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Toxicology 308 (2013) 1– 9

Contents lists available at SciVerse ScienceDirect

Toxicology

jo u r n al homep age: www.elsev ier .com/ locate / tox ico l

uantum dots induced monocyte chemotactic protein-1 expression viayD88-dependent Toll-like receptor signaling pathways in macrophages

hia-Chi Hoa, Yueh-Hsia Luob, Tsung-Hsien Chuangc, Chung-Shi Yangd,ong-Chien Linga,e,∗, Pinpin Linb,∗∗

Institute of NanoEngineering and Microsystems, National Tsing Hua University, Hsinchu, Taiwan, ROCDivision of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Taiwan, ROCImmunology Research Center, National Health Research Institutes, Zhunan, Taiwan, ROCCenter for Nanomedicine Research, National Health Research Institutes, Zhunan, Taiwan, ROCDepartment of Chemistry, National Tsing Hua University, Hsinchu, Taiwan, ROC

a r t i c l e i n f o

rticle history:eceived 19 December 2012eceived in revised form 8 March 2013ccepted 8 March 2013vailable online 15 March 2013

eywords:D705

a b s t r a c t

Quantum dots (QDs) are nano-sized semiconductors. Previously, intratracheal instillation of QD705sinduces persistent inflammation in mouse lungs. In our present study, QD705-COOH and QD705-PEGactivated NF-�B and increased monocyte chemotactic protein-1 (MCP-1) expression in macrophagesRAW264.7 via MyD88 dependent Toll-like receptor (TLR) signaling pathways. MyD88 is an adapter pro-tein for most TLRs to activate NF-�B. Silencing expression of MyD88 or p65 with siRNA or co-treatmentwith a NF-�B inhibitor tremendously abolished QD705s-induced NF-�B activity and MCP-1 expression.The involved TLRs might locate either on the cell surface or inside of cells. Co-treatment with a TLR4

nflammationCP-1F-�ByD88

LRs

inhibitor completely prevented MCP-1 induction by QD705-PEG. Nevertheless, QD705-COOH readilyentered cells, and co-treatment with either inhibitors of endocytosis or intracellular TLRs preventedMCP-1 induction. These findings indicate that, depending on their surface modification, OD705s acti-vate MyD88 dependent-TLRs at the surface or inside of the cells, which is an important mechanismfor nanoparticles-induced inflammatory responses. But other MyD88-independent pathways may also

s.

involve in these response

. Introduction

Quantum dots (QDs) are semiconductor nanocrystals with unique autofluorescent property that have great potential iniomedicine in the areas of diagnosis, drug delivery, and imag-

ng (Zhang et al., 2008; Mulder et al., 2010; Obonyo et al., 2010).D705 emits stable near-infrared fluorescence and is approxi-ately 12–20 nm in diameter; it consists of a metalloid crystalline

ore (Cd/Se/Te) surrounded by a thin shell of ZnS. The near-infrareduorescence of QD705 allows for improved photon penetrationhrough tissue and minimizes the effects of tissue autofluorescenceHilderbrand and Weissleder, 2010). The surface of QD705 can be

odified for specific applications, such as fluorescence efficiency,olubility in biological media or bio-conjugation to antibodies; as aesult the modified QD705s can be used for specific diagnostic and

∗ Corresponding author at: Department of Chemistry, National Tsing Hua Univer-ity, Hsinchu, Taiwan, ROC. Tel.: +886 3 5715131x33393; fax: +886 3 5711082.∗∗ Corresponding author at: Division of Environmental Health and Occupational

edicine, National Health Research Institutes, Zhunan, Taiwan, ROC.el.: +886 37 246166x36508; fax: +886-37-587406.

E-mail addresses: ycling@mx.nthu.edu.tw (Y.-C. Ling), pplin@nhri.org.tw (P. Lin).

300-483X/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.tox.2013.03.003

© 2013 Elsevier Ireland Ltd. All rights reserved.

therapeutic purposes (Gao et al., 2004; Mulder et al., 2010). QD705-COOH has carboxyl groups conjugated onto the surface of QD705.QD705-PEG has been surface modified with methoxy-polyethyleneglycol, which is an inert biologically compatible polymer. PEGmodification of nanoparticles has been reported to increase theirhalf-life and to reduce their accumulation in the liver and bonemarrow (Ballou et al., 2004).

Although QDs have great potential in terms of clinical applica-tions, they have been shown to cause adverse effects in vitro andin vivo, especially inflammatory responses. Romoser et al. (2011)reported that QDs treatment activated NF-�B and upregulatedapoptotic, inflammatory and immunoregulatory proteins such asTNF-�, IL-1� and IL-10 in human skin cells. Fischer et al. (2010)showed that QD treatment increased IL-6 expression in Kupffercells. QDs also induced TNF- ̨ expression in macrophages J774A.1cells (Clift et al., 2010). Recently, we demonstrated that intravenousadministration of QD705-PEG increased TNF- ̨ and IL-6 expressionin mouse liver (Lin et al., 2011). Intratracheal instillation of QD705-PEG and QD705-COOH induced persistent severe inflammation,

including immune cell infiltration, cytokines expression and a gran-ulomatous reaction in the mouse lung (Ho et al., 2013). However,the mechanisms by which QDs induce inflammatory responsesremain unclear.

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Toll-like receptors (TLRs) in mammals are the essential effec-ors for the activation of inflammation, innate immunity, and theubsequent activated adaptive immunity that recognize differ-nt pathogen or danger associated molecular patterns (Iwasakind Medzhitov, 2004). TLRs consist of ten and thirteen distinctamilies in humans and mice, respectively. The cellular locationf the TLR families in cells varies a lot. TLR1, TLR2, TLR4, TLR5,LR6 are mainly expressed on the cell surface where they recog-ize microbial components such as LPS (Kumar et al., 2009). Onhe other hand, TLR3, TLR7, TLR8, and TLR9 are located withinntracellular vesicles such as the endoplasmic reticulum (ER), endo-omes, and lysosomes, and were originally shown to detect nucleiccids (Blasius, 2010). Activation of TLRs results in the inductionf immune responses via their coupling with intracellular adap-or molecules of the MyD88 family, including, MyD88, Mal/TIRAP,RIF/TICAM-1, TRAM/TIRP and SRAM (Cook et al., 2004; Michelsent al., 2004). All TLRs, except TLR3, signal through a MyD88-ependent pathway. In this pathway, activation of the TLR triggersormation of a MyD88/IRAK1/IRAK4/TRAF6 complex, which leadso the activation of various transcription factors including activat-ng protein-1(AP-1) and NF-�B, and thence to the production ofhemokines and inflammatory cytokines, such as TNF-�, IL-1�, IL-6nd MCP-1(Takeda and Akira, 2004; Premkumar et al., 2010).

Previously, we have observed that QD705s induce cytokineroduction and inflammation, which are characteristics of TLRctivation (Lin et al., 2011). Thus, we hypothesized that MyD88nd TLRs might be involved in the inflammatory responses andhe granulomatous reaction that are induced by nanoparticlesuch as QD705 in mouse lungs. Macrophages are the first innatemmune cells recruited that come into contact with foreign agentsn the lungs. Monocyte Chemoattractant Protein-1 (MCP-1) is anmportant chemokine under pathological conditions that involve

onocyte/macrophage and memory T cells infiltration (Hasegawat al., 1999). QD705s persistently elevated MCP-1 mRNA levelsn mouse lungs (Ho et al., 2013). In our present study, we uti-ize the production of MCP-1 in RAW264.7 cells as a model tonvestigate the role of MyD88 and TLRs in QD705 induced inflam-

atory responses. Our findings indicate that MyD88 dependent TLRignaling plays a key role in QD-induced NF-�B activation and MCP-

induction, which provides important new insights regarding theechanisms by which these nanoparticles induced inflammation.

. Method and materials

.1. Materials

CLI-095, Imiquimod, R848 and CpG-2006 were purchased from InvivoGen (Saniego, CA, USA). Chloroquine, polymyxin B, cytochalasin B and dynasore were pur-hased from Sigma (St Louis, MO, USA). Bay117085 was purchased from BIOMOLnternational, L.P. (Plymouth Meeting, PA, USA).

.2. Quantum dot 705 (QD705)

The QD705 nanoparticles used in our experiments were purchased fromnvitrogen, Inc. (Hayward, CA, USA) as Qtracker 705 non-targeted quantum dotsQD705-PEG) and Qdot® 705 ITKTM carboxyl quantum dots (QD-COOH). QD705-PEGnd QD705-COOH contained a Cd/Se/Te core covered with a ZnS shell. QD705-PEGas modified with a methoxy-PEG-5000 coating; QD705-COOH was modified with

arboxyl groups.

.3. Physicochemical characterization of the QD705s

QD705s were characterized in terms of their size distribution, zeta potential,ize, and shape. The hydrodynamic diameters and zeta potentials of QD705s wereeasured with the Zetasizer Nano system (Zetasizer Nano ZS, Malvern Instru-ents, Worcestershire, UK). The zeta potential of QD705-PEG was −6.4 ± 0.6 mV and

D705-COOH was −22.7 ± 0.4 mV. Dynamic laser light scattering measurementsere checked using the single-scattering regime with l = 633 nm and at an angle of

73◦ . The suspension was put into a cuvette at 25 ◦C to enable particle size and zetaotential analysis. The shape and size of the nanoparticles were assessed by trans-ission electron microscopy (TEM) (H-7650, Hitachi, Japan). To do this a suspension

gy 308 (2013) 1– 9

of each QD705 type was dripped on a copper grid for TEM. All of the copper gridswere preserved in a dry cabinet.

2.4. Microdialysis system for separating cadmium ion from intact QD705s

A microdialysis (MD) system was purchased from the Carnegie Medicine Asso-ciates (CMA, Stockholm, Sweden). The MD sampling system, which included amicroinjection syringe pump (CMA/400) and a 10 mm long MD probe (CMA/20)were purchased from CMA (Carnegie Medicine Association, Solna, Sweden). TheMD system was perfused with 50 mM borate buffer solution at a flow rate of1 �L/min. Dialysate samples were collected every hour for 24 h and their Cd con-centrations were analyzed using inductively coupled plasma mass spectrometry(ICP-MS) (Elan6100; Perkin-Elmer, Shelton, CT, USA).

2.5. The Gel-clot Limulus amebocyte lysate (LAL) assay

The LAL assay was performed in duplicate using commercial LAL reagent con-taining Limulus amoebocyte lysate (Charles River Endosave, Charleston, SC, USA).The assay was performed in pyrogen-free test tubes to which 0.2 ml of 30 �g/mlQD705s and 0.2 ml LAL reagent were added. Following 1 h of incubation at 37 ◦C, thetest tubes were examined by 180◦ inversion for the presence of a stable solid clot.A clotted incubation mixture was considered to be a positive result. LPS standard(0.125 EU/ml) and pyrogen-free LAL reagent water, both provided by the manufac-turer, were used as a control.

2.6. Cell culture

The mouse macrophage RAW264.7 cells were purchased from American TypeCulture Collection (Manassas, VA) and were maintained in Dulbecco’s ModifiedEagle’s Medium (GIBCOTM) with 4 mM l-glutamine, 1.5 g/L sodium bicarbonate, and10% fetal bovine serum (FBS), and incubated in a 37 ◦C incubator with a humidifiedmixture of 5% CO2 and 95% air. The medium was changed twice a week, and cellswere sub-cultured by scraping every week.

2.7. Cell viability

Cell viability was determined by the dimethylthiazoldiphenyltetrazolium bro-mide (MTT) assay. RAW264.7 cells were seeded in 24-well plates for 24 h and thenincubated with vehicle, QD705-PEG and QD-COOH for 48 h in serum free DMEMmedium. Subsequently, 1 mg/ml MTT was added to the medium and cells were incu-bated for an additional 2 h. Precipitated formazan was dissolved in 1 ml DMSO andthe absorbance was measured at 535 nm. The data are presented as a percentage ofcontrols. All vehicles, including 1% borate buffer and 0.03% mouse serum, showedno significant cytotoxicity.

2.8. Quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) assay

RAW264.7 (1 × 106 cells per well) were seeded into 6 cm dishes and then treatedwith vehicle, QD705-PEG and QD705-COOH for 24 h in serum free medium. TotalRNA was prepared using RNAzol reagent (Life Technologies, Rockville, MD), andthen total RNA was purified via extraction with double distilled H2O. Synthesisof cDNA was performed using the High-Capacity cDNA Archive kit (P/N4322171,Applied Biosystems, Foster City, CA, USA) using 3.0 �g total RNA. Quantitative poly-merase chain reaction was carried out using the TaqMan Universal PCR Master Mix(Applied Biosystems, Foster City, CA, USA) and analyzed on ABI StepOneTM andStepOnePlusTM Real-time PCR Systems (Perkin-Elmer Applied Biosystems, FosterCity, CA, USA). The PCR primers for monocyte chemotactic protein-1 (MCP-1), andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) were included in the Assay-on-Demand Gene Expression Assay Mix (Applied Biosystems, Foster City, CA, USA).The PCR program: 95 ◦C for 10 min followed by 40 cycles of 60 ◦C for 1 min with 95 ◦Cfor 15 s. Quantitative values were obtained from the threshold cycle (Ct) number.Target gene expression level was normalized against GAPDH mRNA expression foreach sample. The relative mRNA levels of the target gene are presented as 2–�Ct ,where �Ct = Cttarget gene − CtGAPDH.

2.9. Enzyme-linked immunosorbent (ELISA) assay

RAW264.7 (1 × 105 cells per well) were seeded into 24-well dishes and thentreated with QD705-PEG and QD705-COOH with or without an inhibitor (10 �g/mlpolymyxin B, 5 �g/ml cytochalasin B, 80 �M dynasore, 20 �M chroloquine or

1.25 �M Bay117085) under serum free medium for 2 days. The culture mediumwas replaced with 0.5 ml serum free DMEM medium per well, and conditionedmedium was collected 48 h later. The MCP-1 concentration in the medium wasdetermined using a mouse MCP-1 ELISA kit (R&D Systems, Inc.) according to themanufacturer’s instructions.

oxicology 308 (2013) 1– 9 3

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.10. Reporter genes assays

For the luciferase assays, RAW264.7 cells (2 × 105) were seeded onto 12-welllates, and co-transfected with the pNF-�B-Luc (5′-GGGGACTTTCC-3′)5 or AP-15′-TGACTAA-3′)7 reporter vector (Stratagene, La Jolla, CA) and pCMV-�-gal usingipofectamine 2000 (Invitrogen, San Diego, CA, USA) according to the manu-acturer’s protocol. After 6 h of incubation, the medium was carefully removednd fresh serum free DMEM medium containing various treatments were addednto the wells. The cells were treated with QD705-PEG or QD705-COOH for 24 h.hese cells were then collected and transcriptional activity was assayed using theuciferase Assay System (Promega, Madison, USA) and a luminometer (Bertholdnalytical Instruments, Nashua, NH). Each experiment was repeated at least four

imes.

.11. RNA interference (RNAi) knockdown in RAW264.7 cells

The MyD88 target sequence used was GCAUUUUAAAGCAACCUGGTT. The siRNAuplexes were transfected using lipofectamine 2000 (Invitrogen) into RAW264.7ells following the manufacturer’s protocol. Briefly, cells were plated at 1 × 106

ell/well in 6 cm dishes. After 24 h, cells were treated with 100 nM of MyD88-iRNAs in a transfection mixture containing 10 �l/well of lipofectamine 2000 and

ml of Opti-MEM I reduced-serum medium (GBICO) for 6 h in 5% CO2 at 37 ◦C. Onhe following day, the MyD88 siRNA transfected cells were transferred to freshrowth medium for 24 h. Transfected cells were then treated QD705 for 24 h tossess the effect of MyD88 gene knockdown using the quantitative real-time RT-PCRssay.

.12. Cellular uptake of QD705s

RAW264.7 (5 × 104 cells per well) were seeded onto glass coverslips and thenncubated with QD705-PEG and QD705-COOH under serum free medium for 3 h.n some cases this was carried out in the presence of an inhibitor (cytochalasin

or dynasore). Next, the cells were washed 3 times with PBS and fixed with0% formaldehyde for 10 min. The cells were then stained with 4′ ,6-diamidino--phenylindole (DAPI) for 2 min. Finally the fluorescence of the cells was imagedy fluorescence microscopy (Leica DMRXA, Wetzlar, Germany). We also quanti-ed cellular uptake of QD705s in RAW264.7 cell. RAW264.7 cells (1 × 106 cells perell) were incubated with 30 �g QD705-PEG or QD705-COOH for 24 h. Media and

ells were separately collected at 24 h later and liquefied with 1 ml nitric acid andubjected to microwave digestion (CEM MARS; CEM Corporation, Matthews, NC,SA). ICP-MS (Elan6100; Perkin-Elmer, Shelton, CT, USA) was used to determine

he 111Cd concentrations. QD705-PEG and QD705-COOH respectively contain 60%nd 50% (weight/weight) Cd. The uptake of QD705s was calculated by dividing thed amounts in cell pellets with the sum of Cd amounts in cell pellets and conditionededia. Total Cd recovered from cell pellets and conditioned media was 90–100%.

.13. Co-localization studies with lysosomes

RAW264.7 cells (2 × 105 cells) were seeded in 3.5 cm �-dish (ibidi; GmbH,artinsried, Germany). Cells were incubated with QD705-COOH and 150 nM Lyso-

racker Green DND-26 (molecular probes) for 2 h in serum free DMEM medium. Theells were rapidly washed with ice cold PBS to prevent the removal of the attachedysoTracker Green, and replaced with fresh medium, then the living cells werebserved under the Olympus FLUOVIEW FV10i laser scanning confocal fluorescenceicroscopy.

.14. Statistical analysis

Treated and control groups were compared using one-way analysis of vari-nce followed by the Tukey’s Honestly Significant Difference test (p < 0.05 ashe significance level). The MTT assays were compared using the Student’s-test (p < 0.05).

. Results

.1. Physicochemical properties of QD705-PEG and QD705-COOH

The shape and size of the nanoparticles were characterizedy TEM and DLS (Fig. 1, Table 1). QD705-PEG and QD705-COOH

able 1hysicochemical characterization of the nanoparticles.

Inorganic Hydrodynamic diameter inDMEM medium (nm)

Size (TEM)(nm)

Cd io(gm/

QD705-PEG Cd/Te/Se 48.9 ± 3.9 10 ± 2 <0.1

QD705-COOH Cd/Te/Se 38.9 ± 8.6 10 ± 2 <0.1

Fig. 1. Sizes and shapes of QD705s. (A) Transmission electron microscopy (TEM)images of (a) QD705-PEG and (b) QD705-COOH. (B) The size distribution by DLS ofQD705-PEG and QD705-COOH.

were spherical-shaped nanoparticles with a core size of 10 ± 2 nm.The diameters of QD705-PEG and QD705-COOH in medium were48.9 ± 3.9 and 38.9 ± 8.6 nm, respectively (Fig. 1B). As shown inTable 1, the free Cd ion contents of QD705-PEG and QD705-COOHwere less than 0.01‰ (gm/gm).

3.2. Effect of QD705-PEG and QD705-COOH on MCP-1 expression

Previously, we have demonstrated that intratracheal instilla-tion of QD705-COOH or QD705-PEG induces acute inflammation,granuloma, and the expression of cytokines/chemokines in thelungs of mice. QD705-COOH induced much higher levels ofcytokines/chemokines than did QD705-PEG (Ho et al., 2013). In thepresent study we investigated the molecular basis of how thesenanoparticles activate the inflammatory responses. To do this, wefirst determined that these two types of nanoparticles were cyto-toxic and that QD705-COOH was more cytotoxic than QD705-PEGin mouse RAW264.7 macrophages; this difference becomes rel-evant when the dose used was higher than 1.5 �g/ml (Fig. 2A).We further investigated whether these nanoparticles inducedcytokines/chemokines at low-cytotoxic doses (0.15–1.5 �g/ml).Both QD705-PEG and QD705-COOH increased the mRNA level ofMCP-1 in the time-dependent manner. In a similar manner to theirinflammation stimulatory effect on mouse lungs (Di Gioacchinoet al., 2011; Kamata et al., 2011), it was found that QD705-COOHshowed a greater inductive effect on MCP-1 at the mRNA level

than QD705-PEG (Fig. 2B). However, we treated with 0.7 �g/ml ofCdCl2 (Cd molecular equivalent to 1.5 �g/ml of QD705s) in mouseRAW264.7 macrophages. Free Cd2+ failed to increase MCP-1 mRNAlevels (data not shown). Furthermore, QD705-COOH was able to

n in QD705gm, ‰)

LAL assay (EU/ml in15 �g/ml QD705)

Cellular uptake of QD705s (%) (Cd incells/Cd in cells and media) (24 h in cells)

<0.5 1.8 (0.3 �g/16.4 �g)<0.5 75.3 (11.9 �g/15.8 �g)

4 C.-C. Ho et al. / Toxicolo

Fig. 2. QD705-PEG and QD705-COOH increase MCP-1 expression in RAW264.7 cells.(A) Cells were treated with vehicle control, and different concentrations of QD705-PEG or QD705-COOH for 48 h. Cell viability was determined by MTT assay. (B) Cellswere treated with vehicle control, and different concentrations of QD705-PEG orQD705-COOH for 24 h. (C) Cells were treated with vehicle control, 1.5 �g/ml ofQomv

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D705-PEG or 1.5 �g/ml of QD705-COOH for different period of times. Expressionf MCP-1 mRNA was determined by real-time PCR. The results are presented as theean ± standard deviation (S.D.) from six or three independent experiments for cell

iability and mRNA, respectively. *p < 0.05 compared with vehicle-treated cells.

nduce an increase in MCP-1 mRNA levels within 48 h in a time-ependent manner (Fig. 2C).

.3. MyD88 is required for QD705-PEG and QD705-COOH

nduced MCP-1 expression and NF-�B activation

MyD88 is required by all TLRs except TLR3 in order to acti-ate NF-�B and cytokine production during the initiation of

gy 308 (2013) 1– 9

inflammatory responses (Takeda and Akira, 2001, 2004;Premkumar et al., 2010). To determine whether the effects ofQD705-PEG and QD705-COOH are mediated by MyD88, we inves-tigated the activities of these two types of nanoparticles in MyD88knockdown macrophages and compared the results with LPS, aTLR4 agonist. Reducing MyD88 expression by siRNA knockdown to30% of control (data not shown) significantly reduced LPS, QD705-PEG and QD705-COOH induced MCP-1 expression in RAW264.7cells (Fig. 3A). NF-�B and AP-1 are downstream transcriptionfactors that regulate the expression of cytokines and chemokinesinduced by MyD88-dependent TLRs (Pahl, 1999; An et al., 2002;Takeda and Akira, 2004; Yamamoto and Akira, 2004). WhileQD705-PEG and QD705-COOH significantly increased NF-�B andAP-1 reporter activity, the increase in NF-�B activity was muchgreater than that in AP-1 activity (Fig. 3B and C). Consistently,reducing MyD88 expression by siRNA knockdown significantlyprevented the induction of NF-�B and AP-1 activity by LPS, QD705-PEG and QD705-COOH (Fig. 3B and C). In addition, preventingNF-�B activation by co-treatment with Bay117085, an I�B kinasealpha inhibitor, or by interfering with the expression of the p65subunit of NF-�B by siRNA knockdown significantly preventedthe induction of NF-�B and AP-1 activity by LPS, QD705-PEG andQD705-COOH (Fig. 3D and E). These results support the hypothesisthat MyD88 is required for QD705-PEG and QD705-COOH inducedNF-�B activation and MCP-1 expression.

3.4. QD705-PEG induces MCP-1 via TLR4 but QD705-COOHactivates TLR4 and other intracellular TLRs

Given that MyD88 is essential for the QD-705 induced cell acti-vation, we reasoned that TLRs are involved in the cellular signalingof these nanoparticles. We further investigated the part played bythe various TLRs in QD705-induced MCP-1 expression. CLI-095 isa TLR4 inhibitor that inhibits TLR4 activation in a ligand stimula-tion in a dependent and in a ligand stimulation in an independentmanner (Kawamoto et al., 2008). As shown in Fig. 4A, co-treatmentof cells with LPS and CLI-095 effectively blocked LPS-inducedMCP-1 expression. In this context, CLI-095 slightly reduced (13%inhibition) the QD705-COOH induced expression of MCP-1, buttremendously reduced (50% inhibition) the QD705-PEG-inducedexpression of MCP-1 (Fig. 4A).

Nanoparticles are frequently contaminated with LPS, whichalso induces the production of inflammatory mediators suchas cytokines and chemokines (Dobrovolskaia et al., 2009). Todemonstrate whether the stimulatory effects of QD705-PEG andQD705-COOH were a result of the LPS contamination, the pres-ence of LPS in these nanoparticles was analyzed by Gel-clot LALassay. This assay is an appropriate test for QDs because it seemsthat QDs might interfere with the chromogenic limulus amoebo-cyte lysate (LAL) assay (Dobrovolskaia et al., 2009). These resultsindicated that the LPS contents in these two type of QD705s werelower than 0.5 EU/ml (Table 1), which would not activate the cellssignificantly. In addition, co-treatment with polymyxin B, a LPSinhibitor (Cooperstock, 1974), also failed to prevent QD705-PEGand QD705-COOH induced MCP-1 production, while polymyxin Balso completely abolished LPS-induced MCP-1 secretion (Fig. 4B).These results confirmed that QD705-induced MCP-1 expressionwas not due to LPS contamination of the nanoparticles. Overall,these findings indicate that QD705-PEG mainly activated TLR4,which in turn induced MCP-1 expression. However, they also showthat TLR4 only plays a minor role in QD705-COOH induced expres-sion of MCP-1 in RAW264.7 cells.

The TLR family includes both cell surface and intracellular recep-tors (Blasius, 2010). TLR4 is a cell surface receptor. We furtherinvestigated whether intracellular TLRs were involved in QD705-induced expression of MCP-1. Chloroquine is an intracellular TLRs

C.-C. Ho et al. / Toxicology 308 (2013) 1– 9 5

Fig. 3. MyD88 is required for QD705-PEG and QD705-COOH induced MCP-1 expression and NF-�B activation. Cells transfected with MyD88 SiRNA or control SiRNA weretreated with 1 ng/ml of LPS, 1.5 �g/ml of QD705-PEG or 1.5 �g/ml of QD705-COOH for 48 h. (A) MCP-1 protein in the media was measured by ELISA assay. (B) NF-�B and (C)AP-1 activity were detected by reporter assay. (D) Cells were treated with 1 ng/ml of LPS, 1.5 �g/ml of QD705-PEG or 1.5 �g/ml of QD705-COOH with or without Bay117085.( l of LPt dent

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E) Cells transfected with p65 SiRNA or control SiRNA and then treated with 1 ng/mhe media was measured by ELISA. The results are the mean ± S.D. for four indepenith LPS–treated cells; §p < 0.05 as compared with the QD705-COOH-treated cells;

nhibitor that prevents endosome maturation (Rutz et al., 2004). Ashown in Fig. 4C, co-treatment with chloroquine effectively blockedntracellular TLRs ligands-induced MCP-1 expression. We foundhat co-treatment of QD705-COOH with chloroquine reduced 60%f the QD705-COOH induced MCP-1 secretion. However, no simi-ar effect was observed with the co-treatment of QD705-PEG andhloroquine (Fig. 4C). These findings suggested that QD705-COOHnduces MCP-1 mainly via intracellular TLRs.

.5. Cellular locations for the functioning of QD705-PEG andD705-COOH

To confirm that QD705-PEG activated the cell surface TLR4 andhile QD705-COOH mainly activated the intracellularly expressed

LRs, we further investigated the cellular localization of functionalD705-PEG and QD705-COOH. The red fluorescence emitted by

hese nanoparticles was detected via fluorescence microscopy. Theptake of QD705-COOH (75.3%) was greater than that of QD705-EG (1.8%) (Table 1). The uptake of QD705-COOH was reducedy the endocytosis inhibitors, cytochalasin B and dynasore inAW264.7 cells (Fig. 5A). Furthermore, cytochalasin B and dyna-ore inhibited 87% and 64% of the QD705-COOH-induced secretionf MCP-1, whereas these two inhibitors did not significantly inhibithe effects of QD705-PEG (Fig. 5B and C). In addition, fluorescence

icroscopy showed that QD705-COOH colocalized with lysosomest 2 h (Fig. 5A). Overall these results further support the hypothesishat the activation of cell surface TLR4 and of intracellular TLRs byD705-PEG and QD705-COOH, respectively, is responsible for the

nduction of a range of immunity related cellular responses.

. Discussion

An acute inflammatory reaction is one of the major adverseesponses to nanoparticles in biological systems (Kagan et al.,

S, 1.5 �g/ml of QD705-PEG or 1.5 �g/ml of QD705-COOH for 48 h. MCP-1 protein inexperiments. *p < 0.05 compared with vehicle-treated cells; #p < 0.05 as compared05 as compared with the QD705-PEG–treated cells.

2005; Tetley, 2007). NF-�B is a major transcription factor involvedin inflammatory responses and can be induced by a varietyof different stimuli (Murray et al., 2009; He et al., 2011).Recently we demonstrated that intratracheal instillation of QD705sinduces cytokine and chemokine expression, persistent inflam-mation and a granulomatous reaction in the mouse lungs (Hoet al., 2013). QD705s-induced granulomas are composed mainly ofmacrophages, and these are mixed with lymphocytes, neutrophils,and other inflammatory cells (Ho et al., 2013). In the presentstudy, we demonstrated that the MyD88-dependent TLR pathwaysare responsible for QD705-induced NF-�B activation and MCP-1expression in macrophages. While both types of QD705-PEG andQD705-COOH were able to induce these various cellular responsesthrough the cell surface TLR4-MyD88 signaling pathway, QD705-COOH mainly acted via MyD88-dependent intracellular TLRs. Wealso noticed that the kinetics of MCP-1 induction was different forQD705-PEG and QD705-COOH. While MCP-1 induction by QD705-COOH persistently increased over 48 h, induction by QD705-PEGdid not. This is consistent with the results of our previous in vivostudy whereby in mouse lungs QD705-COOH induced more persis-tent inflammation with granulomatous reaction than QD705-PEG(Ho et al., 2013).

The surface chemistry of nanoparticles has been shown to influ-ence the toxicity and biodistribution of nanoparticles in biologicalsystems (Ryman-Rasmussen et al., 2006). PEGylation is known toprevent phagocytosis (Kohler et al., 2006). PEG-coated nanopar-ticles show an increased half-life and altered biodistribution inbiological systems (Owens and Peppas, 2006). On the other hand,QD705-COOH accumulated inside the cell. Which of the intracel-lular TLRs are responsible for QD705-COOH activation remains

unclear at the moment. When nanoparticles enter a biological fluid(such as the intracellular fluid, blood or interstitial fluid), theyare usually coated with proteins that then may undergo confor-mational change, which can lead to adverse responses (Cedervall

6 C.-C. Ho et al. / Toxicology 308 (2013) 1– 9

Fig. 4. QD705-PEG induces MCP-1 via TLR4 and QD705-COOH induces MCP-1 viaboth TLR4 and intracellular TLRs. (A) Cells were treated with vehicle control, 1 ng/mlof LPS, 1.5 �g/ml of QD705-PEG or 1.5 �g/ml of QD705-COOH with or without0.5 �g/ml of CLI-095. (B) Cells were treated with vehicle control, 1 ng/ml of LPS,1.5 �g/ml of QD705-PEG or 1.5 �g/ml of QD705-COOH with or without 10 �g/ml ofpolymyxin B. (C) Cells were treated with vehicle control, 1 ng/ml of LPS, 2.5 �g/mlImiquimod (TLR7 ligand), 2 �M R848 (TLR8 ligand), 2.5 �g/ml CpG-2006 (TLR9ligand), 1.5 �g/ml of QD705-PEG or 1.5 �g/ml of QD705-COOH with or without20 �M of chloroquine. MCP-1 protein in the media was measured by ELISA after48 h. The results are the mean ± S.D. for four independent experiments. *p < 0.05compared with vehicle-treated cells; #p < 0.05 as compared with LPS-treated cells;&p < 0.05 as compared with Imiquimod-treated cells; p < 0.05 as compared withR † §

ct

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Fig. 5. Cellular localization of QD705-PEG and QD705-COOH. (A) Cells were treatedwith vehicle control, 1.5 �g/ml of QD705-PEG or 1.5 �g/ml of QD705-COOH withor without 10 �g/ml of cytochalasin B (CB) or 80 �M of dynasore for 3 h. Localiza-tion of QD705-PEG and QD705-COOH in the RAW264.7 cells were determined usingred fluorescence via fluorescence microscopy. Co-localization of QD705-COOH withlysosomes in the RAW264.7 cells were determined using red fluorescence of QD705-COOH and green fluorescence of LysoTracker via confocal fluorescence microscopy.(B) Cells were treated with vehicle control, 1 ng/ml of LPS, 1.5 �g/ml of QD705-PEGor 1.5 �g/ml of QD705-COOH with or without 10 �g/ml of cytochalasin B. (C) Cellstreated with vehicle control, 1 ng/ml of LPS, 1.5 �g/ml of QD705-PEG or 1.5 �g/ml ofQD705-COOH with or without 80 �M of dynasore. After 48 h, MCP-1 protein concen-

848-treated cells; p < 0.05 as compared with CpG-2006-treated cells; p < 0.05 asompared with QD705-COOH-treated cells; ‡p < 0.05 as compared with QD705-PEG-reated cells.

t al., 2007; Lundqvist et al., 2008). For example, internalizedold nanoparticles are known to accumulate in lysosomes; they

hen bind to high-mobility group box-1 (HMGB-1) in the lyso-omes and HMGB-1 then interacts with the TLR9 signaling pathwayTsai et al., 2012). It has been reported that QD is translocatednto the endosomes of cells (Hanaki et al., 2003; Xiao et al.,

tration in the media was quantified by ELISA. The results are the mean ± S.D. for fourindependent experiments. *p < 0.05 compared with vehicle-treated cells; #p < 0.05 ascompared with LPS-treated cells; §p < 0.05 as compared with QD705-COOH-treatedcells; ‡p < 0.05 as compared with QD705-PEG–treated cells.

2010). Thus it is reasonable that this intracellular accumulation ofQD705-COOH might interact with one or more intracellular TLRs,namely TLR3, TLR7, TLR8 and TLR9. Consistent with the above,our results show that chloroquine blocks the induction of MCP-1expression by QD705-COOH. Chloroquine is an inhibitor that tar-gets the activation of these TLRs.

Many nanoparticles have been reported to induce inflammatory

responses in vivo and in vitro (Lam et al., 2004; Muller et al., 2005).However, few studies have investigated the effect of nanoparticleson TLR activation and only a few papers have shown the effect ofnanoparticles on the mRNA levels of a range of TLRs. Lucarelli et al.

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2004) showed that TiO2 and ZrO2 nanoparticles increased TLR7 andLR10 mRNA levels in human macrophages U-937 cells. Cui et al.2011) reported that TiO2 nanoparticles increased TLR2 and TLR 4

RNA levels in the mouse liver. Castillo et al. (2008) demonstratedhat modified silver nanoparticles enhanced the TLR3 ligand-nduced and TLR9 ligand-induced IL-6 secretion by RAW264.7 cells.n contrast, SiO2 nanoparticles decreased TLR9 mRNA levels in U937ells (Lucarelli et al., 2004). Romoser et al. (2011) reported that QDailed to increase TLR3, TLR4 and TLR6 mRNA levels in human dermalbroblasts. In our present study, although QD705-PEG and QD705-OOH failed to increase the mRNA level of any tested TLR (TLR2,LR4, TLR7, TLR8 and TLR9) (data not shown), however, QD705 didctivate various TLR-mediated pathways in RAW264.7 cells. Thust would appear that QD705 does not activate TLR pathways viancreasing TLR expression.

NF-�B has been reported to be involved in mediating nanopar-icles induced inflammatory responses (Castillo et al., 2008). TiO2anoparticles have been found to increase the mRNA levels of TLR2nd TLR4 in mouse liver via NF-�B activation (Cui et al., 2011).omoser et al. (2011) showed that QD induced expression of IL-� and MCP-1 at the mRNA level via the NF-�B pathway. However,he precise mechanisms of NF-�B activation by these nanoparticlesemain unclear. Ligation of TLRs is known to activate NF-�B and AP-

through MyD88 (Makela et al., 2009). Following TLR activation,yD88 interact with IL-1 receptor-associated kinase, tumor necro-

is factor receptor-associated factor 6, TGF-�-activated kinase andAK1-binding proteins; this subsequently activates NF-�B and AP-1Wang et al., 2001). In our present study, we showed that a numberf different MyD88-TLR pathways were involved in cellular activa-ion by QD705s. This helps to fill the gap in our knowledge related toow nanoparticles activate NF-�B. In addition, in our study, QD705sere more effective at activating the NF-�B reporter than the AP-1

eporter, which suggests that NF-�B may play a more important

ole than AP-1 in cell activation by QD705s.

In our study, although QD705s mainly induced MCP-1 expres-ion via TLR-MyD88 signaling pathway in RAW264.7 cells, but otherathways may also involve in QD705s-induced MCP-1 expression.

ig. 6. A model explaining the molecular mechanisms by which QD705 induces inflammaanoparticles. QD705-PEG activates various transcription factors including NF-�B and AP-LR4-MyD88 signaling pathway. QD705-COOH mainly activates the same cellular responsctivity is mediated by TLR4. These responses result in inflammation at the nanoparticle

gy 308 (2013) 1– 9 7

TLR-TRIF signaling pathway is known to stimulate production ofboth type I interferon, including interferon (IFN) �, IFN�, IFN�-inducible 10-kd protein as well as proinflammatory cytokinesincluding IL-6, MCP-1 (Abdollahi-Roodsaz et al., 2012; Xu et al.,2012). For example, production of IL-6 and MCP-1 was significantlyincreased through the MyD88-independent TLR4/TRIF mechanismin macrophages or NK cells in early allogeneic bone marrow cell(BMC) rejection (Xu et al., 2012). In our present study, QD705s alsomoderately increased IFN� expression (data not shown). Therefore,TLR-TRIF signaling pathway might also involve in QD705s-inducedMCP-1 expression. We observed that QD705s slightly increasedHeme oxygenase-1 (HO-1) mRNA levels and accumulation of radicaloxygen species (ROS) (data not shown). Xu et al. (1996) reportedthat MCP-1 gene expression is also regulated by redox-sensitivemechanisms. Thus, QD705s-induced ROS might partially involvein QD705s induced expression of MCP-1 in RAW264.7 cells. Theinflammasome plays a key role in innate immunity by participat-ing in the activation of the pro-inflammatory cytokines IL-1� andIL-18 (Lamkanfi and Dixit, 2011; Lupfer and Kanneganti, 2012). Theactivation of inflammasome by nanoparticles has been reportedin monocytes, macrophages primed with TLR ligands, such as LPS(Yazdi et al., 2010). However, MCP-1 are inflammasome indepen-dent cytokines (Maslanik et al., 2012). Furthermore, the treatmentof LPS-primed RAW264.7 cells with QD705s failed to increase theMCP-1 secretion in comparison with cells treated with LPS alone(data not shown). It appears that inflammasome may not directlyregulate QD705s-induced MCP-1 expression. However, TNF-� andIL-1� induced MCP-1 expression in human airway smooth musclecells (Patel et al., 2012). In our present study, QD705s also inducedother inflammatory cytokines such as TNF-�, IL-1� and IL-8 (datanot shown). It is also possible that QD705s-induced TNF-� andIL-1� expression might indirectly modulate MCP-1 expression inRAW264.7 cells.

In summary, as shown in Fig. 6, the present study shows that,in a manner that is dependent on their surface modification,different QD705s are able to activate cells via either the cellsurface TLR-MyD88 signaling pathway or intracellular TLR-MyD88

tory responses. The mechanism depends on the surface modification of the QD7051, and thence cytokine production including MCP-1; it does this via the cell surfacees via the intracellular TLR-MyD88 signaling pathways, although a small part of its

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ignaling pathway; this results in NF-�B andAP-1 activation andhen, in turn, MCP-1 induction. Thus activation of the MyD88ependent TLRs pathways would seem to be the major mechanismy which nanoparticles induce inflammation. Furthermore, PEGoating not only reduced the uptake of QD705s, but also preventedhe activation of intracellular TLRs pathways by QD705s. Thesendings will greatly help when evaluating and assessing the safetyf nanoparticles.

onflict of interest

None.

cknowledgements

This work was supported by a research grant, 100A1-NM-PP08-07 from the Center of Nanomedicine Research and the Division ofnvironmental Health and Occupational Medicine of the Nationalealth Research Institutes, Taiwan, ROC. The Microdialysis System

MD) and Gel-clot Limulus amebocyte lysate (LAL) assay were sup-orted by Dr. Jen-Kun Chen.

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