gold nanoparticles cellular toxicity

Nanotoxicology, March 2010; 4(1): 120137

Gold nanoparticles cellular toxicity and recovery: Effect of size,concentration and exposure time

TATSIANA MIRONAVA1, MICHAEL HADJIARGYROU2, MARCIA SIMON3,VLADIMIR JURUKOVSKI3, & MIRIAM H. RAFAILOVICH1

1Department of Materials Science and Engineering, 2Department of Biomedical Engineering, and 3Department of OralBiology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, New York, USA

(Received 19 May 2009; accepted 5 November 2009)

AbstractGold nanoparticles (AuNPs) are used in many applications; however, their interactions with cells and potential health risk(s) are not fully known. In this manuscript, we describe the interactions of AuNPs with human dermal broblasts andshow that they can penetrate the plasma membrane and accumulate in large vacuoles. We also demonstrate that the uptakeof the AuNPs is a function of time, their size and concentration. Specically, we demonstrate that 45 nm AuNPs penetratecells via clathrin-mediated endocytosis, while the smaller 13 nm enter mostly via phagocytosis. Furthermore, we provideevidence of cytoskeleton lament disruption as a result of AuNPs exposure and reconstitution during recovery (followingAuNP removal), despite no changes in actin or beta-tubulin protein levels. In contrast, the expression of the extracellularmatrix (ECM) proteins, collagen and bronectin, was diminished in the cells exposed to AuNPs. We also examined theproliferation rates of cells exposed to AuNPs and show that its diminution is a function of apoptosis and speculate thatapoptosis results from the number of vacuoles present in the cells, which is probably the main factor that disrupts thecytoskeleton causing cell area contraction and decreases in motility. Lastly, we also present data that indicates that AuNPsdamage to cells is not permanent and that the cells can completely recover as a function of AuNPs size, concentration andexposure time. Taken together, our data suggest that AuNPs exert detrimental effects on cell function that could reversefollowing AuNPs removal.

Keywords: Gold nanoparticles, human dermal broblast, endocytosis, apoptosis, recovery

Introduction

Nanotechnology is becoming an increasingly com-mon technique for the fabrication of products rangingfrom consumer items, and electronics to biomedicaldevices. Because of their large surface to volume ratio,nanometer-sized metallic and semiconductor parti-cles are central to many of these applications.Although, nanoparticles are currently and widelyused, their effects on cells are still under intenseinvestigation. Over the last decade, many reportsbrought forth the fact that such nanoparticles exhibitphysical properties that allow them to penetrateunusually deep into skin and other organs(Lademann et al. 1999; Kreilgaard 2002; Hostynek2003; Kato et al. 2003; Tinkle et al. 2003; Hoet et al.2004). A fundamental question exists, whether thetoxicity of these particles arises from known chemical

interactions, whose effectiveness is magnied by theincreased surface area or from new phenomena whicharise simply due to the decreased particle size.AuNPs play important roles in the fabrication of

nanowires, in nanomedicine, and nanoelectronics,where the tolerance for oxidative reactions is verylow (Chan 2007; Narayanan and El-Sayed2007; Huang et al. 2007a; Corma and Garcia 2008;Nanotech Full Report 2008). Due to the increasingdemand, numerous commercial suppliers of AuNPsexist where these particles are manufactured in largequantities where there is signicant exposure to work-ers. Yet, recent reports (Hoet et al. 2004; NanotechFull Report 2008) indicate that AuNP penetrationinto tissue and their effects on cells is still unclear. It isalso extremely important since AuNPs are easilyfunctionalized which allows the production of newdrugs with chemical groups that target cancer cells,

Correspondence: Dr Tatsiana Mironava, Department of Materials Science and Engineering, State University of New York at Stony Brook, Stony Brook,NY 11794-2275, USA. Fax: +1 631 632 8052. E-mail: [email protected]

ISSN 1743-5390 print/ISSN 1743-5404 online 2010 Informa UK Ltd.DOI: 10.3109/17435390903471463

Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

and due to the high electron density of Au, allow forenhanced imaging (Eghtedari et al. 2004; Huang et al.2006, 2007b; Durr et al. 2007; Rayavarapu et al.2007). Other specic biomedical applications of theseparticles involve chemotherapy (via drug delivery) anddirected thermal irradiation of tumors, with bothapproaches representing a promise for cancer treat-ment (Mukherjee et al. 2005; El-Sayed et al. 2006;Harjanto 2006; Paciotti et al. 2006; Dixit et al. 2007;Huang et al. 2007c, 2007d, 2007e; Oyelere et al.2007; Gannon et al. 2008). Further, the majority ofreports dealing with the effects of AuNPs on normalcells have focused on the short term effects (212 h) ofparticle exposure instead of more long term conse-quences that can affect overall cellular function. Col-lectively, results from these studies are thoroughlydiscussed in detail in recent reviews (Lewinskyet al. 2008; Murphy et al. 2008). The detrimentaleffects cannot be detected simply by general prolifer-ation or apoptosis assays, since the presence of theAuNPs inside the cells may be interfering with theproper execution of other specic functions. Further-more, the short experimental times (less than 24 h)frequently reported in the literature (Goodman et al.2004; Connor et al. 2005; Khan et al. 2007; Haucket al. 2008; Ponti et al. 2008) are not sufcient toobserve many of these effects, which were showed toappear after 72 h, and several rounds of cell division(Pernodet et al. 2006). Even after impaired function isdetected, none of the previous studies has addressedthe essential question of possible recovery onceAuNPs are removed. Lastly, our laboratory has pre-viously shown that human dermal broblasts aresignicantly affected by the presence of 13 nm sizeAuNPs, with the most dramatic effect being a largedecrease in cell area which correlated with decreasedcell proliferation and migration, as well as ECMorganization (Pernodet et al. 2006).Since the skin is the primary source of contact for

nanoparticles, and since the interaction with nano-particles is cell specic (Goodman et al. 2004; Connoret al. 2005; Chithrani et al. 2006; Cornell2006; De la Fuente et al. 2006; Krpetic et al. 2006;Takahashi et al. 2006; Patra et al. 2007; Ryan et al.2007), we chose to continue our studies using humandermal broblasts and explore: (a) Whether thedecreased cell area was associated with a concomitantchange in cytoskeletal protein expression; (b) whethera critical exposure concentration or time exists forwhich recovery was possible once AuNPs wereremoved; (c) the mechanism for particle penetrationinto the cells and (d) whether signicant differencescould be discerned in these phenomena betweenAuNPs of two different sizes (13 nm and 45 nm),synthesized with identical surface chemistry.

Materials and methods

Synthesis of Au/citrate nanoparticles

13 nm Au/Citrate nanoparticles were synthesizedaccording to Turkevich (1985a, 1985b) protocol.HAuCl4 (0.1% ml, solution in HCl 30% [Aldrich])in 95 ml MilliQ water in a tree-necked ask withcondenser and thermometer was heated to boilingpoint while stirring, after that sodium citrate (200 mg,dehydrated, 99%, [Aldrich]) in 5 ml of water wasadded to the solution. Over a couple minutes, thecolor of solution changed from yellow to grey andnally to purple. The solution was gently boiled for4050 min and then cooled down at room tempera-ture. For 45 nm Au/citrate nanoparticles synthesiswas carried out in a three-necked ask under con-denser. 25 mg of KAuCl4 was boiled in 98 ml MilliQwater following by addition of 2.5 ml of 1.5% sodiumcitrate under vigorous stirring, after changing thecolor from greyish to purple nanoparticles solutionwas cooled down at room temperature. In both casesthe resulting particles were coated with negativelycharged citrate and, therefore, were uniformlysuspended.

Cell culture

Primary human dermal broblasts (CF-31, Cauca-sian female, 31 years old, National Institute on Aging(NIA) Bank, passage 714 only) were plated at celldensity 2,500 cells per well and cultured in 24-welldishes. Dulbeccos Modied Eagles Medium(DMEM) was used with 1% of penicillin-streptomy-cin (PS) and 10% of fetal bovine serum (FBS) (allpurchased from Sigma). 1 ml of medium containingAuNPs (withconcentrations in the rangeof0189mg/mlin case of 13 nm and 026 mg/ml in case of 45 nm)was added to each well 24 h after plating. The wellswere incubated with AuNPs for the chosen timepoints (up to 6 days) and then counted or xed,stained and imaged. All incubations were performedat 37C and 5% CO2. Each experiment had acontrol (cells grown in medium without AuNPs),and was performed in triplicate and repeated at leastthree times.

Cell counting

To determine the cell number during the growthcurve experiments cells were plated at an initial den-sity of 2,500 cells per well and counted using hemo-cytometer at the specic time point (up to eight days).

Gold nanoparticles toxicity 121

Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

Each grid square of the hemocytometer slide repre-sents a volume of 10-7 m3, and cells were counted in10 squares in 1 ml of the cell suspension. Each con-dition had triplicates and all experiments were con-ducted three times. Cell suspensions were mixed foruniform distribution and were diluted enough so thatthe cells did not aggregate.

Cell staining for confocal microscopy

Cell area and overall morphology as a function of timeand concentration was monitored using a Leica con-focal microscope. For these experiments, cells werexed with 3.7% formaldehyde for 15 min followingexposure to AuNPs for three and six days. Alexa Fluor488-Phalloidin was used for actin ber staining andPropidium Iodide for nuclei staining. In addition, aset of images was obtained using an Hg lamp with theexcitation lter of 450490 nm and the emission lterof long pass (LP) at 515 nm.

TEM

TEM analysis was used to assess the size distributionof the AuNPs as well as the fate of internalizedparticles. One drop of the original AuNPs solution(95 mg/ml 13 nm and 13 mg/ml 45 nm particles) wasplaced on 300 mesh copper grip, which was coatedwith formvar lm. The sample was then dried out atroom temperature. Gaussian distributions of dia-meters were calculated from the samples with morethan 170 nanoparticles. After exposure to AuNPs forthree and six days at 142 mg/ml (13 nm) and 20 mg/ml(45 nm), the cells were xed in a solution of 2%paraformaldehyde and 2.5% glutaraldehyde in 0.1 MPhosphate Buffered Saline (PBS), stained in 2%uranyl acetate, dehydrated with ethanol, then embed-ded in Propylene oxide. The specimen was cut intoultrathin sections (90 nm) with Reichart UltracutEultramicrotome and stained on the grid with uranylacetate and lead citrate. The samples were imagedusing a FEI Tecnai12 BioTwinG2 transmission elec-tron microscope. Digital images were acquired withan AMT XR-60 CCD Digital Camera System andcompiled using Adobe Photoshop program.

SEM

SEM analysis was used to assess the uptake of par-ticles by exposing cells to 20 mg/ml 45 nm AuNPs andto 142 mg/ml 13 nm AuNPs for three days, followingby xing (3.7% formaldehyde in PBS) and then multi

step ethanol dehydration. Samples were coated with athin layer of atomic Au for conductivity and imagedby SFEG SEM LEO 1550.

Apoptosis induction and quantication

Induction of apoptosis was induced by the use ofRecombinant Human Tumor Necrosis Factor-alpha(TNF-a, Biosource). An anti-ACTIVE Caspase-3pAb (Promega) and Cy3 conjugated secondary anti-body was used to determine whether cells underwentapoptosis after exposure to AuNPs for three and sixdays at different concentrations (13 nm AuNPs: 95,142and190mg/ml:45nmAuNPs:13,20and26mg/ml).Cells were also stained with DAPI to reveal thenuclei (Invitrogen). The percentage of apoptoticcells per sample was determined using the imagescaptured during uorescent microscopy. For thisexperiment, 15 random images were captured foreach sample (samples were in triplicate and theexperiment was repeated twice).

Western blotting

Proteins were extracted with RIPA Lysis and Extrac-tion Buffer (25 mMTrisHCl, pH 7.6, 150 mMNaCl,1%NP-40, 1% sodium deoxycholate, 0.1% SDS) andwere separated by SDS-PAGE (0.9 mg of protein wasapplied per lane) and blotted onto nitrocellulosemembrane (Millipore, Beverly, MA, USA). Themembranes were blocked with 5% non-fat milk andprobed with diluted monoclonal antibodies (Anti-actin Clone AC-40, obtained from Sigma and Anti-b-tubulin [E7]) developed by Klymkowsky andobtained from the Developmental Studies Hybrid-oma Bank (Department of Biological Sciences, TheUniversity of Iowa, Iowa City, IA, USA) at 4C for1 h. After washing, bound antibodies were detectedwith a goat anti-rabbit IgG or anti-mouse IgG coupledto HRP (1:10,000) at room temperature for 1 h. Thesignal was visualized by using ECL (Amersham Phar-macia Biosciences, Piscataway, NJ, USA).

Clathrin-mediated endocytosis inhibition

Cells were cultured in 24-well dish for 24 h, after thatPAO (Gibson et al. 1989) solution in 1% DMSO wasadded to maintain a nal concentration 20 mM. After20 min of pre-treatment with PAO, the AuNPs wereadded to medium and the cells were cultured foranother 2 h, rinsed with PBS, and xed for TEMas described above.

122 T. Mironava et al.

Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

Endocytosis inhibition

Cells were plated in tissue culture asks (75 cm2)under normal conditions (37C, DMEM, 5% CO2)for 24 h. The cells were then exposed to AuNPs for24 h either at low temperature (4C, DMEM, 5%CO2) or at 37C as control. The cells were thencounted and sent to Columbia Analytical Inc. forgold content analysis by atomic absorptionspectroscopy.

Collagen and Fibronectin expression

Collagen and Fibronectin were analyzed in super-natants of cultured CF-31 cells by ProcollagenType I C-Peptide EIA Kit (Takara, MK101) and

human Fibronectin EIA Kit (Takara, MK115) asdescribed in instructions provided by the manufac-turer. Samples with high concentration of collagenand bronectin were appropriately prediluted withSample Diluent.

Results

Particle characteristics

Two different sizes of citrate AuNPs were synthesizedand were characterized by TEM (Figure 1a, 1b).Quantitative measurements shown by histograms(Figure 1c, 1d) reveal that the average particles sizeswere 13 1.1 nm and 45 3.2 nm.

35 40 45 50 55 600

20

40

60

80

100

120

140

Nan

opar

ticle

s nu

mbe

r

AuNPs diameter, nm

B.

50nm

A. C.

D.

50nm

10 11 12 13 14 150

20

40

60

80

100

120

140

AuNPs diameter, nm

Nan

opar

ticle

s nu

mbe

r

Figure 1. AuNPs imaged by TEM and their Gaussian size distribution histograms. (a) and (c) 45 5.1 nm particles, (b) and (d) 13 1.8 nmparticles.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

Size and time-dependent uptake of AuNPs

Since gold is more electron dense than the surround-ing organic material it was clearly visible under SEM(Figure 2). The images shown in Figure 2 clearlyreveal the outline of the cell and the brighter regions,indicative of the presence of gold, especially aroundthe nuclei (Figure 2ad). This observation was con-rmed by EDS/X-ray Microanalysis, which shows theAu Ka peak at 2.3 keV, indicating the presence ofAuNPs clusters inside the cells (Figure 2e). In thecontrol cells (not exposed to AuNPs), the Au peak is

very small, probably resulting from the gold thin lmcoating used for conductivity (Figure 2g).

The impact of AuNPs on cell proliferation

Results in Figure 3 show that for both particle sizes,the cell doubling time is increasing with higherAuNPs concentration and longer exposure. If wecompare the concentrations for which the cell dou-bling time increases from 3545 h, we nd that theconcentration of the 13 nm particles, on average, is

A.

C. D.

B.

E. F. G.

200 nm 200 nm

10 m 10 m

keV

KCnt

2.0

1.6

1.2

0.8

0.4

0.0

C

O

ZnZnTi

K

Si

AuAu

0 1 2 3 4 5 6 7 8 9 10keV

1.1

0.9

0.6KCnt0.4

0.2

0.0

CO Zn

ZnTiKCa

Si

Au

Au

0 1 2 3 4 5 6 7 8 9 10keV

1.2

0.9

0.7

0.5

0.2

0.0

KCnt

0

CO

Zn K Ca

Si

Au

ZnAu

1 2 3 4 5 6 7 8 9 10

Figure 2. (a) and (c). SEM images of cells exposed to 45 nmAuNPs (20 mg/ml); (b) and (d) SEM image of cells exposed to 13 nmAuNPs (142mg/ml); (e), (f) and (g) EDAX spectrum of cells with 13 nm, 45 nm and control, respectively.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

seven times higher than that of the 45 nm particles (atday 2 and 4) and increases to nearly a factor of 10 aftersix days of incubation. The inset graphs show that thedoubling time increases as a function of time for bothparticles sizes suggesting that saturation is not occur-ring, and for a specic concentration of AuNPs(20 mg/ml 45 nm and 142 mg/ml 13 nm), the effectis almost equivalent at day 4 and 6.

Intracellular fate of internalized AuNPs

The data clearly shows that for both particle sizes theAuNPs are sequestered inside large vacuoles and donot penetrate either the nucleus or the mitochondria(Figure 4). The vacuoles are distributed uniformlyacross the cytoplasm (Figure 4ah), consistent withthe particle distribution observed in the dry samplesimaged with SEM (Figure 2). Closer examination alsoshows that even after six days, no particles weredetected inside the nucleus or mitochondria(Figure 4c, 4g). We also present higher magnicationimages revealing that neither the 13 nm nor the 45 nmparticles are uniformly distributed within vacuoles.Rather, we nd that both types of particles areattracted to the vacuole membrane, leaving the inte-rior almost empty (Figure 4l, 4m). In the case of the13 nm particles, their density around the membrane isgreater and we can see sections of the membraneprotruding inside the vacuole with particles still

attached to it (Figure 4l). In contrast, with the45 nm particles, only a monolayer string of particlescovers the membrane surfaces and no particles wereseen attached to the thinner tendrils.Furthermore, with increasing incubation time addi-

tional AuNPs (both sizes) enter the cell, causing theformation of greater number of vacuoles as well asincreasing the diameter of the existing vacuoles(Figure 4i, 4j). Specically, we nd that the numberand diameter of vacuoles per cell increases at the samerate for both types of AuNPs (average of seven cellsfor each conditions were examined). The major dif-ference between samples exposed to the two AuNPsizes is the number of particle clusters per vacuole,which is roughly ve times greater with the 13 nmparticles (Figure 4k).Finally, close examination of TEM cross sections

obtained after three and six days of incubation withboth types of AuNPs indicates that in all cases thevacuoles clustered inside the cytoplasm. Magniedregions near the cell membrane show that eventhough occasional particle clusters are observed, novacuoles are present. Hence, we have no direct evi-dence of exocytosis occurring to prevent accumula-tion of particles within the cells. Rather, the numberof particles within the cells nearly doubles for bothtypes of particles, when the incubation time isincreased from 36 days.In order to determine whether the mode of pene-

tration of the AuNPs in the cells is a function of

0 20 40 60 80 100 120 140 160

35

40

45

50

55

60

65

70

75

80

85

90

95

Control

2 4 650607080

45 nm

13 nm

Day of exposureDoub

ling

time,

hou

rs

AuNPs:

13 nm (2 days) 13 nm (4 days) 13 nm (6 days) 45 nm (2 days) 45 nm (4 days) 45 nm (6 days)

Dou

blin

g tim

e, h

ours

Figure 3. Doubling time for cells exposed to different concentrations of AuNPs 13 nm and 45 nm for 2, 4 and 6 days; inset: Doubling timeversus time of exposure for samples exposed to 20 mg/ml 45 nm and 142 mg/ml 13 nm AuNPs.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

3 days 6 days0

50

100

150

20045 nm AuNPs 13 nm AuNPs

Part

icle

s nu

mbe

r per

vac

uole

C.

2 microns

2 microns

A.

1 micron

D.

1 micron

B.

G.

E.

2 microns

2 microns

1 micron

H.

F.

1 micron

I.

J.

3 days 6 days0.0

0.3

0.6

0.9

1.2

1.5

1.8

Vacu

ole

size

, mic

ron 45 nm AuNPs

13 nm AuNPs

K.

M.

3 days 6 days0

50

100

150

200

25045 nm AuNPs 13 nm AuNPs

Vacu

ole

num

ber p

er c

ell

L.

100 nm 100 nm

N.

2 microns

Figure 4. TEM section of cells exposed to AuNPs; (a) and (b) 142 mg/ml 13 nm particles three days exposure; (c) and (d) 142 mg/ml 13 nmnanoparticles six days exposure, (e) and (f) 20 mg/ml 45 nm AuNPs three days exposure; (h) and (g) 20 mg/ml 45 nm gold six days exposure;(i) control; (j) vacuole size distribution; (k) number of vacuoles per cell; (l) number of particles/clusters per vacuole, (m) and (n) highmagnication of cell vacuole led with to 13 and 45 AuNPs, respectively.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

particle size, i.e., do some particles penetrate bypassive diffusion through the cell membrane(Geiser et al. 2005) or do they enter via endocytosis(Connor et al. 2005), clathrin mediated endocytosiswas inhibited using PAO (Gibson et al. 1989). Fol-lowing treatment of cells with PAO prior to AuNPexposure, the cells were xed, sectioned, and exam-ined with TEM (Figure 5). The results were quanti-ed by measuring the ratio of AuNPs adjacent to thecell membrane versus the number in the cytoplasm.Figure 5 shows that the number of AuNPs clusteredin the vicinity of the cell membrane, and the numberthat penetrated the cells in the absence of PAO isnearly the same for both particle sizes. With exposureto PAO the ratio is nearly unchanged for the 13 nmparticles (Figure 5e), indicating that clathrin-mediated endocytosis is not the predominant particlepenetration pathway. In Figure 5fi we show cross-sections for cells treated with and without PAO andexposed to 45 nm AuNPs, revealing that almost noparticles are present inside the cytoplasm of the trea-ted cells, while a signicant number is present in theuntreated control cells. In fact, a strong accumulationof AuNPs is clearly seen outside of the membrane inthe treated cells (Figure 5i) and as a result, the ratio isreduced to nearly zero for the 45 nm particles, ascompared to that of the control cells (Figure 5k),indicating that clathrin-mediated endocytosis is thepredominant mode of entry.To further investigate if the major pathway for

13 nm AuNPs penetration is diffusion through mem-brane driven concentration gradients or non-receptormediated endocytosis (phagocytosis), we utilized anapproach involving temperature. Specically, whenwe decreased the incubation temperature to 4C,almost 85% of 13 nm AuNPs penetration rate intothe cells was inhibited (Figure 5j). In contrast, only73% of penetration was inhibited in case of 45 nmAuNPs (Figure 5l), indicating that phagocytosisappears to be the prime mode of 13 nm AuNPspenetration, but also is partly responsible for thecell penetration of the 45 nm AuNPs.

Cell recovery

Based on these results, an interesting question arises:Do cells have a mechanism for eliminating AuNPsand thereby recover from their adverse effects? In thesimplest scenario the cells can pass the nanoparticlesto daughter cells, therefore in the absence of particlesin the media, the AuNPs concentration inside the cellbecomes increasingly diluted. Hence, if the nano-particles are removed from the environment, canthe cells eventually recover from AuNP exposure?

Thus, we tried to address these questions by specif-ically investigating whether cells containing AuNPsare still capable of dividing and passing the particles tothe daughter cells.In Figure 6 images of cells immediately before the

removal of the particles, and ve days later show thatafter a three-day incubation AuNPs uptake is greaterfor the 13 nm particles (Figure 6b) than for 45 nm(Figure 6e). After ve days of incubation withoutAuNPs, a drastic reduction in the average amountof particles per cell is observed in cells exposed to bothparticle sizes (Figure 6c, 6f). Thus, the major mech-anism for AuNPs reduction appears to be cell division(Figure 6d), where the particles are seen to be dividedbetween the two daughter cells.

Particle-mediated apoptosis

To investigate whether exposure to AuNPs leads todetrimental effects for the cells, we examined the rateof apoptosis. Figure 7 shows the percentage of cellsundergoing apoptosis exposed to different AuNPsconcentrations and incubated for three and sixdays. Specically, we nd that in the presence ofthe 45 nm AuNPs there is a higher rate of apoptosiswith either longer exposure or higher particle con-centrations in comparison to that with the 13 nmAuNPs. Whereas the apoptotic rate for cells exposedto 13 nm AuNPs at day 3 starts at 23% and increasesto 42% at the highest concentration, with the 45 nmAuNPs, it ranges from 4475%. Similarly, after sixdays, apoptosis further increases to 61% with thelowest and increases to 97% with the highest concen-tration of 13 nm, whereas with the 45 nm AuNPs therate is 91100% at the different concentrations(Figure 7). It is clear that for the 45 nm AuNPsthat apoptosis: (1) Occurs at nearly one tenth ofthe concentration of that of the 13 nm particles, (2)increases steeply with higher concentration, and (3) isnearly 100% for all concentrations after six days ofincubation. Hence, even though the damage mayappear to be equivalent, i.e., approximately 40% for142 mg/ml of 13 nm particles, versus 13 mg/ml for the45 nm particles, the rate of increase with concentra-tion and incubation time is much faster with the largerAuNPs.Next we tested to see if removal of AuNPs can

rescue the cells from apoptosis. Figure 8 shows thateven after removal of the AuNPs, the rate of cellgrowth in the exposed cells is slower than that ofthe control unexposed cells, with the slowest beingin cells exposed to the 45 nm particles. In the inset weplotted the percentage recovery after a total ofan eight-day incubation (three days with AuNPs


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

not inhibited inhibited with PAO0.0

0.2

0.4

0.6

0.8

1.0 13 nm

AuN

Ps in

side

/out

side

cel

l rat

io

37C 4C 0

102030405060708090

100110120 13 nm

AuN

Ps n

g/ce

ll, 1

04A

uNPs

insi

de/o

utsi

de c

ell r

atio

not inhibited inhibited with PAO0.0

0.2

0.4

0.6

0.8

1.0 45 nm

37C 4C 05

10152025303540

AuN

Ps n

g/ce

ll, 1

04

45 nm

1 micron 200 nm

1 micron 200 nm

1 micron 200 nm

1 micron 200 nm

A. B.

C. D.

E.

F. H.

G. I.

J

K.

L.

Figure 5. (ab) TEM sections of the cells exposed to 142 mg/ml 13 nm gold; (cd) cells treated with PAO (20 mM) and exposed to 142 mg/ml13 nm AuNPs; (e) 13 nm AuNPs ratio inside and outside of the cell with PAO and without inhibition (p = 0.9461); (fh) cells exposed to20 mg/ml 45 nm nanoparticles; (gi) cells treated with PAO and exposed to the same concentration of 45 nm gold; (j) amount of gold per cell forsamples exposed to 13 nm AuNPs at 37C and 4C (p < 0.001); (k) 45 nm AuNPs ratio inside/outside cell (p < 0.0001); (l) amount of gold percell for samples exposed to 45 nm AuNPs at 37C and 4C (p = 0.0123).


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

incubation, ve days of recovery), which is calculatedby dening control as undergoing 100% recovery.With the 13 nm AuNPs the rate of cell proliferationincreases immediately after removal of the particlesand recovery reaches 61%, 48% and 37% for increas-ing concentration (Figure 8a inset). With the 45 nm

AuNPs the rates of recovery are much slower withrespect to increasing particle concentrations (42%,22% and 11%).Since no division occurs during apoptosis, recovery

is also not possible, and thus the number of AuNPs inthe cells is not reduced. Therefore, we replotted the

A. B. C.

D. E. F.

Figure 6. (a) Control; (b) Cells exposed to 142 mg/ml of 13 nm AuNPs for three days; (c) Cells for the same concentration of 13 nm AuNPsafter ve days recovery following three days exposure; (d) Cell transmitting nanoparticles to daughter cell upon dividing; (e) Cells exposed to20 mg/ml of 45 nm AuNPs for three days; (f) Cells for the same concentration of 45 nm AuNPs after ve days recovery.

0 25 50 75 100 125 150 175 200 2250.0

0.5

1.0

1.5

20

40

60

80

100 3 days exposure 6 days exposure

Apo

ptos

is, %

13 nm AuNP concentration, mg/ml 0 5 10 15 20 25 30

0.0

0.5

1.0

1.5

20

40

60

80

100

Apo

ptos

is, %

45 nm AuNP concentration, mg/ml

3 days exposure 6 days exposure

A. B.

Figure 7. Apoptosis rate of cells exposed to (a) 13 nm for three and six days at different concentrations (p = 0.0527 and 0.0143, respectively)and (b) 45 nm AuNPs for three and six days at different concentrations (p = 0.0315 and 0.0083, respectively).


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

data by subtracting the fraction of cells that haveundergone apoptosis after three day incubation,and thus no longer divide. The data shows that thecell number (as a function of incubation time aftercorrecting for the fraction of non-dividing apoptoticcells on day 3) for both types of particles is nearlyidentical to that of the control cells indicating that thedoubling time of the exposed cells, even if they stillcontain nanoparticles is similar.

Particle-mediated microlament disruption

In Figure 10ah we show confocal images of cells afterexposure to different concentrations of both 13 nm

and 45 nm AuNPs for three and ve days andfollowing recovery (Figure 10ip). Figure 10eand Figure 10m shows the average cell aspect ratiocorresponding indicating that it is bigger in theexposed cells than control. Furthermore, we canclearly see that there are fewer cells for the samplesexposed to 45 nm AuNPs (Figure 10fh), which arealso more elongated and appear ready to lift off thesubstratum, as compared to cells exposed to the 13nm AuNPs (Figure 10bd). Removing the AuNPsand allowing the cells to recover for another ve days,has a dramatic effect; the 13 nm AuNPs exposed cellshave the same appearance as the unexposed controlcells even at the highest concentration. In contrast, forthe larger particles, the recovery is much slower, and

5000

10000

15000

20000

25000

30000

35000

40000

190 g/ml

142 g/ml

95 g/ml

Control

RecoveryExposure

Num

ber o

f cel

ls

Day

13 nm AuNPs

75 100 125 150 175 2000

102030405060

Rec

over

y, %

AuNPs, g/ml

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 80

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

26 g/ml

20 g/ml

13 g/ml RecoveryExposure

Num

ber o

f cel

ls

Day

Control

10 15 20 250

102030405060

45 nm AuNPs

AuNPs, g/ml

Rec

over

y, %

B.A.

Figure 8. Cell recovery. (a) and (b) dermal broblast cells CF-31 exposed to different AuNPs concentrations for three days and then allowed torecover for ve days. Time where nanoparticles were removed is outlined with dashed line; control is equal to 100% recovery.

10000

100000

Control 95 g/ml AuNPs 13 nm 142 g/ml AuNPs 13 nm 190 g/ml AuNPs 13 nm

Cell

num

ber

Cell

num

ber

Days4 5 6 7 8 4 5 6 7 8

10000

100000

Control 13 g/ml AuNPs 45 nm 20 g/ml AuNPs 45 nm 26 g/ml AuNPs 45 nm

Days

Figure 9. Cell recovery. Growth curves for the recovery after AuNPs exposure for three days (cell number was corrected by apoptotic cellsubtraction on day 3). Dermal broblast cells CF-31 were exposed to different concentrations of 13 nm (a) and 45 nm (b) AuNPs, respectively.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

3 days exposure

0 50 100 150 20002468

1012

Exposure Recovery

Cell

aspe

ct ra

tio

13 nm AuNPs, mg/ml

5 days following recovery

0 5 10 15 20 25 300

2

4

6

8

10

12 Exposure Recovery

Cell

aspe

ct ra

tio

45 nm AuNPs, mg/ml

A.

I.

B. C. D.

F. G. H.

J. K. L.

N. O. P.

E.

M.

170.6 m

Figure 10. Human dermal broblasts CF-31 imaged with confocal microscopy after three days for (a) control and cells exposed to 13 nmAuNPs at the following concentrations, (b) 95 mg/ml, (c) 142 mg/ml, and (d) 190 mg/ml and to 45 nm AuNPs at (f) 13 mg/ml, (g) 20 mg/ml and(h) 26 mg/ml. The cells were also monitored following recovery after ve days of AuNPs removal (jp), compared to control. (i) Cell aspect ratioafter exposure and recovery (e) 13 nm AuNPs, (m) 45 nm AuNPs.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

even after ve days many cells still appear elongatedand their density remains low (Figure 10np).We further show a magnied view of cells, that

shows how the actin bers are well extended acrossthe cell cytoplasm in the control conditions(Figure 11ac). On the other hand, after three daysof AuNPs exposure to either 13 or 45 nm AuNPs, theactin laments are broken or appearing disruptedand thinner, appearing as an aspiration of dots(Figure 11d, 11g). After ve days of recovery thedisruption of the actin laments are less obvious inthe case of 13 nm AuNPs (Figure 11e) as comparedto those still present with the 45 nm particles(Figure 11h). Stretched actin laments were observedagain with the 13 nm AuNPs indicating that the cells

are in the process of recovery while for the largerparticles, the damaged actin was still visible, eventhough some stretched laments were also observed.We also nd, that following 14 days of recovery, thecytoskeleton of the cells exposed to both size particlescompletely recover (Figure 11f, 11i) and resemblethat of the control cells (Figure 11c).In order to examine whether these effects on the

cytoskeleton were a direct result of less production ofactin or tubulin, we examined the expression of theseproteins from the particle exposed and controlcells. Figure 12 shows no difference between thecontrol and any of the exposed samples, indicatingthat the amount of actin and beta tubulin isunaffected.

3 days exposure 5 days recovery 14 days recoveryA.

Control

B. C.

D.

45 nm AuNPs

E. F.

13 nm AuNPs

G. H. I.

11.24 m

Figure 11. CF-31 imaged with confocal microscopy after three days of exposure and 5 and 14 days following recovery. (a), (b), and (c) control;(d), (e) and (f) cells exposed to 142 mg/ml of 13 nm nanoparticles; (g), (h), and (i) cells exposed to 20 mg/ml of 45 nm.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

Another possible cause for the observed reductionin cell area may be a decrease in the expression ofECM proteins. Therefore, we measured the amountof collagen (type I) and bronectin expressed by thecells after incubation for three days with 20 and 142mg/ml of 13 nm and 45 nm AuNPs, respectively, andthen after recovery for ve and 14 days. The magni-tude of the reduction after three days of exposure forboth proteins is within one standard deviation for thetwo particle sizes and appears to be mostly a functionof the exposure conditions (Figure 13). It is alsoimportant to notice, that collagen is reduced at agreater level than bronectin and indicating thatECM proteins produced after exposure to AuNPsis altered to a low collagen and higher bronectincomposition (a ratio contains 20% less collagen thanbronectin when compared with control compositionfor cells exposed to 13 nm AuNPs and 35% lesscollagen in case of cells exposed to 45 nm particles).Furthermore, the data shows that the production ofbronectin partially recovers after ve days and fullyrecovers after 14 days, whereas the production ofcollagen only slightly recovers after ve days and isstill not fully recovered after 14 days. These results areconsistent with those of the actin bers, shownin Figure 11.

Discussion and conclusion

In this manuscript we attempted to elucidate some ofthe fundamental aspects regarding the effects ofAuNPs on primary dermal broblasts. In a typicaltissue or organ, cells are organized in different layers,interact with each other, and damage in one layer canhave an impact on the other cells in an unpredictablefashion. We chose to study dermal broblasts sincetheir interactions within skin are well known (i.e., interms of gene expression, protein deposition etc.).Therefore, our approach is to rst understand theeffects of AuNPs on the function of cells in vitro, andthen to try to model the effects at the tissue level.While the most common approach in studying theeffects of nanoparticles reported in numerous pub-lications (Goodman et al. 2004; Connor et al.2005; Khan et al. 2007; Hauck et al. 2008; Lewinskyet al. 2008; Ponti et al. 2008) is to cell proliferation,our results allow a deeper understanding of additionaleffects on cells, in the context of how living cellstolerate the particles once they penetrate the cell.In attempt to directly compare the effects of the

13 nm and 45 nm size AuNPs, we chose exposureconcentrations based on their effect on doubling time(Figure 2). We observed that for a 26 day timeinterval, the concentration difference for two nano-particle sizes (for the same doubling time increase)varies by 6- to 13fold. This signicant variationoccurs because of the concentration-doubling timenon-linearity over time. The average concentrationthat causes the same increase in cell doubling time forcells exposed to 13 nm particles is larger than theconcentration of the 45 nm particles by roughly afactor of eight. Therefore, a set of three

3 8 170

20

40

60

80

100

Days

Control 13 nm AuNPs 45 nm AuNPs

% o

f fib

rone

ctin

/ ce

ll

3 8 170

20

40

60

80

100

Control 13 nm AuNPs 45 nm AuNPs

% c

olla

gen

/ cel

l

Days

Figure 13. Fibronectin (a) and collagen (b) expression, cells treated with 142 mg/ml 13 nm and 20 mg/ml 45 nm AuNPs for three days,recovered for ve and 14 days after exposure.

a b cBeta-tubulin 55 kDa

actin 45 kDa

Figure 12. Western blot. (a) control, cells cultured for three dayswith AuNPs; (b) 142 mg/ml 13 nm; (c) 20 mg/ml 45 nm.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

concentrations: 13, 20 and 26 mg/ml was chosen for45 nm AuNPs, and a set of 95, 142 and 190 mg/ml for13 nm AuNPs.In this study we found that the nanoparticle uptake

by cells was dependent on exposure time, particle sizeand concentration. We also found that the 45 nmparticles, at much lower concentrations result in amore signicant increase of the doubling time thanthe 13 nm particles. These results were surprising inview of the ndings of Pan et al (2007) who reportedthat maximum toxicity occurs for a particle diameterof 1.4 nm (Pan et al. 2007). In order to understandthe basis of the observed toxic effects between thedifferent particles, we performed several experimentswhere the distribution of AuNPs in the cell could bemeasured and have shown that they penetrate the cellmembrane and accumulate in large vacuoles.Even though the mechanism of sequestration for

both particles sizes appears to be similar, the methodof penetration into the cytoplasm was determined tobe different. In previous studies investigating nano-particle penetration, gold particles of different shapesor sizes were coated with transferrin (Yang et al.2005; Chithrani et al. 2006; Chithrani andChan 2007) for which known membrane recognitionpathways exist. In our case, the particles were stabi-lized with citrate for which no specic recognition site(s) on the plasma membrane is known. Also, to ruleout any independent effects of citrate on cells, weexposed cells to citrate and found no differences incell growth when compared to control cells. Further,most of the previous work was also performed withcancer cells or cell lines whose characteristics, to anextent, are known to differ from primary cells.The data described herein shows that we have

identied the AuNPs cell penetration pathways indermal broblasts; for 45 nm nanoparticles clathrinmediated endocytosis is the major pathway, while forthe 13 nm it is phagocytosis. Another possibility maybe that the particle size affects the amount of proteinadsorbed, which in turn can affect the recognitionpathway triggered when the particles attach to the cellmembrane. Since the radius of gyration of albumin isapproximately 3.5 nm, more molecules are likely to beadsorbed on the larger particles, with radii of 22 nmand surface area of ~ 1200 nm than on the smallerparticles with radii of 6 nm and surface area ~ 120 nm.Also, the dependence of the uptake on temperaturemay help to clarify whether non clathrin mediatedendocytosis is responsible for particle penetration.Sudhakaran et al. (2007) have shown that edocytosisin human dermal broblasts decreases to approxi-mately 15% with decreasing the temperature to16C; a similar decrease was observed by Mamdouhet al. (1996) in epithelial cells, who also found that at

4C endocytosis in epithelial cells is reduced by nearly95%. Our results show that both small and largeAuNPs uptake was inhibited by 85% and 73%,respectively. Although the amount of inhibition issignicant, it is still smaller that that reported forendocytosis, indicating that other pathways mayalso play a role in overall particle uptake. Furtherwork is in progress, which will hopefully clarify theissue, of the involvement of both the amount andnature of the adsorbed proteins as a function ofparticles size. Lastly, the ratio of particles insideand outside the cell, in the untreated control cellsis an indication of the efciency of the particularpenetration mechanism used by the cells to bringthe AuNPs across the cell membrane to the interiorof the cell (PAO data). From this data it appears thatthe penetration process used to bring in the smallerAuNPs is more efcient than that used for entry of thelarger particles.Regardless, with both particles, once inside the cell,

they are sequestered in vacuoles, where they localizeto the membrane. These vacuoles are considered fullwhen almost all available membrane surfaces arecovered with particles. As a result, the concentrationof AuNPs that they can hold is inversely proportionalto the cross sectional area of the particles. Comparingcross-sections obtained from cells incubated for threeand six days, we nd that the mean diameter of thevacuoles increases by approximately a factor of 2 whilethe number of particles per vacuole increases by afactor of 45. Hence the number of particles within avacuole increases in a manner proportional to theavailable increased area which the particles couldsubtend on the membrane surface. On the otherhand, the size of the vacuoles is similar for the twotypes of particles after equivalent incubation times,and the number of vacuoles is nearly the same after sixdays of incubation. Thus, it is the number of vacuoles,rather than the absolute concentration of particleswithin the cell, that plays the largest role in disruptingnormal cellular function. However, we do observeloose particles that are visible in the cytoplasm ofcells exposed to 45 nm AuNPs for six days. Thisobservation might be the result of vacuole structuralinstability at certain local particle concentrations. Inessence, it is more difcult for the inner surface of avacuole to equilibrate larger 45 nm AuNPs, leading tovacuole collapse and nanoparticle release in the cyto-plasm. Therefore, our observations that toxicity cor-relates with vacuole number must be qualied by thefact that some vacuoles ruptured when exposed to 45nm AuNPs, and thus were not counted. With thesame intact vacuole number, the higher toxicity ofcells exposed to the 45 nm AuNPs probably arisesfrom additional vacuoles that collapsed and release


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

the AuNPs in the cytoplasm leading to interferencewith normal cellular function and ultimatelydamaging the cell.Furthermore, we also determined the apoptotic

rates for cells exposed to different AuNPs sizes andconcentrations and showed that the previouslyreported decrease in cell proliferation was erroneouslyinterpreted as an increase in doubling time. We mea-sured the fraction of cells undergoing apoptosis byassaying the production of caspase 3, which is aprotein produced in the early stage of apoptosis. Cellsproducing this protein are adherent, but no longerdivide, therefore for obtaining the precise doublingtime, the number of cells have to be corrected bysubtraction of apoptotic cells. AuNPs were previouslyreported to have no toxic effects (Patra et al. 2007),but others found that they induce apoptosis in certaincell types (Cornell 2006; Pan et al. 2007; Patra et al.2007), clearly indicating cell type dependence ofAuNPs cytotoxicity. However, even though Patraet al. (2007) and Pan et al. (2007) reported apoptosisin melanoma, broblast and macrophage cell lines,they did not test it with apoptosis specic assays.Furthermore, we found that for the 13 nm particles,the fraction of cells undergoing apoptosis increaseslinearly with concentration at both incubation at threeand six days, however, for 45 nm AuNPs it increasesin exponential fashion with higher concentration forthree days and almost 100% for all concentrationstested for six-day exposure.Even though numerous papers have explored the

effects of AuNPs exposure in different cell culturesystems (Connor et al. 2005; Chithrani et al.2006; Cornell 2006; Krpetic et al. 2006; Pernodetet al. 2006; Takahashi et al. 2006; Patra et al.2007; Ryan et al. 2007), no data has been reportedon the ability of any cell type to recover once theparticles have been removed from themedia. Our datafor cell proliferation where we found a decrease ingrowth rate with increasing AuNPs concentration arein agreement with other studies (Goodman et al.2004; Cornell 2006; Krpetic et al. 2006;Pernodet et al. 2006) regardless of nanoparticle coat-ing. The second observation we made is cell areareduction that was previously also detectedby Pernodet et al. (2006). In order to try to providea possible explanation for this reduction in cell areawe looked at the integrity of the cytoskeleton. Actinbers are responsible for distributing internal forcesby attaching one end the integrins receptors expressedon the cell membrane, which adhere to proteinssecreted by the cell in the extra cellular matrix.We show that the actin bers are well extended

across the cell cytoplasm in the control cells, whereasin those exposed to both 13 nm and 45 nm particles,

actin laments were disrupted and appear as smalldots.Aftervedays of recovery, the cells exposed to the13 nm particles appear to generate large and strongactin bers, whereas in the cells exposed to the 45 nmparticles, a signicant amount of disrupted actin la-ments even after ve days are present. By day 14 allcells had recovered with an intact actin cytoskeleton.This type of ber disruption due to the presence of

AuNPs can occur either due to down regulation ofcytoskeletal proteins or triggering a signaling pathwaywhich somehow interferes with the self assembly ofthe actin, possibly due to physical interference by thelarge amounts of vacuoles. Interestingly, Western blotanalyses reveals that the amount of actin and beta-tubulin formed by the cells is not affected by thepresence of the nanoparticles, indicating that it isprobably the latter that is responsible for the observedcytoskeletal disruption. We reasoned that the largenumber of vacuoles as well as loose particles foundin samples exposed to 45 nm AuNPs interferes withthe ability of actin bers to effectively form linkageswith the ECM via integrins. As a result the cell area isdecreased and disrupted bers are observed in thecells. In order to carry proper tension, the actin bersmust also be anchored to integrins that are bound toArg-Gly-Asp (RGD) domains found on the secretedECM proteins. Therefore, the lack of tension in thebers may also be caused by a disruption in theexpression of the ECM proteins, which for broblastsare primarily collagen and bronectin. When weinvestigated this possibility, we found that signi-cantly less collagen and bronectin were expressedby the cells exposed to nanoparticles, which mayexplain the observed drastically reduced cell area.Furthermore, since collagen reduction is higherthan bronectin, the composition of the ECM isaltered. Loss of collagen is generally associated withincreased ECM rigidity and aging. Hence, this shift incomposition may explain the increased modulus ofthe bers previously reported by Pernodet et al.(2006) while the decreased overall quantity is consis-tent with the smaller and thinner bers for cellscultured with AuNPs.Lastly, we also found that as the cells divide the

concentration of particles is decreased, which in turnallows the cells to reduce the number of vacuoles andform normal actin bers and increase their productionof ECM proteins. Hence, after 14 days near fullrecovery occurs for cells expose to both types ofAuNPs. Taken together, these data indicate thatAuNPs are toxic for human dermal broblasts andthe toxicity rate depends on the concentration andsize of nanoparticles, as well as the time of exposure.Even though the internalized fate of 13 nm and 45 nmAuNPs is similar, the major penetration pathways are


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

different. In addition, our results indicate that overalltoxicity of different sized AuNPs does not depend ontotal gold concentration in the cell. Rather, the vac-uole number and stability play a signicant role in theinduction of and the disruption of the cytoskeleton.

References

Chan WC. 2007. Bio-applications applications of nanoparticles.Adv Exp Med Biol 620.

Chithrani D, Ghazani AA, Chan WC. 2006. Determining the sizeand shape dependence of gold nanoparticle uptake into mam-malian cells. Nano Lett 6:662668.

Chithrani DB, Chan CW. 2007. Elucidating the mechanism ofcellular uptake and removal of protein-coated gold nanoparticlesof different sizes and shapes. Nano Lett 7:15421550.

Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. 2005.Gold nanoparticles are taken up by human cells but do not causeacute cytotoxicity. Small 1:325327.

Corma A, Garcia H. 2008. Gold nanoparticles as catalysts fororganic reactions. Chem Soc Rev 37:20962126.

Cornell E. 2006. Cytotoxicity of gold nanoparticles in mast cells.Biolog Applicat 23.

De la Fuente JM, Berry CC, Riehle MO, Curtis AS. 2006.Nanoparticle targeting at cells. Langmuir 22:3286293.

Dixit V, Bossche JV, Sherman DM. 2007. Synthesis and grafting ofthioctic acid-PEG-folate conjugates onto Au nanoparticles forselective targeting of folate receptor-positive tumor cells.Bioconjugate Chem 17:603609.

Durr NJ, Larson T, Smith DK, Korgel BA, Sokolov K,Ben-Yakar A. 2007. Two-photon luminescence imaging of can-cer cells using molecularly targeted gold nanorods. Nano Lett7:941945.

Eghtedari M, Motamedi M, Popov VL, Kotov NA, Oraevsky AA.2004. Optoacoustic imaging of gold nanoparticles targeted tobreast cancer cells. Photons Plus Ultrasound: Imaging Sensing5320:2128.

El-Sayed I, Huang X, El-Sayed M. 2006. Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR anti-body conjugated gold nanoparticles. Cancer Lett 239:129135.

Gannon CJ, Patra CR, Bhattacharya R, Mukherjee P, Curley SA.2008. Intracellular gold nanoparticles enhance non-invasiveradiofrequency thermal destruction of human gastrointestinalcancer cells. J Nanobiotechnol 6:19.

Geiser M, Rothen-Rutishauser B, Kapp N, Schurch S, Kreyling W,Schulz H, Semmler M, Hof VI, Heyder J, Gehr P. 2005.Ultrane particles cross cellular membranes by nonphagocyticmechanisms in lungs and in cultured cells. Environ HealthPerspect 113:15551560.

Gibson AE, Noel RJ, Herlihy JT, Ward WF. 1989. Phenylarsineoxide inhibition of endocytosis: Effects on asialofetuin internal-ization. AJP Cell Physiol 257:182184.

Goodman CM, McCusker CD, Yilmaz T, Rotello VM. 2004.Toxicity of gold nanoparticles functionalized with cationic andanionic side chains. Bioconjugate Chem 15:897900.

Harjanto D. 2006. Gold nanoparticle-assisted assisted delivery ofTNF-a in thermal treatments of cancer. Biol Appl 89.

Hauck TS, Ghazani AA, Chan WC. 2008. Assessing the effect ofsurface chemistry on gold nanorod uptake, toxicity, and geneexpression in mammalian cells. Small 1:153159.

Hoet PH, Bruske-Hohlfeld I, Salata OV. 2004. Nanoparticles known and unknown health risks. J Nanobiotechnol 2(1):1217.

Hostynek JJ. 2003. Factors determining percutaneous metalabsorption. Food Chem Toxicol 41:327345.

Huang X, El-Sayed IH, El-Sayed MA. 2006. Cancer cell imagingand photothermal therapy in the near-infrared region by usinggold nanorods. J Am Chem Soc 128:21152120.

Huang X, Jain PK, El-Sayed IH, El-Sayed MA. 2007a. Goldnanoparticles: Interesting optical properties and recent applica-tions in cancer diagnostics and therapy.Nanomedicine 2:681693.

Huang X, El-Sayed IH, QianW, El-SayedMA. 2007b. Cancer cellsassemble and align gold nanorods conjugated to antibodies toproduce highly enhanced, sharp, and polarized surface Ramanspectra: A potential cancer diagnostic marker. Nano Lett7:1591597.

Huang X, Qian W, El-Sayed IH, El-Sayed MA. 2007c. Thepotential use of the enhanced nonlinear properties of gold nano-spheres in photothermal cancer therapy. Lasers Surg Med39:747753.

Huang X, Jain PK, El-Sayed IH, El-Sayed MA. 2007d. Goldnanoparticles and nanorods in medicine: From cancer diagnos-tics to photothermal therapy. Nanomedicine 2:681693.

Huang X, Jain PK, El-Sayed MA. 2007e. Plasmonic photothermaltherapy (PPTT) using gold nanoparticles. LasersMed Sci23:217228.

Kato T, Yashiro T,Murata Y, Herbert DC, Oshikawa K, BandoM,Ohno S, Sugiyama Y. 2003. Evidence that exogenous substancescan be phagocytized by alveolar epithelial cells and transportedinto blood capillaries. Cell Tissue Res 311:4751.

Khan JA, Pillai B, Das TK, Singh Y, Maiti S. 2007. Moleculareffects of uptake of gold nanoparticles in HeLa cells. Commu-nication 8:12371240.

Kreilgaard M. 2002. Inuence of microemulsions on cutaneousdrug delivery. Adv Drug Deliv Rev 54:S7798.

Krpetic Z, Porta F, Scari G. 2006. Selective entrance of goldnanoparticles into cancer cells. Gold Bull 39:6668.

Lademann J, Weigmann H, Rickmeyer C, Barthelmes H,Schaefer H, Mueller G, Sterry W. 1999. Penetration of titaniumdioxide microparticles in a sunscreen formulation into the hornylayer and the follicular orice. Skin Pharmacol Appl Skin Physiol12:247256.

Lewinsky N, Colvin V, Drezek R. 2008. Cytotoxicity of nanopar-ticles. Small 1:2649.

Mamdouh Z, Giocondi MC, Laprade R, Le Grimellec C. 1996.Temperature dependence of endocytosis in renal epithelial cellsin culture. Biochimica et Biophysica Acta. 1282:171173.

Mukherjee P, Bhattacharya R, Mukhopadhyay D. 2005. Goldnanoparticles bearing functional anti-cancer drug and anti-angiogenic agent: A 2 in 1 system with potential applicationin cancer therapeutics. J Biomed Nanotechnol 1:224228.

Murphy CJ, Gole AM, Stone JW, Sisco PN Alkilany AM,Goldsmith EC, Baxter SC. 2008. Gold nanoparticles in biology:Beyond toxicity to cellular imaging. Acc Chem Res 41:17211730.

Nanotech Full Report. 2008. Regulating emerging technologies insilicon valley and beyond. A Report by the Silicon Valley ToxicsCoalition (SVTC). April 2, 2008.

Narayanan R, El-Sayed MA. 2007. Catalysis by metallic nanopar-ticles: The good and the bad. Chimica Oggi 25:8486.

Oyelere AK, Chen PC, Huang X, El-Sayed IH, El-Sayed MA.2007. Peptide-conjugated gold nanorods for nuclear targeting.Bioconjugate Chem 18:1490497.

Paciotti GF, Kingston DG, Tamarkin L. 2006. Colloidal goldnanoparticles: A novel nanoparticle platform for developingmultifunctional tumor-targeted drug delivery vectors. DrugDevelop Res 67:4754.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

Pan Y, Neuss S, Leifert A, FischlerM,Wen F, SimonU, SchmidG,Brandau W, Jahnen-Dechent W. 2007. Size-dependent cytotox-icity of gold nanoparticles. Small 3:19411949.

Patra HK, Banerjee S, Chaudhuri U, Lahiri P, Dasgupta AK. 2007.Cell selective response to gold nanoparticles. Nanomed:Nanotechnol, Biol Med 3:111119.

Pernodet N, Fang X, Bakhtina A, Ulman A, Rafailovich M. 2006.Effects of citrate/gold nanoparticles on human dermalbroblasts. Small 2:766773.

Ponti J, Colognato R, Franchini F, Gioria S, Simonelli F, Abbas K,Rossi F. 2008. Uptake and cytotoxicity of gold nanoparticles inMDCK and HepG2 cell lines. Accessed 12 May 2009 from thewebsite: www.aist-riss.jp/projects/nedo-nanorisk/nanorisk_symposium2008/pdf/03_122_en_ROSSI.pdf

Rayavarapu RG, Petersen W, Ungureanu C, Post JN, VanLeeuwen TG, Manohar S. 2007. Synthesis and bioconjugationof gold nanoparticles as potential molecular probes for light-based imaging techniques. Int J Biomed Imaging 2007:110.

Ryan JA, Overton W, Speight ME, Oldenburg CN, Loo L,Robarge W, Franzen S, Feldheim DL. 2007. Cellular uptakeof gold nanoparticles passivated with BSA-SV40 large T antigenconjugates. Anal Chem 79:91509159.

Sudhakaran P, Prinz R, Von Figura K. 2007. Effect of temperatureon endocytosis and degradation of sulphated proteoglycans bycultured skin broblasts. J Biosci 4:413418.

Takahashi H, Niidome Y, Niidome T, Kaneko K, Kawasaki H,Yamada S. 2006. Modication of gold nanorods using phos-phatidylcholine to reduce cytotoxicity. Langmuir 22.

Tinkle SS, Antonini JM, Rich BA, Roberts JR, Salmen R,DePree K, Adkins EJ. 2003. Skin as a route of exposure andsensitization in chronic beryllium disease. Environ HealthPerspect 111:12021208.

Turkevich J. 1985a. Colloidal gold. I. Gold Bull 18(3):8692.Turkevich J. 1985b. Colloidal gold. II. Gold Bull 18(4): 125131.Yang PH, Sun X, Jen-Fu C, Sun H, Qing-YuH. 2005. Transferrin-

mediated gold nanoparticle cellular uptake. Bioconjugate ChemChem 16:494496.


Nan

otox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f S.A

Lib

119

257

on 1

1/22

/12

For p

erso

nal u

se o

nly.

gold nanoparticles cellular toxicity

Documents