 2014]. Ziem�ınska et al. [2014]investigated the role of Ag NPs in the induction of exci-

totoxicity, a pathological process by which nerve cells are

damaged and killed by excessive stimulation of neuro-

transmitters such as glutamate. Excitotoxicity is linked to

alterations of intracellular calcium levels and deregulation

of intracellular calcium signaling pathways, leading to

ROS production, mitochondrial dysfunction, and ulti-

mately cell death. To this end, primary cultures of rat cer-

ebellar granule cells exposed to Ag NPs activated the

glutamatergic N-methyl-d-aspartate receptors (NMDAR)

and induced calcium imbalance, changes in mitochondrial

membrane potential and significant ROS production, thus

suggesting that Ag NPs have neurotoxic potential

[Ziem�ınska et al., 2014]. Interestingly, Ziem�ınska et al.


12 Migliore et al.

[2014] showed that the toxic effects exerted by Ag NPs

were attenuated in the presence of MK-801, a non-

competitive inhibitor of NMDAR.

In vivo studies have demonstrated that Ag NPs accu-

mulate in liver [Kim et al., 2008, 2010] and lungs [Sung

et al., 2009; Song et al., 2013], but Ag NPs are also able

to translocate into the CNS. In fact, 25 nm Ag NPs were

detected by autometallography in the olfactory bulb and

in the lateral brain ventricles of C57BL/6J mice [Genter

et al., 2012], and mass spectrometry showed size-related

internalization of Ag NPs in young ICR mice, with 22–

71 nm particles distributed into the brain, whereas

300 nm Ag NPs were not detected in the tissue after 14

days of oral administration [Park et al., 2010].

Ag NPs (50–60 nm) administered into systemic circula-

tion or brain ventricular spaces of rats and mice showed

severe BBB leakage, formation of brain edema and

decrease in local CBF, as well as glial activation and loss

of myelinated fibers [Sharma et al., 2009]. Size-dependent

BBB breakdown, NOS upregulation, neuronal damage,

and glial fibrillary acidic protein upregulation was

observed in inbred male Sprague-Dawley rats: small Ag

NPs (20–30 nm) induced more severe damage in young

(9–10 weeks old) and old (30–35 weeks old) rats com-

pared with mid-age (18–20 weeks) animals, and the effect

was significantly reduced in the presence of 50–60 and

130–150 nm Ag NPs [Sharma et al., 2013b]. The evi-

dence that very young and old rats showed the most

severe neurodegeneration led Sharma et al. [2013b] to

suggest that children and elderly might be more suscepti-

ble to Ag NPs-induced brain damage.

Altered expression of mouse oxidative stress and anti-

oxidant genes was observed in different regions of

C57BL/6N mice exposed by injection to Ag NPs, sug-

gesting that 25 nm Ag NPs are able to induce oxidative

stress and oxidatively damaged DNA, and could be

involved in

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