cytotoxicity due to nanomaterials

Cytotoxicity

DOI: 10.1002/smll.200700595

Cytotoxicity of NanoparticlesNastassja Lewinski, Vicki Colvin, and Rebekah Drezek*

From the Contents

1. Introduction..............27

2. Cytotoxicity Assays.. .28

3. Carbon Nanoparticles..................................30

4. Metal Nanoparticles. 34

5. SemiconductorNanoparticles............39

6. Summary and Outlook..................................45

Keywords:· biocompatibility· cytotoxicity· nanomaterials· nanoparticles· toxicology

26 www.small-journal.com � 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim small 2008, 4, No. 1, 26 – 49

reviews R. Drezek et al.

Human exposure to nanoparticles is inevitable as nanoparticles becomemore widely used and, as a result, nanotoxicology research is now gainingattention. However, while the number of nanoparticle types and applicationscontinues to increase, studies to characterize their effects after exposure andto address their potential toxicity are few in comparison. In the medical fieldin particular, nanoparticles are being utilized in diagnostic and therapeutictools to better understand, detect, and treat human diseases. Exposure tonanoparticles for medical purposes involves intentional contact or adminis-tration; therefore, understanding the properties of nanoparticles and theireffect on the body is crucial before clinical use can occur. This Reviewpresents a summary of the in vitro cytotoxicity data currently available onthree classes of nanoparticles. With each of these nanoparticles, different datahas been published about their cytotoxicity due to varying experimentalconditions as well as differing nanoparticle physiochemical properties. Fornanoparticles to move into the clinical arena, it is important thatnanotoxicology research uncovers and understands how these multiplefactors influence the toxicity of nanoparticles so that their undesirableproperties can be avoided.

1. Introduction

The presence of nanoparticles in commercially availableproducts is becoming more common. Nanoparticles, accord-ing to the ASTM standard definition, are particles withlengths that range from 1 to 100 nanometers in two or threedimensions.[1] It is projected that production of nanoparti-cles will increase from the estimated 2 300 tons producedtoday to 58 000 tons by 2020.[2,3] With this increase in manu-facturing of nanoparticle-containing merchandise along withthe constant discovery of new applications of nanoparticles,it is surprising that knowledge on the health effects of nano-particle exposure is still limited. However, the number of ef-forts aimed at determining the health risks associated withnanoparticle exposure continues to grow. This is essential aspublic perception of nanotechnology can be jeopardized byevents such as the “nano scare” in 2006 in Germany, involv-ing the aerosol glass protective Magic-Nano,[4] or thesunscreen controversy after the United States Environmen-tal Protection Agency released findings that nanometer-sized titanium dioxide particles found in sunscreens couldcause brain damage in mice.[5] These two incidents exempli-fy why the need to verify the safety of nanoparticles is in-creasingly more pertinent.

In contrast to nanoparticle exposure through use of con-sumer products, emerging biomedical applications of nano-particles as drug-delivery agents, biosensors, or imaging con-trast agents involve deliberate, direct ingestion or injectionof nanoparticles into the body. For biomedical purposes, es-pecially in vivo applications, toxicity is a critical factor toconsider when evaluating their potential. Nanoparticles forimaging and drug delivery are often purposely coated withbioconjugates such as DNA, proteins, and monoclonal anti-

bodies to target specific cells. As these nanoparticles are in-tentionally engineered to interact with cells, it is importantto ensure that these enhancements are not causing any ad-verse effects. More significant is whether either naked orcoated nanoparticles will undergo biodegradation in the cel-lular environment and what cellular responses degradednanoparticles induce. For example, biodegraded nanoparti-cles may accumulate within cells and lead to intracellularchanges such as disruption of organelle integrity or gene al-terations.

While in vitro nanoparticle applications afford less strin-gent toxicological characterization, in vivo use of nanoparti-cles requires thorough understanding of the kinetics andtoxicology of the particles. In vitro cytotoxicity studies ofnanoparticles using different cell lines, incubation times, andcolorimetric assays are increasingly being published. How-ever, these studies include a wide range of nanoparticle con-centrations and exposure times, making it difficult to deter-mine whether the cytotoxicity observed is physiologicallyrelevant. In addition, different groups choose to use various

[*] N. Lewinski, Prof. R. DrezekDepartment of Bioengineering MS-142Rice UniversityPO Box 1892, Houston, TX 77251-1892 (USA)Fax: (+1) 713-348-5877E-mail: [email protected]

Prof. V. ColvinDepartment of Chemistry MS-60Rice UniversityPO Box 1892, Houston, TX 77251-1892 (USA)

small 2008, 4, No. 1, 26 – 49 � 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 27

Cytotoxicity of Nanoparticles

cell lines as well as culturing conditions, which makes directcomparisons between the available studies difficult.

Despite these issues, general trends in the pool of exist-ing data can be extracted. This Review examines the cyto-toxicity of several classes of nanoparticles currently beingdeveloped for biomedical applications. The nanoparticles in-cluded are: 1) Carbon-based nanoparticles, such as ful-

lerenes and single- and multi-walled carbon nanotubes;2) metal-based nanoparticles, such as gold colloids, nano-shells, nanorods, and superparamagnetic iron oxide nanopar-ticles, and 3) semiconductor-based nanoparticles such asquantum dots.

2. Cytotoxicity Assays

The first step towards understanding how an agent willreact in the body often involves cell-culture studies. Com-pared to animal studies, cellular testing is less ethically am-biguous, is easier to control and reproduce, and is less ex-pensive. In the case of cytotoxicity, it is important to recog-nize that cell cultures are sensitive to changes in their envi-ronment such as fluctuations in temperature, pH, and nu-trient and waste concentrations, in addition to theconcentration of the potentially toxic agent being tested.Therefore, controlling the experimental conditions is crucialto ensure that the measured cell death corresponds to thetoxicity of the added nanoparticles versus the unstable cul-turing conditions. In addition, as nanoparticles can adsorbdyes and be redox active, it is important that the cytotoxici-ty assay is appropriate. Conducting multiple tests is advanta-geous to ensure valid conclusions are drawn.

One simple cytotoxicity test involves visual inspection ofthe cells with bright-field microscopy for changes in cellularor nuclear morphology. Fiorito et al. used this techniquewhen evaluating the cytotoxicity of single-walled carbonnanotubes (SWNTs).[6] However, the majority of cytotoxici-ty assays used throughout published nanoparticle studiesmeasure cell death via colorimetric methods. These colori-metric methods can be further categorized into tests thatmeasure plasma membrane integrity and mitochondrial ac-tivity.

Exposure to certain cytotoxic agents can compromisethe cell membrane, which allows cellular contents to leakout. Viability tests based on this include the neutral red andTrypan blue assays. Neutral red, or toluylene red, is a weakcationic dye that can cross the plasma membrane by diffu-sion. This dye tends to accumulate in lysosomes within thecell. If the cell membrane is altered, the uptake of neutralred is decreased and can leak out, allowing for discernmentbetween live and dead cells. Cytotoxicity can be quantifiedby taking spectrophotonic measurements of the neutral reduptake under varying exposure conditions.[7] Two studies byFlahaut et al. and Monterio-Riviere et al. exploring the cy-totoxicity of carbon nanotubes utilized the neutral redassay.[8,9] Trypan blue, a diazo dye, is only permeable to cellswith compromised membranes; therefore, dead cells arestained blue while live cells remain colorless. The amount ofcell death can be determined via light microscopy.[10] Thisassay was used by Bottini et al. and Goodman et al. to de-termine the cytotoxicity of SWNTs and gold nanoparti-cles.[11,12]

The LIVE/DEAD viability test, which includes twochemicals calcein acetoxymethyl (calcein AM) and ethidiumhomodimer, is another assay measuring the number of dam-aged cells. This method has been used to test fullerenes and

Prof. Vicki Colvin was recruited by RiceUniversity in 1996 to expand its nano-technology program. Today, she servesas Professor of Chemistry and ChemicalEngineering at Rice University as well asDirector of the Center for Biological andEnvironmental Nanotechnology (CBEN).CBEN was one of the nation’s first Nano-science and Engineering Centers fundedby the National Science Foundation.Prof. Colvin has received numerous acco-lades for her teaching abilities, includingPhi Beta Kappa’s Teaching Prize for

1998–1999 and the Camille Dreyfus Teacher Scholar Award in 2002.In 2002, she was also named one of Discover Magazine’s “Top 20Scientists to Watch” and received an Alfred P. Sloan Fellowship. Dr.Colvin received her Bachelor’s degree in chemistry and physics fromStanford University, and obtained her Ph.D. in chemistry from theUniversity of California, Berkeley. She is a frequent contributor to Ad-vanced Materials, Physical Review Letters, and other peer-reviewedjournals, and holds patents of four inventions.

Prof. Rebekah Drezek is currently an As-sociate Professor in the Departments ofBioengineering and Electrical and Com-puter Engineering at Rice University. Shehas been on the faculty at Rice since2002 where she conducts basic, ap-plied, and translational research at theintersection of medicine, engineering,and nanotechnology towards the devel-opment of minimally invasive photonics-based imaging approaches for detection,diagnosis, and monitoring of cancer. Herresearch has been supported by grants

worth over $ 7M from the Whitaker Foundation, Welch Foundation,Coulter Foundation, Beckman Foundation, NSF, NIH, DOD CDRMP,and the Center for Biological and Environmental Nanotechnology.Prof. Drezek is the recipient of the HSEMB Outstanding Young Scien-tist Award, the MIT TR100’s Top 100 Young Innovators Award, theAmerican Association for Medical Instrumentation Career Achieve-ment Award, and the DOD Era of Hope Scholar Award.

Nastassja Lewinski graduated from RiceUniversity with a BS in Chemical Engi-neering in 2006. She is currently a Ph.D.candidate in the Department of Bioengi-neering at Rice University under theguidance of Prof. Rebekah Drezek. Herresearch interests include assessing theuse of quantum dots as optical contrastagents for enhancing in vivo diseasescreening and detection, and addressingpublic policy concerns of nanoparticlesafety.



gold nanoshells.[13,14,15] Calcein AM, an electrically neutral,esterfied molecule, can easily enter cells by diffusion. Oncewithin cells, it is converted to calcein, a green fluorescentmolecule, by intracellular esterases. In contrast, damaged ordead cells are stained by ethidium homodimer, a mem-brane-impermeable molecule, and fluoresce red when thedye binds to nucleic acids. When excited at 495 nm, calceinAM and ethidium homodimer emit distinct fluorescence sig-natures at 515 nm and 635 nm, respectively.[16]

A third cytotoxicity assay used in several carbon-nano-particle studies is lactate dehydrogenase (LDH) releasemonitoring.[13,17,18] In this assay, LDH released from dam-aged cells oxidizes lactate to pyruvate, which promotes con-version of tetrazolium salt INT to formazan, a water-solublemolecule with absorbance at 490 nm. The amount of LDHreleased is proportional to the number of cells damaged orlysed.[19]

In addition to distinguishing between live and dead cellsby detecting compromised plasma membranes, other colori-metric cytotoxicity assays attempt to determine the mecha-nism behind the induced cell death. Mitochondrial activitycan be tested using tetrazolium salts as mitochondrial dehy-drogenase enzymes cleave the tetrazolium ring. Only activemitochondria contain these enzymes; therefore, the reactiononly occurs in living cells.[20] The most widely used test isthe MTT viability assay.[8,9,13,21–26] MTT, 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl tetrazolium bromide, is pale yellow insolution but produces a dark-blue formazan product withinlive cells. A variation of this is the Cell Titer 96 AqueousOne Solution Cell Proliferation Assay distributed by Prom-ega, which has been em-ployed by several groups.[27–29]

Here MTS, 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophen-yl)-2H-tetrazolium, and phen-azine ethosulfate, is used in-stead. The number of livingcells can be determined simi-larly by quantifying the pro-duction of formazan by meas-uring the absorbance at492 nm.[30] Another tetrazoli-um-based assay used to testthe cytotoxicity is the WSTassay.[31] WST-1 or WST-8converts to a yellow–orange-colored formazan product,the concentration of whichcan be quantified at450 nm.[32] Resazurin orAlamar blue has also beenused to ascertain cytotoxici-ty.[33–35] This test is also a col-orimetric assay where thenonfluorescent alamar bluedye is reduced to a pink fluo-rescent dye by cell metabolicactivity, mainly by acting as

an electron acceptor for enzymes such as NADP andFADH during oxygen consumption.[36]

As not all disruptive effects result in membrane or meta-bolic function defects, more extensive cytotoxicity studieshave attempted to determine the sub-lethal effects of nano-particles. Oxidative stress can be detected using a gluta-thione assay. Glutathione (GSH) is a major antioxidantcompound that is oxidized to glutathione disulfide (GSSG)in the presence of reactive oxygen species. In order to sus-tain its protective role against oxidative stress, a high GSH/GSSG ratio is required, which is maintained by the enzymeglutathione reductase. The glutathione assay detects levelsof glutathione using EllmanFs reagent, 5,5’-dithio-bis-2-nitro-benzoic acid (DTNB), which reacts with the sulfhydrylgroup of GSH to produce a yellow-colored product, 5-thio-2-nitrobenzoic acid (TNB). Glutathione reductase also recy-cles GSH from the GSH-TNB complex producing moreTNB. Since the rate of TNB production is directly propor-tional to the concentration of GSH in the sample, the ab-sorbance of TNB can be measured at 405 or 412 nm to de-termine the level of GSH.[37]

Lipid peroxidation of the plasma membrane can be de-tected using GSH or a variety of other methods includingthe widely used thiobarbituric acid (TBA) assay. In theTBA assay, malondialdehyde (MDA), a toxic byproduct oflipid peroxidation, when heated at acidic pH reacts with 2-thiobarbituric acid to form a fluorescent pink chromagen,which can be measured colorimetrically when excited at532 nm. Other methods of lipid peroxidation are listed in anextensive review by Halliwell et al.[38]

Figure 1. Structures and human dermal fibroblast Live/Dead cell viability assay results for C60 and deriva-tives. Reprinted with permission from Ref. [13]. Copyright American Chemical Society, 2004.

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Inflammation is also a possible adverse effect of nano-particle exposure. Commonly tested pro-inflammatory cyto-kines or protein signals of inflammatory response includeIL-1b, IL-6, and TNF-a plus the chemokine IL-8.[39,40] Thesecytokines are detected using enzyme-linked immunosorbantassay (ELISA) and can be quantified by measuring the ab-sorbance from either alkaline phosphatase or strepavidin-horseradish peroxidase labeled antibodies at 405 or 620 nm,respectively.[41]

More extensive cytotoxicity studies have attempted todetermine the genotoxic potential of nanoparticles by exam-ining the extent of DNA damage using several methods.One test that has been used extensively in studying theeffect of carbon-nanoparticle exposure is flow cytometry.[42–

45] This technique utilizes a laser beam that differentiatescells based on their size and density. Using DNA intercalat-ing dyes, the cellular DNA content can also be used to de-termine the proportion of cells undergoing apoptosis. Onesuch dye is propidium iodide, a membrane-impermeable reddye, which undergoes a fluorescence change proportional tothe number of damaged cells. This occurs as binding of thedye to nucleic acids increases with increased membrane per-meability.[46] In addition to flow cytometry, the comet assayhas also been used to detect DNA damage in individualcells using gel electrophoresis. Cells with damaged DNAappear as “comets” with intact DNA residing in the headportion and broken DNA pieces migrating away, formingthe tail. A DNA-specific dye such as propidium iodide isused to read the gel, and the amount of DNA found in thetail is proportional to the amount of DNA damage.[47] Morerecently, to determine which specific genes are up- or down-

regulated due to nanoparticle exposure, some groups haveconducted preliminary DNA microarray studies.[48–51]

For nanoparticles, the major biological effects involve in-teractions with cellular components such as the plasmamembrane, organelles, or macromolecules. As differentnanoparticles can trigger distinctive biological responses, itis important that cytotoxicity studies are conducted for eachnanoparticle type. The following sections review the existingcytotoxicity literature on carbon, metal, and semiconductorbased nanoparticles.

3. Carbon Nanoparticles

Carbon nanoparticles are materials composed mainly ofcarbon with one or more dimensions at 100 nm or less.These include, but are not limited to, carbon dots,[52] ful-lerenes, nanodiamonds,[53] nanofoam,[54] nanohorns,[55] andnano-onions.[46] However, as fullerenes are the most estab-lished of the carbon nanoparticles, the focus is on this type.Fullerenes, as defined by IUPAC, encompass C60, SWNTs,and multi-walled (MW) NTs.[56] These three types are themost widely used and well developed of the carbon nano-particles. Their unique physiochemical properties (lightweight, high tensile strength, thermal/chemical stability andconductivity) have generated several applications includinguse in biomedical materials and devices such as tissue scaf-folds, drug-delivery agents, and fluorescent-contrastagents.[57–59] In terms of cytotoxicity, a major factor influenc-ing potential toxicity is the carbon nanoparticlesF complexityand variety in size, shape, charge, methods of production,

Table 1. Cytotoxicity studies on C60. d=diameter.

Cell Line Surfacecoating

Exposureconditions

NP concentration(average size)

Test Exposureduration

[h]

Toxicity Author Year

Human dermal fibro-blasts, HDF; humanliver carcinoma, HepG2

COOH,OH, Na

70% confluency 0.24–2400 ppb(d=100 nm)

MTT, Live/Dead,LDH

48 LD50=20 ppb for bare C60 onHDF; no cytotoxicity observedwith C60(OH)24

Sayes[13]

2004

Guinea pig alveolarmacrophages

pristine 2<105 cells mL�1

in 24-well plates8.36<104

NP mg�1,1.41–226 mg cm�2

MTT 3 No significant toxicity up to226 mg cm�2

Jia[21]

2005

Human dermal fibro-blasts, HDF; humanliver carcinoma,HepG2; neuronalhuman astrocytes, NHA

COOH,OH, Na

70% confluency 0.24–2400 ppb(d=100 nm)

MTT, Live/Dead,LDH

48 Nano-C60 is cytotoxic at20 ppb level; after 30 h cellsbegin to have leaky mem-branes and lipid oxidation

Sayes[49]

2005

Monocyte-derivedmacrophages

pristine 3<105 cells mL�1 30 and60 mg mL�1

NuclearMorph, PI

1, 24,48

Did not induce damage ordeath of macrophages

Fiorito[6]

2006

Human monocytemacrophages

pristine 2<106 cells well�1

for 24-well plate,0.5–1<106 for96-well plate

0.16–10 mg mL�1

(d=60–270 nm)Neutralred

48 No significant toxicity Porter[62]

2006

Human epidermalkeratinocytes, HEK

N-Boc-Baa

70% confluency,96-well plate

0.00004–0.4 mg mL�1

MTT 24, 48 Cytotoxicity at 0.04 and0.4 mg mL�1; IL-8, IL-6, andIL-1b levels increased

Rouse[63]

2006

Human umbilical veinendothelial cells,HUVEC

C60(OH)24 90% confluencyon 6-well plates

1–100 mg mL�1

(d=7.1�2.4 nm)LDH, WST,microarray

24 100 mg mL� inhibit cellgrowth; 10 mg mL�1 inhibitcell attachment

Yamawaki[50]

2006



chemical compositions, surface chemistry/functionalization,and aggregation tendency. A few reviews have been publish-ed that look at the biocompatibility of carbon nanoparti-cles.[60,61] This section looks at the studies that have beenconducted to elucidate the safety of these three major typesof carbon nanoparticle. Summaries of the experimentalsetup and results on C60, SWNTs, and MWNTs are providedin Tables 1–3, respectively.

3.1. C60

Several groups have studied the effect of C60 exposureunder various experimental conditions with different celllines and have yielded different results. The most significantfactor influencing cytotoxicity in this class of carbon nano-particles seems to be cell type. The groups that reportednoncytotoxic effects studied C60 exposure in macrophage

Table 2. Cytotoxicity studies on SWNTs. l= length.

Cell line Surfacecoating

Exposureconditions

NP Concentration(average size)


[h]


3T3 cells FITC N/A 1–10 mm (d=1 nm,l=300–1000 nm)

flow cytome-try (annexin,PI)

1 5 mm, 90% viability,10 mm, 20% viability

Pantarot-to [43]

2003

Immortalized humanepidermal keratino-cytes, HaCaT

none(w/30% Fecat)

80% confluencyon 96 wellplates or 75-cm2

flasks

0.06, 0.12, or0.24 mg mL�1

Alamar blue,GSH

2, 4, 6,8, 18

viability decreased after4h, 0.24 mg mL�1 ~65%viability

Shvedova[33]

2003

Mouse peritonealmacrophage-likecells, J774.1A

pristine 107 cells 0–7.3 mg mL�1

(d=1 nm, l=1 mm)microscopy 4, 8, 12,

18, 24Cells ingest NT withouttoxic effects

Cherukuri[68]

2004

Human embryonickidney, HEK293

pristine 24 well plates MTT: 0.7812–200 mg mL�1;others: 25 mg mL�1

(CAS 7782-42-5)

MTT, westernblot, flowcytometry,microarray

24–120 Cytotoxicity dose- andtime-dependent; 43.5%in G1 cell cycle arrestafter 1 day

Cui[44]

2004

Human promyelcyticleukemia cells, HL60;Jurkat T cells

COOH,biotin, fluo-rescein,streptavidin

3<106

cells mL�10.05 mg mL�1

(d=1–5 nm,l=0.1–1 mm)

PI, flowcytometry

1 No significant toxicityfor nonstretavidin-modi-fied SWNTs

Kam[45]

2004


pristine 2<105

cells mL�1 in24-well plates

1.41–226 mg cm�2

(d=1.4 nm,l=1 mm)

MTT 3 Cytotoxic effects seen at0.38 mg cm�2; necrosisat 3.06 mg cm�2

Jia[21]

2005

Human keratinocytes,HaCaT; HeLa cells;Lung carcinoma(A549, H1299) cells

pristine 5000cells well�1,96-well plate

0.1, 0.5, 1, 5, 10,20 mg mL�1

MTT, Live/Dead

72 Cytotoxic effects seen at0.5 mg mL�1, NFkB path-way activated by SWNT

Manna[65]

2005

Monocyte-derivedmacrophages

pristine 3<105 cellsmL�1

30 and 60 mg mL�1 nuclear mor-phology, PI

1, 24, 48 Did not induce damageand death of macro-phages

Fiorito[6]

2006

Human dermal fibro-blasts, HDF

phenyl-(SO3H,SO3Na, or(COOH)2),pluronicF108

70% confluency 0.2–2000 mg mL�1

(d=1 nm,l=400 nm)

MTT, Live/Dead

24, 48 Cytotoxicity decreasedw/ decreased C/phenyl-SO3H ratio; LD50 couldnot be obtained

Sayes[66]

2006


pristine 5x103 cellswell�1

0.8–100 mg mL�1

(d=2 nm,l= 500 nm)

MTT 24–120 Strongest adverse effectw/ SWNT; 100 mg mL�1

gave 79%, 50% and31% viability after1, 3 & 5 days

Tian[23]

2006

Lung epithelial-likecells, A549

pristine 25 000 cellswell�1,96-well plate

50 mg mL�1

(d=1.4 nm)MTT, WST,LDH, MMP

24-96 MTT gave different re-sults from WST, LDHand MMP

Worle-Knirsch[69]

2006

Rat alveolar macro-phage cells, NR8383;human alveolar epithe-lial cells, A549

pristine 105 cells well�1

in 96-wellplates;2.5<104 cellswell�1 in 96-wellplates (human)

5–100 mg mL�1

(d=1–2 nm,l=100 nm)

MTT, WST 24–96 Cytotoxicity dosedependent; 100 mgmL�1, 60–80% reduc-tion

Pulskamp[64]

2007

Mesothelioma cells,MSTO-211H

pristine 3000 cellswell�1 in 24-well plates

7.5, 15, 30 mg mL�1 MTT 72 Cytotoxicity dose depen-dent; agglomeratedworse than well-dis-persed

Wick[67]

2007



cell lines. Fiorito et al. found non-toxic responses for pris-tine C60 in their studies with murine macrophages.[6] Theyreported that C60 had low cellular uptake, did not stimulatenitric oxide release, and did not induce apoptosis in compar-ison to graphite and SWNTs. From this, C60 was consideredto be fairly noncytotoxic. C60 was also deemed the leasttoxic of the carbon nanoparticles by Jia et al.[21] The groupstudied alveolar macrophage response to incubation withC60 and found that, after six hours of exposure to as muchas 226 mg cm�2 of C60, no significant toxicity resulted.Porter et al. studied the effect of C60 in human monocytemacrophages and also found no significant cytotoxicity.[62]

However, looking at the subcellular level, C60 was found toaggregate into hexagonal units along the plasma membrane.In addition, they were found to accumulate intracellularly inlysosomes, cytoplasm, along the nuclear membrane andinside the nucleus. In contrast, the groups that studied C60

exposure in other cell lines found a dose-dependent cytotox-icity relationship. Incubating four different C60 derivatives,as illustrated in Figure 1, for up to 48 hours with humandermal fibroblast and liver carcinoma cells, Sayes et al.found, for all four types, the lowest concentration (0.24 ppb)was relatively nontoxic while the highest concentration(2400 ppb) was more cytotoxic.[13]

Between the four types, the addition of surface chemis-tries for water solubility decreased the in vitro cytotoxicity,with pristine C60 being more cytotoxic while the more hy-droxylated C60, C60(OH)24, had no apparent cytotoxicitywith an LD50 value of >5 mg mL�1. It has been suggestedthat this difference is due to the generation of reactiveoxygen species associated with C60. Additionally, particle ag-gregation was also determined to cause some of the cytotox-ic effects. A more extensive study by Sayes et al. reportedthat cell apoptosis due to exposure to C60 was caused bycell-membrane lipid peroxidation from oxygen radicals.[49]

This was confirmed when the addition of an antioxidant, L-ascorbic acid, preventedmembrane damage resultingin cell viability comparable tothe control. Rouse et al. stud-ied the effect of amino acid-derivatized fullerenes inhuman epidermal keratino-cytes (HEK).[63] The levels ofpro-inflammatory cytokinecytotoxicity indicators, IL-8,IL-6, TNF-a, and IL-1b, weremeasured. After 24 and 48hours of incubation, a dose-dependent decrease in cell vi-ability was found as well ashigher phagocytosis of parti-cles observed in cells exposedto concentrations above0.004 mg mL�1. Yamawakiet al. tested the effect of hy-droxyl fullerene, C60(OH)24,on human umbilical vein en-dothelial cells and found cyto-

toxicity after 24-hour incubation with concentrations of be-tween 1 and 100 mg mL�1.[50] Morphological changes, in-creases in LDH release, and growth inhibition were report-ed. In addition, the fullerenes were found to aggregate andinternalize in autophagosomes, suggesting autophagic celldeath.

3.2. Single-Walled Carbon Nanotubes

Compared to the mixed reports on C60, SWNTs havetypically been labeled as having cytotoxic effects at highconcentrations. In addition, in a few comparison studiesSWNTs were reported as more toxic than the other twomajor types of carbon nanoparticle.[21,23,64] In a study expos-ing human embryo kidney cells to SWNTs for one to fivedays, Cui et al. found dose- and time-dependent decreasesin cell-adhesion ability, cell proliferation, and increases ininduction of apoptosis.[44] In addition, flow cytometry analy-sis revealed altered cell-cycle regulation such as G1 phasearrest due to SWNT exposure. Testing four different celllines (human keratinocytes, HeLa cells, and two lung carci-noma lines: A549, H1299), Manna et al. found oxidativestress and inhibition of proliferation increased in a dose-and time-dependent manner.[65] They also studied the NFkBpathway, which they found was activated by SWNT expo-sure either via MAPK or IKK kinase activation.

While pristine SWNTs were found to exhibit some cyto-toxic effects, a few groups found these effects were mitigat-ed by functionalizing the SWNT surface. Kam et al. provideflow cytometry analysis that revealed no significant toxicitydue to carboxyl-, biotin-, and fluorescein-coated SWNTs inHL60 and Jurkat T cells after one hour.[45] Sayes et al.looked at the cytotoxicity of water-soluble, SWNT-treatedhuman dermal fibroblasts over a range of concentrations(3–30 mg mL�1) for up to 48 hours.[66] As illustrated inFigure 2, cell death was highest in the cultures exposed to

Figure 2. Structures and human dermal fibroblast cytotoxicity data for SWNTs and derivatives. Reprintedwith permission from Ref. [66]. Copyright Elsevier, 2007.



pristine SWNTs while, of the three functionalized SWNTs,the SWNT containing the most functional groups yieldedthe best cell viability.

Several hypotheses have been postulated to explain thecytotoxicity observed with SWNTs. One is related to themode of production, as the synthesis of SWNTs requires theuse of metal catalysts, which can be toxic themselves. Shve-dova et al. reported dose- and time-dependent cytotoxicityin human epidermal keratinocytes exposed to SWNTs.[33] Athigher concentrations and longer incubation times, in-creased oxidative stress, reduced glutathione levels, and nu-clear and mitochondrial changes were found. They alsonoted that the addition of a metal chelator reduced cytotox-icity, suggesting that residual iron catalyst in solution mayplay a role in the cytotoxicity observed. In addition, Jiaet al. found more than a 20% growth inhibition at thelowest SWNT dose of 1.41 mg cm�2.[21] This cytotoxicity wasalso speculated to be due to the 90% purity of the SWNTsolution as the presence of metallic catalysts could confoundthe results.

Particle aggregation has also been suggested to be afactor in nanoparticle cytotoxicity. Wick et al. aimed to de-termine how agglomeration influenced SWNT cytotoxicityand tested four different SWNT solutions: the raw materialinvolved in SWNT production, the SWNT-agglomerates re-sulting from the synthesis, and the SWNT bundles and theSWNT pellets (devoid of nanotubes) produced from centri-fuging the SWNT agglomerate.[67] Aggregation occurred inall SWNT fractions except the well-dispersed SWNT bun-dles. Correspondingly, the SWNT bundles did not induceadverse cellular effects, and as this was the only solutionwhere agglomerates were not formed, this corroborates thehypothesis that SWNT agglomeration leads to cytotoxic ef-fects. However, an earlier study by Tian et al., testing an un-refined SWNT solution and a SWNT solution with themetal catalysts removed, found lower cytotoxicity with theunrefined SWNTs.[23] The group proposed that the lower cy-totoxicity of the unrefined SWNTs was a result of their ag-gregation into larger, and therefore less toxic, particles. Thiscontradicts the reasoning of Wick et al., who hypothesizedthat the agglomerated SWNTs were cytotoxic due to thestiffness and larger size, making the nanotubes emulate theeffects of asbestos fibers. While the conflicting results maybe due to the use of two different cell lines and asbestos-in-duced lung-cancer cells versus keratinocytes, the effect ofSWNT aggregation is still questionable.

3.3. Multi-Walled Carbon Nanotubes

Studies on MWNTs have yielded results similar to thoseof SWNTs. Monteiro-Riviere et al. reported that cells incu-bated with higher concentrations of MWNTs for longer ex-posure times contained more MWNTs.[9] The percentage ofcells with MWNTs inside increased from 59% at 24 hours to84% after 48 hours. In addition, a dose- and time-dependentdecrease in cell viability was observed, coupled with an in-crease in release of cytokine IL-8 at the higher MWNT con-centrations. While the complimentary study by Shvedova

et al. on SWNTs proposed that the cytotoxicity may be dueto trace amounts of catalyst in the solution, the lack of cata-lyst particles in these MWNT solutions suggests theMWNTs alone were potentially hazardous. Instead, Mon-teiro-Riviere et al. hypothesize that the cytotoxicity is dueto MWNT attachment to the cell membrane or MWNT in-ternalization, as MWNTs were seen in the cytoplasm andnear the nucleus. Sato et al. also found aggregates ofMWNT in cytoplasm in their studies.[70] Bottini et al. alsosaw dose- and time-dependent cytotoxicity in T lymphocyteand Jurkat leukemia cells.[11] In addition, comparing the ef-fects of different surface coatings, hydrophobic MWNTswere less toxic than ones coated with hydroxyl or carboxylgroups. The same conclusions were made by Magrez et al.after studying MWNT in lung carcinoma cells.[22] A dose-de-pendent decrease in cell viability was also evident after ex-posing alveolar macrohages to >95% purified MWNTs,conducted by Jia et al.[21] However, as they tested differentdiameters of MWNT (10–20 nm) this dose dependencecould be due to particle mass, size, or both. Interestingly,while these groups found a dose-dependent trend in cyto-toxicity, Flahaut et al. found a decrease in viability inhuman umbilical vein endothelial cells (HUVEC) with dilu-tion of their MWNT solution.[8] Although they concludedthat the MWNTs were nontoxic as metabolic activity wasmaintained above 75%, HUVEC viability seemed to de-crease with exposure to decreasing concentrations ofMWNTs with large surface areas. The group suspects this isa result of the aggregation of MWNTs or their enhanced in-teractions with the cells due to their higher dispersion atlower concentrations.

Few groups have studied the inflammatory response toMWNTs. One group, Ding et al. , looked at the genetic ef-fects of MWNTs and found that high concentrations in-duced immune and inflammatory gene overexpression.[48]

Witzmann et al. considered the protein-expression changesafter human epidermal keratinocyte exposure to MWNTsand noted upregulation of proteins related to irritation andcell apoptosis.[71] Muller et al. incubated peritoneal macro-phages for up to 24 hours containing purified MWNTs andground MWNTs at concentrations of 20–100 mg mL�1.[17]

They determined that ground MWNTs had a capacity forinducing dose-dependent cytotoxicity and up-regulatingTNF-a expression that is similar to that of asbestos andcarbon black. However, the unground MWNT sample ex-hibited lower effects than the ground sample, which they at-tributed to the increased agglomeration found in the un-ground sample preventing cellular uptake. Murr et al. alsofound that the cytotoxicity of MWNTs was similar to asbes-tos.[72] Chlopek et al. investigated the viability and stimula-tion of fibroblasts and osteoblasts exposed to purifiedMWNTs.[27] The group deemed MWNTs to be biocompat-ible with the tested cell types, as they found unchangedlevels of osteocalcin, cytokine IL-6, and oxygen-free radi-cals.



4. Metal Nanoparticles

4.1. Gold Nanoparticles

Gold colloids, now often referred to as gold nanoparti-cles, have been used in medical applications during clinicaltesting of heavy metals to treat rheumatoid arthritis as earlyas the 1920s.[73] This precedence suggests that current appli-cations of gold nanoparticles should not be limited by theirbiocompatibility. While gold nanoparticles refer to particlesspherical in shape, other geometries, such as gold nanorods,tripods, tetrapods,[74] and nanocages,[75] have been synthe-sized, with gold nanorods discussed in further detail in a

later section. Gold nanoparticles exhibit an intense color inthe visible spectroscopic region and since gold can easilybind functionalizing or targeting ligands, it has great prom-ise as a contrast agent for bioimaging.

Due to their small size, gold nanoparticles have beenfound to easily enter cells. Early studies with cytotoxicitydata were focused on utilizing this property for nucleartransfection and targeting. In their work to find nonviralgene-delivery devices, Thomas et al. found polyethylenimine(PEI)-modified gold nanoparticles could transfect monkeykidney (COS-7) cells six times better than PEI alone.[76] Cellviability was recorded after exposure to PEI–gold nanopar-ticle complexes, and 80% of the cells were still metabolical-

Table 3. Cytotoxicity of MWNTs.

Cell Line Surfacecoating

Exposureconditions

NP concentration (aver-age size)


[h]



pristine 80% confluency,7000 cells well�1,96-well plates

0.1, 0.2, 0.4 mg mL�1 Neutral red 1, 4, 8,12, 24,48

~73% viability at0.4 mg mL�1; IL-8increases withMWNT conc.

Monteiro-Riviere[9]

2005

Human skin fibro-blasts, HSF42;human embryoniclung fibroblasts(IMR-90)

pristine 70% confluency,96-well plates

0.06–0.6 mg L�1 Hoechst33342,YO-PRO 1,PI, BrdU,microarray

24, 48 Cytotoxicity dose-dependent for puri-fied MWNT

Ding[48]

2005


pristine 2<105 cells mL�1 in24-well plates

1.41–226 mg cm�2

(d=10–20 nm,l=0.5–40 mm)

MTT 3 Necrosis seen at3.06 mg cm�2

Jia[21]

2005

Sprague–Dawley ratperitoneal macro-phages

pristine direct lunginjection

20–100 mg mL�1

0.5–2 mg rat�1LDH 3, 15

daysLDH doubled from20 to 100 mg mL�1

ground nanotubes

Muller[17]

2005

Murine alveolarmacrophages(RAW267.9)


in 96-well plates0.005–10 mg mL�1,(d=5–30 nm,l=0.03–3 mm)

MTT, ELISA 48 Cytotoxicity beginsat 2.5 mg mL�1;similar to asbestos

Murr[72]

2005

Human acute mono-cytic leukemia cells(THP-1) Wister malerats


in 96-well plates5–500 ng mL�1 0.1 mg(d=20–40 nm,l=0.5–5 mm)

HU TNF-aFlexia histolo-gy, microscopy

16 TNF-a productiondose dependent,aggregates in sev-eral cell types

Sato[70]

2005

T lymphocytes, JurkatT leukemia cells

Hydroxyl,carboxyl

4.4x104 cells mL�1 40–400 mg mL�1

(d=20–40 nm,l=1–5 mm)

Trypan blue 24–24 Cell death >80%in oxidized, <50%in pristine at400 mg mL�1

Bottini[11]

2006

Human osteoblasticline hFOB 1.19;Human fibroblasticline HS-5

Poly-sulfone(PS)

2 cm3 cells/12 well plate

N/A (d=10–15 nm) Cell titer 96 24, 48, 7days

Small viability de-crease inPS+MWNTs vs.pure PS

Chlopek[27]

2006

Human umbilicalvein endothelialcells, HUVEC

pristine 6000 cells cm�2

in 96-well platesMax: A: 0.5 mg mL�1

(d=1.1–3.2 nm),B: 0.64 mg mL�1

(d=1.1–4.3 nm)C: 0.9 mg mL�1

(d=0.7–6.3 nm)

MTT, Neutralred

24 None were cytotox-ic but error bars ofsamples A & Bbelow threshold

Flahaut[8]

2006

Human lung-tumorcell lines, H596,H446, and Calu-1

Carbonyl(CdO),carboxyl(COOH),hydroxyl(OH)

N/A 0.002–0.2 mg mL�1

(d�20 nm, aspectratio=80–90 nm)

MTT 24–96 Cell viability de-creased 33% at0.2 mg mL�1; func-tionalized havelower survival

Magrez[22]

2006

Human neonatalHEKs

pristine 80% confluency,6-well plates

0.4 mg mL�1 protein array 24, 48 Irritation and cellapoptosis proteinsupregulated

Witzmann[71]

2006



ly active. While PEI–gold nanoparticles with dodecyl–PEIcomplexes achieved even better transfection, cell viabilitydecreased to 70%. This complex was mainly found insidethe cell suggesting that internalization is a factor in cytotox-icity. However, as the gold nanoparticles were conjugated toPEI, whether or not the observed decrease in cell viabilitywas due to the gold nanoparticles is unclear. Anothergroup, Tkachenko et al., looked first at the nuclear targetingability of gold nanoparticles alone, and then at gold nano-particles with a full-length peptide containing both the re-ceptor-mediated endocytosis and nuclear localization signalsegments from an adenovirus in HepG2 cells.[77] The groupfound that plain gold nanoparticles were readily taken upinto the cytoplasm; however, they did not enter the nucleus.Experiments conducted at 48C indicated that cell entry wasenergy dependent since a decrease in the number of parti-cles inside the cells was observed. The gold nanoparticleswere determined to be able to enter the cell by receptor-mediated endocytosis but unable to leave the endosomes,hindering nuclear targeting. However, the nanoparticle-pep-tide complex incorporating both transport signals was foundto enter the nucleus. Despite this nuclear exposure, cell via-bility was greater than 95% after 12 hours of incubation.

Tkachenko et al. conducted another study examiningfour different peptide–BSA–gold nanoparticle conjugates inthree cell lines (HeLa, 3T3/NIH, and HepG2).[78] Here theyreported differing effects of the nanoparticles between thethree cell lines. The four peptide–BSA–gold nanoparticleswere able to enter HeLa cells, escape the endosomes, and,except for the particle with the HIV Tat protein, enter thenucleus. In contrast, the four peptide–BSA–gold nanoparti-cles were found clustered together in endosomes within the3T3/NIH cells. The HepG2 cells did not seem to uptake thepeptide–BSA–gold nanoparticles except for the gold nano-particle with the integrin-binding domain. The LDH cyto-toxicity assay also confirmed these cell-line differences.After three hours of incubation, the peptide–BSA–goldnanoparticles conjugated with the adenovirus fiber proteincaused 20% cell death in HeLa cells while only 5% in the3T3/NIH cells. This suggests that the nuclear delivery of thepeptide–BSA–gold nanoparticles influences cell viabilitydue to particle interactions with cellular DNA. Goodmanet al. also tested the effect of gold nanoparticle exposure inmultiple cell lines.[12] Cationic (ammonium-functionalized)and anionic (carboxylate-functionalized) gold nanoparticleswith concentrations of 0.38–3 mm were incubated with COS-1 cells, red blood cells, and Escherichia coli cultures for24 hours. While the cationic nanoparticles were clearly morecytotoxic than the anionic, a small variation was observed intheir LC50 values between cell types, showing that differentcell types experience similar toxicity. This contradicts thefindings of Tkachenko et al. as they found a difference be-tween cell lines; however, this could be due to the use ofdifferent surface coatings.[78] In addition, Goodman et al. [12]

proposed that the nanoparticles interact with the cells pas-sively rather than by energy-dependent processes, as sug-gested by Tkachenko et al. [78] since mammalian and bacteri-al cells exhibited similar nanoparticle uptake. However, re-duced-temperature incubation studies were not reported.

As the results from Tkachenko et al. and Goodmanet al. show, the type of surface coating can play an impor-tant role in the cytotoxicity of gold nanoparticles. Connoret al. studied the effect of size and different surface modifi-cations on uptake and acute toxicity in human leukemia(K562) cells.[24] The sizes ranged between 4 and 18 nm withsurface modifiers including biotin, CTAB, cysteine, citrate,and glucose. After three days of exposure, the largest nano-particle with citrate and biotin surface modifiers did notappear to be toxic at concentrations up to 250 mm. In con-trast, a similar concentration of the gold-salt (AuCl4) solu-tion was found to be over 90% toxic. Glucose and cysteinewere found to be less effective in rendering the nanoparti-cles nontoxic. The gold nanoparticle concentration droppedwithin the first hour of exposure, suggesting rapid uptake ofnanoparticles into cells. The consumed nanoparticles werefound clustered in endocytic vesicles and maintained theirsize after being taken up by the cells.

Cytotoxicity may not be the only adverse effect of nano-particles; nanoparticles may also affect the immunologicalresponse of cells. Shukla et al. tested the effect of goldnanoparticles on the proliferation, nitric oxide, and reactiveoxygen species production of RAW264.7 macrophagecells.[25] After 48 hours of up to 100 mm gold-nanoparticletreatment, RAW264.7 macrophage cells showed greaterthan 90% viability with no increase in pro-inflammatory cy-tokines TNF-a and IL-1b. Cell viability decreased to 85%after 72 hours, which was attributed to depletion of medianutrients since the media was not changed in those 72 hours.The group found that cells take up gold nanoparticles inter-nalizing them in lysosomes, which move in a time-depen-dent manner toward the nucleus but do not enter the nu-cleus. They also corroborate the findings of Goodman et al.that gold nanoparticles were not present in cells kept atcold (48C) temperatures.[25] Fu et al. and Shenoy et al. incu-bated bare gold nanoparticles and gold nanoparticles func-tionalized with methoxy-PEG-thiol or coumarin-PEG-thiolwith breast cancer (MDA-MB-231) cells for 24 hours.[28,29]

They found that the functionalized nanoparticles were inter-nalized, by what they suggest is nonspecific endocytosis,within the first hour and localize mainly in the cytoplasmand perinuclear region. This is in agreement with the find-ings of Shukla et al.[25]

Other noncytotoxic effects of nanoparticles, such as theinfluence of nanoparticle exposure on the proliferation,morphological structure, spreading, migration, and proteinsynthesis of human dermal fibroblast cells, were examinedby Pernodet et al.[79] They found that with increasing con-centration of gold nanoparticles, cell area decreased alongwith cell number and density of actin fibers. Although nocytotoxicity tests were conducted, the decreased number ofcells indicates some cytotoxic effects. The number of va-cuoles present within the cells increased with time, and thecells were filled with vacuoles by the sixth day. The goldnanoparticles accumulated inside the cells, entering not byendocytosis but rather through diffusion facilitated by theirsmall size (average size ~13 nm). The observed cellularchanges were both dose- and time-dependent. The groupalso notes that the gold nanoparticles are not digested in



the lysosomes, and even though the vacuoles accumulatenear the nucleus, no nuclear penetration was seen. A sum-mary of the experimental setup and results on gold nanopar-ticles is provided in Table 4.

4.2. Gold Nanoshells

Typically, gold nanoshells are composed of a silica die-lectric core coated with an ultrathin metallic gold layer. Thiscore/shell structure allows for the gold nanoshells to bemade by either preferentially absorbing or scattering byvarying the relative core and shell thicknesses. Because ofthe “tunability” of their optical properties, nanoshells arebeing developed for imaging contrast and photothermaltherapeutic medical applications.[80,81]

A few studies have been published with cytotoxicity re-sults on gold nanoshells. The first to suggest that gold nano-shells are nontoxic was Hirsch et al. While the focus of thestudy was on the photothermal ablative ability of the nano-shells, they mention that exposure to nanoshells did notcause cell death.[14] In later studies by Loo et al., SKBR2breast-cancer cells exposed for one hour to 8 mg mL�1 or3K109 nanoshells mL�1 of anti-HER2 bioconjugated nano-shells exhibited no difference in viability compared to con-trol cells.[15,82] A more recent study by James et al. studiedthe biodistribution of gold nanoshells in female albinomice.[83] A 100 mL nanoshell solution with a 2.4K1011 nanoshells mL�1 concentration was injected into the tailveins of 30 mice. Five mice were sacrificed at several timepoints up to 28 days, and the accumulation of nanoshells inthe blood and major organs, such as the liver, kidneys,spleen, lungs, muscle, brain, and bone was measured. The

nanoshells were found to quickly clear the blood circulationand predominantly accumulated in the liver and spleen. De-spite the lack of complete nanoshell clearance from thebody after 28 days, the mice are reported to have shown nophysiological complications from the residual presence ofnanoshells.

Cytotoxicity of a gold/copper nanoshell has also beenstudied by Su et al. Au3Cu nanoshell concentrations be-tween 0.001 and 200 mg mL� -1 were incubated with Verocells for 6 or 24 hours.[84] Using the WST assay, the groupfound cell damage to be dose dependent with cell viabilitydecreased to 15% at the highest concentration after24 hours of incubation with the nanoshells. The in vivo ef-fects were also tested in male BALBc mice and, after30 days, a dose dependence in viability rates was also foundwith 100% viability in the low-dose mice but 67% viabilityin the high-dose mice. Urine was collected from the micethree hours after injection, and the amount of gold andcopper found suggested the nanoshells were being excretedfrom the body. The loss of MRI signal after four hours cor-roborated this finding. A summary of the experimentalsetup and results on gold nanoshells is provided in Table 5.

4.3. Gold Nanorods

The advantage of gold nanorods is that they have both atransverse and longitudinal plasmon. As the optical proper-ties of materials depend both on the type and shape of themetal, the unique properties of these rod-shaped particlescan be utilized in several potential applications. While veryfew groups have published data on the cytotoxicity of goldnanorods, the results are similar to those seen for gold nano-

Table 4. Cytotoxicity of gold nanoparticles.

Cell line Surface coating Exposureconditions




COS-7 cells PEI2 3x105 cells/well

N/A MTT 6 h +42 h

70-80% viability aftertransfection

Thomas[76]

2003

Human liver carci-noma, HepG2

BSA, 4 targetingpeptides

85%confluency

N/A(d=20-25 nm)

LDH 12 h Viability slightly compromised(<5%)

Tkachenko[77]

2003

COS-1, Red bloodcells, E.coli

NH3, COOH 80% conflu-ency, 96-well plate

0.38, 0.75, 1.5, or3 mm

MTT,Trypanblue

1, 2.5,6, 24 h

LD50 (Cos-1): anionic ~1 mMand cationic >7.37 mm; similarfor other cell types

Goodman[12]

2004

HeLa, 3T3/NIH,HepG2

BSA, 4 targetingpeptides

75% conflu-ency

150 pm

(d=22 nm)LDH 3 h Cell viability reduced by 20%

in HeLa cells, but only 5% in3T3/NIH

Tkachenko[78]

2004

Leukemia cell line,K562

citrate, biotin, L-cysteine, glu-cose, CTAB

104 cells/well

0-250 mm Au atoms(d=4, 12, 18 nm)

MTT 3 days No apparent toxicity at 250 mm,glucose and cysteine modifiednot toxic up to 25 mm

Connor[24]

2005

Human breastcarcinoma xeno-graft cells,MDA-MB-231

coumarin-PEG-thiol, mPEG-thiol (neg. con-trol)

105 cells/well in96-wellplates

50-200 mg mL�1

(d=10 nm)CellTiter96

24 h Nanoparticles are internalizedbut essentially non-toxic up to200 mg mL�1

Fu/Shenoy[28/29]

2005

RAW264.7macrophage cells

lysine, PLL, FITC 105 cells/well in96-wellplates

10, 25, 50, and100 mm (d=3-8 nm)

MTT 24, 48,72 h

100 mm - after 72 hr cell viabilityto decreased to 85%

Shukla[25]

2005

Human dermalfibroblasts

citrate N/A 0-0.8 mg mL�1

(d=13+/-1 nm)micros-copy

2-6days

Dose-dependent decrease in cellarea & density; many vacuoles

Pernodet[79]

2006



particles. An early study by Salem et al. tested feasibility ofgold/nickel nanorods as gene-delivery agents.[31] A concen-tration of 44 mg mL�1 was used to test transfection ofhuman embryonic kidney (HEK293) cell line with Au/Ninanorods functionalized with GFP and luciferase reportergenes. The group notes that this is significantly below theLD50 value that was determined by the WST assay to be750 mg mL�1. Transmission electron microscopy (TEM) re-vealed that the nanorods localized in vesicles or in the cyto-plasm but not the nucleus. While no biodistribution analysiswas done during their preliminary in vivo studies, no com-plications due to skin and muscle exposure to nanorodswere reported. While examining the photothermal capabili-ties of gold nanorods, Takahashi et al. found the viability ofcells incubated with nanorods, but without laser irradiation,did not decrease significantly.[85]

More recently, other groups have found that the chemi-cals involved in the synthesis of gold nanorods play a role intheir potential cytotoxicity. Niidome et al. looked at theeffect of poly(ethylene glycol) (PEG)-modified gold nano-rods on HeLa cells after 24 hours of incubation.[86] Strongcytotoxicity was associated with a low concentration ofCTAB-stabilized gold nanorods. They proposed that freeCTAB in solution was the source of the cytotoxic effect.This was corroborated when removal of excess CTAB fromthe PEG-modified gold-nanorod solution yielded 90% cellviability at the highest concentration tested (0.5 mm). Taka-hashi et al. , the same group, published another study thattested the cytotoxicity of gold nanorods extracted fromCTAB using a phosphatidylcholine (PC)-containing chloro-form.[26] Concentrations from 0.09 to 0.72 mm exhibited littlecytotoxicity; however, at higher concentrations of 1.45 mm,cell viability was reduced by approximately 20%. This celldeath was proposed to be due to nanorod aggregation. Incomparison, twice-centrifuged gold-nanorod solutionsshowed significant cytotoxicity with the lowest tested con-centration, 0.09 mm, reducing cell viability by about 15%after 24 hours of incubation. Cytotoxicity was found to bedose dependent with almost 0% cell viability at 1.45 mm.Therefore, the group concluded that extraction processusing PC was a better method than twice centrifugation.

Huff et al. exposed KB cells to gold nanorods to examinetheir internalization, whether by endocytosis or by CTABinteraction on cell membranes.[87] This group found that KBcells internalized the majority of CTAB-coated nanorodswhile mPEG-DTC-coated nanorods were internalized at re-duced levels. The CTAB-coated nanorods were found local-ized near the perinuclear region within the KB cells and,after five days, the cells appeared unaffected by the inter-nalized nanorods as they grew to confluence over thatperiod. This study suggests that CTAB promotes nanoroduptake by cells, which could explain the cytotoxicity ob-served by Niidome et al. with CTAB stabilized nanorods. Asummary of the experimental setup and results on goldnanorods is provided in Table 6.

4.4. Super-Paramagnetic Iron Oxide Nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs)are engineered g-Fe2O3 or Fe3O4 particles that exhibit mag-netic interaction when placed within a magnetic field. In ad-dition, when encountered by an alternating magnetic field,the particles heat up, allowing for both imaging and therapyapplications. Specifically, their utilization as an MRI con-trast agent has been extensively studied.[88–92]

In terms of cytotoxicity, while bare iron oxide nanoparti-cles exert some toxic effects, coated SPIONs have beenfound to be relatively nontoxic. Gupta et al. showed thatPEG-coated nanoparticles were biocompatible as exposedcells remained more than 99% viable relative to control atan upper concentration of 1 mg mL�1.[93] On the other hand,bare iron oxide nanoparticles induced a 25–50% loss in fi-broblast viability at 250 mg mL�1. In a more extensive study,Gupta et al. found SPION cytotoxicity to be dose depen-dent. SPIONs caused a 20% reduction in cell viability at thelowest concentration tested (0.05 mg mL�1).[94] Further re-ductions were seen at higher concentrations, with the high-est concentration tested (2.0 mg mL�1) resulting in about60% loss of cell viability. However, using a different PEG-based coating, Yu et al. found PMAO-PEG-coated SPIONs,illustrated in Figure 3, to be relatively nontoxic, with cell vi-

Table 5. Cytotoxicity of gold nanoshells.


Exposureconditions




Human breastcarcinomaSK-BR-3 cells

PEG N/A 4.4x109 nanoshellsmL�1 (core=55 nm,shell=10nm)

Live/Dead 1 h No differences in viability Hirsch[14]

2003

HER2-positiveSKBr3 breastadenocarci-noma cells

antibody-PEG-thiol

N/A 3x109 nanoshellsmL�1 (core=120 nm,shell=10nm)

Live/Dead 1 h No differences in viability Loo[15,82]

2004-2005

Female albinomice

PEG tail veininjection

2.4x1011 nanoshellsmL�1 (core=110 nm,shell=10 nm)

observation 4 h - 28days

Limit in mice muscle tissue about70pg, accumulating in the RESorgans, 1–10 ppm levels found inbone, muscle, kidney, lung

James[83]

2007

Vero cells,BALBc mice

PEI/PAA(in mice)

4x103 cells/well in96-well plates

0.001-200 mg mL�1

(core=48.9�19.1 nm,shell=5.8�1.8 nm)

WST 6, 24 h Cell viability decreased 15% at200 mg mL�1

Su[84]

2007



ability decreasing by only 9% at the 400 nm exposurelevel.[95]

In addition, other groups testing bare iron oxide nano-particles considered them to be biocompatible. Hussainet al. had similar findings as the highest dose they tested,250 mg mL�1, resulted in approximately 30% decrease incell viability but this was judged as exhibiting little to notoxicity.[96] Higher concentrations were tested by Chenget al. and at 23.05 mm of nanoparticles the group found nosignificant difference between the exposed cells and thecontrol. However, this may be due to the relatively short ex-posure time of four hours.[97]

In addition to PEG, several groups have studied the cy-totoxicity of different surface-coated iron oxide particlesand found little cytotoxicity. Gupta et al. looked at pullulan(Pn)-coated SPIONs and found no cytotoxic effects, withthe cells remaining more than 92% viable at 2.0 mg mL�1.[94]

The group attributed the low toxicity of Pn-SPIONs to thepullulan coating, which prevents the iron oxide core frominteracting with cells. Petri-Fink et al. observed no cytotox-icity in melanoma after two hours of exposure to amino-SPION for all polymer/iron ratios tested.[98] After 24 hours,cytotoxicity became apparent for high polymer concentra-tions. A similarly coated SPION tested by Cengelli et al.was found to be nontoxic as N11 microglial cells only tookup aminoPVA-coated SPIONs, and as no nitric oxide wasproduced.[99] Wan et al. tested the effects of three surfacecoatings on iron oxide cytotoxicity and found MPEG–Asp3-NH2-coated iron oxide nanoparticles had almost no cytotox-

icity at the concentrationstested.[100] In comparison,MPEG–PAA- and PAA-coated iron oxide nanoparti-cles significantly reduced cellviability with only 16% of thecell remaining at an iron con-centration of 400 mg mL�1. Asbare iron oxide nanoparticlesadsorbed to the cell surface,MTS analysis was infeasible;however, cell counts after in-cubation indicated that un-coated iron oxide nanoparti-

cles also significantly reduced cell viability.The mechanism for SPION cytotoxicity, when it does

occur, has been linked to both cellular uptake and ROS pro-duction. Hu et al. found PACHTUNGTRENNUNG(PEGMA)-immobilized nanopar-ticles were relatively nontoxic, as exposed cells had greaterthan 93% viability.[101] However, pristine iron oxide nano-particles had a viability reduced to 70% in the first twodays, increasing to about 90% by day five. The group sug-gests that this increase in viability is due to the decrease innanoparticle concentration with the increase in cells aftermitosis. This was seen as cell uptake of particles went from154 pg cell�1 on the first day to 58 pg cell�1 after five days.PACHTUNGTRENNUNG(PEGMA) nanoparticles were taken up at 2 pg cell�1 sug-gesting that their lower toxicity is due to their lack of celluptake. Brunner et al. found a cell-specific response to bareiron oxide nanoparticle exposure.[102] 3T3 cells remainedproliferative with the addition of up to 30 ppm iron oxide;however, human mesothelioma cells exhibited significant re-duction in cell viability at only 3.75 ppm iron oxide. Thegroup attributed the observed toxicity to iron-induced free-radical production via the Fenton or Haber–Weiss reactionsin addition to internalization of the iron oxide particles. Pi-sanic et al. showed anionic dimercaptosuccinic acid(DMSA)-coated iron oxide nanoparticles are readily endo-cytosed by rat pheochromocytoma cells and are foundeither in the cytoplasm, inside endosomes, or accumulatedin the perinuclear region within the cells.[103] Most of the celldeath occurred during the first 48 hours of exposure with cy-totoxicity and cell detachment being dose dependent.

Table 6. Cytotoxicity of gold nanorods.





Human embry-onic kidney,HEK293

AEDP, plasmid,rhodamine,transferrin

3x105 cells/well in 24-wellplates

44 mg mL�1

(w=100 nm,l=200 nm)

WST 4 h LD50=750 mg mL�1 Salem[31]

2003

Hela Cells PEG, CTAB 5x103 cells/well in 96-wellplates

0.01-0.5 mm

(w=11�1 nm,l=65�5 nm)

WST 24 h Cell death: ~80% at 0.05 mm

w/CTAB nanorods; only ~10%at 0.5 mm w/PEG nanorods

Niidome[86]

2006

Hela Cells phosphatidyl-choline

5x103 cells/well in 96-wellplates

0.09-1.45 mm

(w=11�1 nm,l=65�5 nm)

MTT 24 h Twice centrifuged more toxicthan PC-NRs; 20% cells died at1.45 mm

Takahashi[26]

2006

KB Cells CTAB, mPEG-DTC N/A(l=50 nm)

100 mL microscopy 5 days Internalized to perinuclearregion w/ CTAB, little uptakew/mPEG

Huff[87]

2007

Figure 3. Example structure and cell viability data for water-soluble iron oxide nanoparticles. Reprintedwith permission from Ref. [95].



Changes in cell morphology were observed with nanoparti-cle exposure with the cells assuming a spherical shape withdisruption of the cell cytoskeleton. Muller et al. consistentlyfound a 20–30% decrease in neutral red uptake in mono-cyte-macrophages with 10 mg mL�1 Ferumoxtran-10 acrossvarious incubation times.[104] Similar results were foundusing the MTT assay. Testing over a longer period showedcell viability remained about the same between Ferumox-tran-10-pretreated cells and control cells after two weeks.The group speculates that the cytotoxicity is due to ROSproduction via the Fenton reaction, which can result in lipidperoxidation, DNA damage, and protein oxidation. Testingfor inflammatory responses, the group found Ferumoxtran-10 did not induce increases in cytokines IL-1b, TNF-a, IL-6

or IL-12, or superoxide anion production. This led to theconclusion that the monocyte-macrophages were not acti-vated by the nanoparticles. A summary of the experimentalsetup and results on superparamagnetic iron oxide nanopar-ticles is provided in Table 7.

5. Semiconductor Nanoparticles

In the case of semiconductor nanocrystals, better knownas quantum dots, the concern of their cytotoxicity is not un-justified as several are composed of known toxic elements.However, despite the potential health risks, promising appli-cations of quantum dots include their use in the medical

Table 7. Cytotoxicity of Fe3O4 nanoparticles.


Exposureconditions




COS-7 cells none 3x104 cells/well,24-well plates

0.92-23.05 mm

(d=9 nm)MTT 4 h No significant difference be-

tween control and exposedCheng[97]

2004

Human fibroblasts MA-PEG 10,000 cells mL�1

in 24-well plates0-1000 mg mL�1

(d=50nm)MTT,Live/Dead

24 h 250 mg mL�1: 25-50% viabilitydecrease for bare; 1 mg mL�1:99% viable for PEG-coated

Gupta[93]

2004

Melanoma cells PVA(amino,caroxyl,thiol)

N/A 0.25 mg mL�1

(core=~9 nm,d=19-54 nm)

MTT 2 h No cytotoxicity after 2 h for allpolymer/iron ratios; after24 hr, cytotoxicity at high poly-mer conc.

Petri-Fink[98]

2004

Primary human fibro-blasts, hTERT-BJ1

pullulan 104 cells/well in96-well plates

0-2 mg mL�1

(d=40-50 nm)MTT 24 h Plain SPION showed signifi-

cant decrease in viability, Pn-SPION showed no cytotoxicityw/ >92% viability

Gupta[94]

2005

Rat liver cells, BRL3A

none confluent in 6 or24 well plates

0-250 mg mL�1

(d=30, 47 nm)LDH,MTT,GSH

24 h EC50 > 250 mg mL�1 Hussain[96]

2005

Human mesothelio-ma MSTO-211H,rodent 3T3 fibroblastcells

none N/A 3.75-15 ppm(d=12-50 nm)

MTT 3, 6 days 3T3 cells viable w/ up to30 ppm; MSTO cell viabilitydecrease at 3.75 ppm, freeradicals via Fenton rxn

Brunner[102]

2006

Rat brain-derived en-dothelial EC219;murine N9 & N11microglial cells

PVA, ami-noPVA, car-oxylPVA,ThiolPVA

96 or 48-wellplates

2.5 mL NPs mL�1,11.3 mg iron mL�1

(core=8-12 nm,d=30 nm)

MTT 48 h Only aminoPVA-SPION upakenby N11

Cengelli[99]

2006

Mouse macrophages,RAW 264.7

PACHTUNGTRENNUNG(PEGMA) 105 cells mL�1 0.2 mg mL�1

(d=6.2�0.7 nm)ratio oftreated/controlcells

1, 4 days Cytotoxicity dose dependent;decrease w/time attributed tocell division

Hu[101]

2006

Human breast carci-noma SK-BR-3 cells,human dermalfibroblasts

PMAO-PEG Confluent 10-400 nm(9.6 nm)

Live/Dead

1, 24,48 h

91% viability at 400 nm after48 h in HDF cells

Yu[95]

2006

Human monocyte-macrophages

dextran 1–2x106 cells/wellin 24-well plates &0.5-1x106 cells/well in 48-wellplates

0.0001-10 mgmL�1 (d=30 nm)

MTT,NeutralRed

24, 48,72 h, 4days +14 daygrow

1 mg mL�1: not toxic after72 h; 10 mg mL�1: mildlytoxic; viability similar over2 wks

Muller[104]

2007

rat pheochromo-cytoma cell linePC12M

DMSA 20,000 cells mL�1

in 6 or 12 wellplates

15, 1.5 mm and150 mm (d=5-12 nm)

Live/Dead

2, 4, 6days

SPION exposure reduced PC12ability to respond to nervegrowth factors

Pisanic[103]

2007

OCTY mouse cells MPEG–Asp3-NH2,MPEG-PAA,PAA

104 cells/well in96-well plates

0-400 mg mL�1

(d=14 nm)CellTiter 96

72 h MPEG–Asp3-NH2 almost nocytotoxicity; MPEG–PAA- andPAA-coated decrease cell via-bility

Wan[100]

2007



field as new drug-delivery and biomedical-imaging agents.This section explores the current research that has beenconducted on quantum-dot toxicity.

Quantum dots (QDs) are nanoscale particles rangingfrom 2 to 100 nm in diameter depending on the types of sur-face coating or functional group added. For biological appli-cations, QDs typically have a core/shell conjugate structure.The core of the QD is composed of atoms from groups II–VI (e.g., CdSe, CdTe, CdS, PbSe, ZnS, and ZnSe) andgroups III–V (e.g., GaAs, GaN, InP, and InAs) on the peri-odic table.[105] Many of these core metals are known to betoxic at low concentrations; examples include cadmium, se-lenium, lead, and arsenic. Therefore, if these QDs are ex-posed to conditions promoting degradation, such as an oxi-dative environment, toxicity related to the release of freemetal ions is expected. Thus the crucial factor in QD toxici-ty is stability. The cytotoxicity of QDs is reduced when their

cores are protected from degradation given that the addedcoatings are biocompatible. To prevent core degradation, anadditional shell layer is added, making the QD more bio-compatible. Additional functionalities or bioconjugates canbe added to the surface to improve bioavailability or intro-duce bioactivity. Since CdSe/ZnS quantum dots are believedto be the most versatile for biological applications, most ofthe published toxicity studies focus on this type.[106,107] Sum-maries of the experimental setup and results on CdSe andCdTe QDs are provided in Tables 8 and 9, respectively.

5.1. Cadmium Selenide Quantum Dots

Historically, QDs were used in animals well before ex-tensive cytotoxicity studies. These results highlighted keyissues, such as biodistribution and coating integrity, now of

Table 8. Cytotoxicity studies on CdSe quantum dots.


NP concen-tration(averagesize)



BALB/c nu/numice

3 peptides (GFE, F3,LyP-1), PEG

tail veininjection

100-200 mgQDs in0.1-0.2 mLsolution

histology 5 or20 min

Specific tissue targeting achievable,accumulate in liver and spleen

Akerman[109]

2002

Xenopusembryos

PEG-PE,phosphatydilcholine

N/A 2.3 mm microscopy At least4 days

At >5x109 QDs/cell abnormalitiesbecame apparent

Dubertret[108]

2002

Vero cells MUA, SSA N/A 0.24mg mL�1

microscopy 2 h 0.4 mg mL�1: no difference in via-bility of MUA-QD/SSA complexes

Hanaki[123]

2003

Hela cells, Dic-tyostelium dis-coideum

DHLA N/A 400-600 nm microscopy 45-60 min

No differences in viability Jaiswal[122]

2003

Mice Micelle tail veininjection

20 nM�1 mm observa-tion

N/A No abnormal behavior observed Larson[110]

2003

BALB/c mice poly(acrylic acid)polymer or mPEG-750 QDs, mPEG-5000 QDs, COOH-PEG-3400 QDs

tail veininjection

50-500 pmolQDs in 50-200 mL saline

histology/microscopy

1-3 h @1 mininterval

QDs in endosomes in liver, spleenand bone; fluorescence at 1 mo.similar to 24 h signal, mPEG-5000QDs have longer circ. time & loweraccumulation

Ballou[111]

2004

Hela cells silane, biotin, STV,peptides, NLS

300 cells/100 mm

1, 10,100 nm

(d=8-10 nm)

colonigenicassay

2 h On average >90% cells survived Chen[126]

2004

Rat primaryhepatocytes

MAA, BSA/EDAC,EGF

5x105 cellson 35 mmwells

0.0625,0.25, 1 mgmL�1

MTT,microscopy

24 h Coating eliminates air oxidation;however, high conc. with 8 h UVexposure still have 95% cell death

Derfus[113]

2004

Humanlymphoblastoid,WTK1

MUA, cysteamine(NH2),thioglycerol (OH)

5x104

cells/wellon 96-wellplates

0-2 mm

(d=9-48 nm)Comet,flow cytom-etry, MTT

12 h Crude QDs exhibited decreased cellactivity, QD fluorescence lost in lowpH oxidation, TOPO is cytotoxic

Hoshino[118]

2004

Vero cells, Helacells, primaryhuman hepato-cytes

MUA, SSA 3x104


0-4 mg mL�1 MTT, flowcytometry

24 h Damaged cells increased sharply at0.2 mg mL�1 but slowly at0.1 mgmL�1

Shiohara[116]

2004

B16F10 melano-ma cells

TOPO, DHLA 5x104


10 mL microscopy 4-6 hr No detectable toxicity Voura[129]

2004

DNA biotin N/A N/A Plasmanickingassay

0-60 minin darkor UV

DNA damage occurs in bothenviron, UV exposure incr damage

Green[127]

2005



concern in cytotoxicity studies. Dubertret et al. injected mi-celle-encapsulated CdSe/ZnS QDs into Xenopus embryos.At 2K109 QDs cell�1, the quantum dot injected and controlembryos displayed similar growth patterns with the QDs re-maining in the injected cells and their progeny. However, at5K109 QDs cell�1, adverse effects were found. The groupspeculated that the abnormalities resulted from the QDs af-fecting the osmotic equilibrium of the cell.[108] The majorityof published in vivo studies were conducted in mouse or ratmodels. Akerman et al. injected three different (GFE, F3,

LyP-1) peptide-coated CdSe/ZnS QDs into normal BALBcmice and studied the tissue distribution after 5 or 20 mi-nutes of circulation. Each of the peptide-coated QDs werefound to accumulate in the liver and spleen in addition tothe targeted tissue; yet, this nonspecific accumulation wasreduced by adding PEG to the QD surface. The group didnot observe any acute toxicity caused by the QDs after 24hours of circulation.[109] Similarly, Larson et al. observed noadverse effects after imaging the mice used in their experi-ment and hypothesized that CdSe/ZnS QDs clear from the

Table 8. (Continued)


NP concen-tration(averagesize)



NRK fibroblasts;MDA-MB-435Sbreast cancercells; Chinesehampster ovary,CHO; RBL cells

MPA, mercapto-carbonic acid

7x104

cells mL�12-10 mm

(core d=2.4-4.5 nm)

ratio ofpre/postadherentcells, Live/Dead

18-48 h NRK cells: poisoning occurs around0.65 mm for MPA-CdSe, 5.9 mm forCdSe/ZnS; other 3 cells: only largerparticles inside cell, polymer betterthan MPA

Kirchner[114]

2005

COS-7 cells,NIH 3T3 cells,Human livercarcinoma,HepG2

SiO2, MAA, PA 2x104 cellsmL�1 in 96-well plates

0-0.6 mm

(cored=5 nm,d=25 nm)

Alamarblue

48 h(cos-7 &hepG2),72 h(3T3)

SiO2

coating better than PA, ZnS coatingdecreases toxicity

Selvan[35]

2005

Human breastcarcinomaSK-BR-3 cells

PEG (750-,6000-Mw)

22,500cells/wellin 96-wellplates

10-150 nM Live/Dead 4 h 150 nm - 0%, 70%, 80% cell viabili-ty for bare, 750- & 6000-Mw PEGcoated QDs

Chang[124]

2006

Human bonemarrow mesen-chymal stemcells (hBMSC)

HIV-derived Tatpeptide

7x105 cellsin 10 cmdishes

1.625 mg WST, flowcytometry,Rt-PCR, mi-croscopy

24 h Growth curve and cell cycledistribution not affected by QDs

Hsieh[121]

2006

Sprague–Dawley rats

LM, BSA jugularvein injec-tion

5 nmol in0.2 mL solu-tion (d=25,80 nm)

histology 90 min QDs uptake in Kupffer cells, majori-ty uptake in liver with some inspleen, lungs, kidneys, lymphnodes, bone marrow

Fischer[112]

2006

Human breastcancer cells,Lung (IMR-90),skin (HSR-42)

silica, thiol, PEG N/A 2-10 nm

Brca, 8-80 nm lung,skin (d=8-10 nm)

microarray 48 h No adverse effects in lung cells,skin cells showed <50 gene ex-pression changes

Zhang[128]

2006

Human breastcancer cells,MCF-7

NAC, Cys, MPA 105 cellscm�2 in24-wellplates

10 mg mL�1 MTT 24 h MPA capped QDs more toxic thanCys capped, cells exhibit reactionsof oxidative stress

Cho[130]

2007

Hela cells PEG-g-PEI, poly-carboxylate

60% con-fluency

1 nm (cored=6.5 nm,PEI coatedd=15.3 nm)

MTT 2 h PEI-coated dots very toxic, toxicityreduced w/ more PEG added

Duan[119]

2007

HepG2 cells,Wister mice

PLA, F-68, SDS,CTAB

6x104 cellson 96-wellplates

10-400 ppm(d=159-266 nm)

MTT 12-72 h >80% cell viability up to 400 ppm Guo[131]

2007

ctDNA MAA 5 mg mL�1

ctDNA3.6x10�7

mol L�1Nucleicacid probe(RuACHTUNGTRENNUNG(bipy)2-ACHTUNGTRENNUNG(dppx)2+)

110 min 1.7x10�5 mol L�1 Cd2+ released,70% DNA damaged by QDs

Liang[132]

2007

primary HEKs PEG, PEG-amine,polyacrylic acid

1.5–2x104

cells cm�2,40-60%confluency

0.2-20 nm

(d=4.6 nm& w=6 nm,h=12 nm)

MTT 24, 48 h Dose–response significant for PEG-amine and carboxylic acid but notPEG alone

Ryman-Rasmussen

[120]

2007



body before the protective coating can breakdown. Howev-er, the group did not report a time line for which the ani-mals were kept, only mentioning that the animals weremaintained for a long-term toxicity study, which diminishestheir findings.[110]

More recently, the role of the particle surface on distri-bution and toxicity has been studied. Ballou et al. also ob-served QD deposition in the liver, spleen, and bone marrowof BALBc mice depending on the surface coating present.Of the four coatings tested (poly(acrylic acid), mPEG-750,mPEG-5000, COOH-PEG-3400), the mPEG-5000 QDswere found to have the longest circulation time in additionto reduced nonspecific accumulation. All QDs were foundlocalized internally in endosomes, primarily within perifol-licular cells for the spleen, and no visible signs of break-down were seen via electron microscopy. A long-term stabil-ity study conducted with mPEG-750 QDs found the QDsremained in the liver, lymph nodes, and bone marrow for amonth. Although the fluorescence decreased after onemonth, the fluorescence distribution was close to what thegroup observed 24 hours after injection.[111] More recently,Fischer et al. injected mercaptoundecanoic acid (MUA),lysine, and BSA-coated CdSe/ZnS QDs in Sprague–Dawleyrats. A difference in biodistribution was also found as theliver took up 40% of the lysine-QDs and 99% of BSA-QDsafter 90 minutes. QDs endocytosed by Kupffer cells, similarto RES processing, were sequestered not excreted. Smallamounts of both QDs appeared in the spleen, kidney, andbone marrow, but no QDs were detected in the feces orurine even after ten days. The group also measured the sizeof the quantum dots within the vesicles and found the QDsretained their size, suggesting no degradation after 90 mi-nutes of exposure.[112] These studies conclude that ZnScapped CdSe QDs are relatively nontoxic as the animalswere not killed, nor did they exhibit abnormal behavior

after QD injection. However, as several of the group found,the QDs are internalized and seem to be retained inside thecells. As clearance from the body is an important aspect ofsafety, this suggests possible toxicity could result from thebioaccumulation.

All of the previously mentioned studies involved ZnScoated CdSe quantum dots; the ZnS coating provides awell-terminated surface with few defects and high quantumyields. A seminal in vitro study conducted by Derfus et al.found that, when incubated with rat primary hepatocytes,bare CdSe QDs undergo surface oxidation, resulting in therelease of free cadmium ions. Cadmium is a known toxicagent that induces cell death via mitochondrial damage andoxidative stress. When QD surface oxidation was preventedwith surface coatings, the cadmium atoms remained boundto selenium atoms and the surface-coating molecules ren-dering them relatively nontoxic. This was demonstratedwith the addition of a ZnS shell; the oxidative degradationof the CdSe core due to exposure to air was significantly re-duced resulting in lower cytotoxicity.[113] Several groupshave confirmed the effectiveness of the ZnS shell in reduc-ing the cytotoxicity of CdSe quantum dots.[34,35,114,115] Chanet al. proposed a mechanism for the bare CdSe QD-inducedcell death. In addition to determining that apoptosis, not ne-crosis, occurred in CdSe exposed cells, the group suggestedthat QDs induced apoptosis by activating Jun N-terminalkinase (JNK) in a dose-dependent manner. In addition, mi-tochondrial-dependent apoptotic processes, involving activa-tion of caspase 9 and 3, increases in Bax protein and de-creases in Bcl-2, were also observed. Mitochondrial-mem-brane potential was reduced with exposure to bare CdSeQDs resulting in an increase in cytochrome c release.[115]

Researchers have also examined the effect of additionalsurface coatings on the cytotoxicity of quantum dots. QDsmust be appropriately encapsulated to prevent cadmium re-

Table 9. Cytotoxicity studies on CdTe quantum dots.

Cell line Surface coat-ing

Exposureconditions

NP concentra-tion (averagesize)



Rat pheochro-mocytoma cells,PC12

anionic, cat-ionic, BSA

105 cellscm�2 on24-wellplates

0.01-100 mgmL�1 (d=2.2-5.7 nm w/ocoating)

MTT 24 h 50% decrease in metabolic activity: 50 mgmL�1 (green) 100 mg mL�1 (red)

Lovric[133]

2005

Human hepato-ma HepG2 cells

none 2x105 cellsmL�1 in96-wellplates

0, 10�8, 10�7,10�6, 10�5 m

MTT 24 h ~50% viability reduction at 10�5 m; simi-lar cytotoxicity found w/ air and N2 ex-posed QDs

Liu[135]

2006

Human breastcancer cells,MCF-7

NAC, Cys,MPA

105 cellscm�2 in24-wellplates

10 mg mL�1 MTT 24 h MPA capped QDs more toxic than Cyscapped; cys-CdTe QDs more cytotoxicthan cys-CdSe/ZnS QDs; cells exhibit oxi-dative stress

Cho[130]

2007

Human neuro-blastoma cells,SH-SY5Y

cysteamine,N-acetylcys-teine (NAC)

105 cellscm�2 in24-wellplates

5 mg mL�1 MTT, flowcytometry

24 h 52% viability w/ cyc-QDs, 85% viability ifpretreated w/NAC

Choi[136]

2007

Human hepato-ma cells,HepG2; Spra-gue-Dawley rats

none 2x105 cellsmL�1 in96-wellplates

0-100 mm; 2mm,1 mL kg�1

in rats (d=2-6nm)

MTT 48 h; 24 h(0, 0.5, 1,2, 4 h) - inrats

IC50: 19.1 mm (red), 4.8 mm (yellow), 3 mm

(green); rats: few signs of toxicity, butchanges in locomotor activity were ob-served

Zhang[134]

2007



lease and subsequent cytotoxicity. At issue for many re-searchers is the best way to accomplish this. One simple ap-proach is to replace the original organic coat with water-soluble ligands. These ligand-exchange reactions yield QDswith smaller hydrodynamic sizes but generally do not pro-vide biocompatible materials. Shiohara et al. studied the cy-totoxicity of three MUA-coated CdSe/ZnS QDs (520-, 570-and 640-nm emission) in three different cell lines (Verocells, Hela cells, and human primary hepatocytes). After in-cubating the cells with quantum-dot concentrations rangingfrom 0 to 0.4 mg mL�1 over 24 hours, a concentration de-pendence of cytotoxicity was found.[116] It is important torecognize that, in this case, the toxicity is due to the surfacecoating rather than the quantum dots. MUA is a compoundthat in previous studies was found to water solubilize CdSe/ZnS quantum dots.[117] This is important in biological appli-cations as hydrophobic compounds have poor bioavailabil-ity. This study reveals that the MUA coating is not appropri-ate for this purpose as it increases the toxicity of the quan-tum dots. Hoshino et al. did a larger study incorporatingmore surface coatings (MUA, cystamine, thioglycerol) onCdSe/ZnS QDs. MUA-coated quantum dots were moretoxic than ones without. Of the three coatings, thioglycerolwas found to induce the least genotoxicity and therefore cy-totoxicity. These results indicate that some hydrophilic sur-face coatings contribute to the cytotoxicity of QDs. BecauseTOPO was also found to be a cytotoxic compound, the com-plete removal of TOPO from the QD samples is importantin reducing toxicity.[118] Selvan et al. looked at SiO2, mercap-toacetic acid (MAA), polyanhydride (PA) surface-coatedCdSe/ZnS quantum dots in three different cell lines (humanliver carcinoma (HepG2), NIH T3T cells, COS-7 cells) andobtained dose-dependent results for all surface coatings ineach cell line. SiO2/CdSe QDs were found to be much lesscytotoxic than MAA or PA coated QDs. SiO2/ZnS-CdSeQDs were less cytotoxic than SiO2/CdSe QDs, suggestingthat the combination of ZnS capping and SiO2 coating pro-vided for the optimal protection against CdSe dissolution.[35]

A better option for biocompatible QDs is to use amphi-philic polymers to encapsulate the inorganic/organic system.Most commercial sources of QDs prepare their systems inthis way. While larger polymeric coatings increase the hy-drodynamic size, they yield very bright and stable materials.Kirchner et al. found PEG to lower cellular uptake of silica-coated QDs resulting in lower cytotoxicity.[114] Duan et al.tested PEI-coated QDs in HeLa cells and found they areendocytosed or macropinocytosed after one to two hours ofincubation. However, since PEI-coated dots are toxic tocells, PEG was added to reduce this toxicity. The two differ-ent forms (PEI grafted with two PEG and PEI grafted withfour PEG) of coated QDs exhibited different distributionpatterns inside the cells and different cytotoxicities. ThePEI-g-PEG4 QDs accumulated in the perinuclear region,which yielded better cell viability while the PEI-g-PEG2QDs were distributed in the cytoplasm and had significantcytotoxic effects. The group believes the cytotoxicity is dueto the PEI polymer and not the presence of cadmiumions.[119] Ryman-Rasmussen et al. tested two different QDs(565- and 655-nm emission) with three different surface

coatings (PEG, PEG-amine and carboxylic acids). All QDswere localized intracellularly by 24 hours with the PEG-coated QDs found in the cytoplasm, perinuclear region, andfor QD 565 within the nucleus. After 24 hours, no cytotoxic-ity was observed, but by 48 hours toxicity became apparentat the largest concentration of 20 nm, indicating time-depen-dent cytotoxicity. Surface coating had an observable effecton IL-1b, IL-6, and IL-8 pro-inflammatory cytokine release.Cytokine levels increased after carboxylic acid coated QDexposure, while there was no increase in cytokine releasewith PEG-coated QDs.[120]

In addition to their coatings, size and concentration caninfluence the toxicity of quantum dots with smaller sizesand higher concentrations being more cytotoxic. The ad-vantage of quantum dots in imaging applications is theirtunability. By changing their size or core diameter, the fluo-rescence emission peak can be shifted to a wavelength ofchoice within a fairly broad range. This is particularly usefulin biological applications as cells contain endogenous fluo-rophores, which can mask the signal emitted from contrastagents with similar emission peaks. However, several groupshave found cytotoxicity to be size dependent with smallerQDs exhibiting larger reductions in cell viability. Kirchneret al. tested the exposure of CdSe/ZnS QDs in several celllines (NRK fibroblasts, MDA-MB-435S breast cancer cells,CHO cells, RBL cells). After 18 hours exposed to the sameconcentration, cytotoxic effects were higher for smallerQDs, which they suggest could be due to the higher surface-to-volume ratio of smaller particles.[114] Interestingly, Hsiehet al. found size-independent internalization of QDs.[121] Inaddition, while high concentrations of QDs can be toxic tocells, the group found that concentration influenced QDsdelivery into cells with a low concentration (15 nm) effec-tively labeling cells while a 10-fold increase in QD concen-tration, resulting in poor cellular uptake. However, this wasnot the typical finding. As with many chemicals, cytotoxicityof QDs was also found by many research groups to be dosedependent with higher concentrations, resulting in signifi-cantly higher cell death.[113,114,116,120]

In vitro cytotoxicity studies report findings similar toin vivo studies that QDs are taken up and sequestered intra-cellularly. Jaiswal et al. demonstrated that targeted CdSe/ZnS QDs could be internalized by HeLa cells and trackedin live cells for more than 10 days with no morphologicalsigns of toxicity.[122] Hanaki et al. studied how long MUA-coated QDs could stay in Vero cells. QD-containing ve-containing vesicles.[123] The number of vesicle-containingcells reduced to half after three days and about 10% of thecells contained QD vesicles after five days. These findingscorrespond to introducing a QD concentration of0.4 mg mL�1, which was found to have no cytotoxicity. Amore long-term study was conducted by Seleverstov et al. ,who found that QD-labeled cells retained their fluorescentsignal for 52 days in both continuous culture or after cellpassaging.[34] The QDs were internalized and observedmainly within endosomes near the perinuclear region withno nuclear involvement. In addition, QD aggregates werefound localized around the mitochondria and after 72 hoursmorphological effects included swollen mitochondria and



enlarged Golgi cisterns. Hsieh et al. confirmed the perinu-clear localization of QDs and found that cells retained QDsfor at least three weeks.[121] While these studies have shownthat cells can survive long after internalizing QDs, Changet al. found with three different QDs (bare, two PEGcoated) that cytotoxicity is similar between the differentlycoated QDs when their intracellular concentration is thesame, as seen in Figure 4. Therefore, while extracellular con-centrations of different QDs suggest dose-dependent cyto-toxicity, cytotoxicity is dependent on the intracellular QDcontent, and biocompatibility can be improved by minimiz-ing QD uptake.[124,125]

The ability of QDs to be internalized by cells has ledsome groups to pursue using CdSe/ZnS QDs for nuclear tar-geting. Chen et al. revealed that silane-coated CdSe/ZnSQDs conjugated with the SV40 nuclear localization signal(NLS) protein only entered the nucleus of 15% of thecells.[126] Perinuclear accumulation was still observed for themajority of the NLS-QDs. However, QDs conjugated to arandom peptide did not enter the nucleus and only localizedrandomly within the cells. Testing for cytotoxicity on HeLacells, the group found most of the transfected cells survivedin all the experiments thus implying negligible toxicity evenwith nuclear exposure to QDs.

Since QDs are capable of entering the nucleus, severalgroups have suggested QD interaction with nuclear DNA orproteins to be a factor in their cytotoxicity. Green et al. re-ported data corroborating this theory as they found biotin-coated CdSe/ZnS QDs were able to nick DNA in an in vi-tro, cell-free assay.[127] Hsieh et al. showed that QDs canalter gene expression in human bone-marrow mesenchymalstem cells. While flow cytometry analysis showed that theinternalized QDs did not change the cell-cycle distributionof hBMSCs compared to the control, an inhibited responseof hBMSCs to osteogenesis was found as ALP activity wassignificantly suppressed, and mRNA expression of osteo-pontin and osteocalcin, two osteogenesis specific markers,

was also inhibited. These effects could be reproduced usingQDs of various sizes.[121] A DNA microarray study was re-cently published by Zhang et al., examining the impact oftreating human lung and skin epithelial cells with two dosesof PEG-silane QDs. No adverse effects were found in lungepithelial cells; however, the skin epithelial cells exhibitedcell-cycle regulator gene repression. Overall, fewer than50 genes showed significant expression changes after PEG-silane QD treatment. However, the group did not find in-volvement of the genes that are associated with heavy metalexposure. In addition, no pronounced difference in pheno-typic response was found between low or high QD doses.Higher QD doses led to more particle uptake made appar-ent from the stronger measured fluorescent signal.[128]

5.2. Cadmium Telluride Quantum Dots

Besides CdSe/ZnS QDs, one group has published severalstudies on CdTe QDs, which also have potential in biomedi-cal applications such as bioimaging. The initial study pre-sented by Lovric et al. tested cell exposure to both red(�5.2 nm) and green (�2.2 nm) CdTe QDs coated withmercaptopropionic acid (MPA) and cysteamine (Cys).[133] Arange of concentrations (0.01–100 mg mL�1) was used totest the effect of exposure on metabolic activity. For bothtypes of QD, a decrease in metabolic activity was found atconcentrations of 10 mg mL�1 or more. Looking at cell mor-phology, the group found that the rat pheochromocytoma(PC12) cells took up the QDs at both the low(3.75 mg mL�1) and high (37.5 mg mL�1) concentrations.However, at the high concentration, chromatin condensa-tion and membrane fragmentation were observed, indicativeof apoptosis. In addition, cytotoxicity was more noticeablewith the smaller QDs than with the larger QDs at the sameconcentrations. To explain this difference, the group notedthat the red QDs were found primarily in the cell cytoplasmwith none entering the nucleus, while the green QDs weremainly found in the cell nucleus. This suggests that since thesmaller QDs could access the nucleus they could causedamage to DNA and induce apoptosis or cell death. As QDcytotoxicity is believed to be due to free-radical formationcaused by the presence of free Cd2+ from the degradationof the QD core, the effect of free-radical scavengers N-ace-tylcysteine (NAC) and Trolox, as well as adding anotherprotective coating, bovine serum albumin (BSA), wastested. Both NAC and BSA but not Trolox significantly re-duced CdTe QD toxicity, suggesting that Cd2+ is a factor inQD-induced toxicity.[133]

Zhang et al. had similar findings after testing green-(�2 nm), yellow- (�4 nm), and red- (�6 nm) light emit-ting, uncoated CdTe quantum dots in human hepatoma cells(HepG2). A size-dependent difference in toxicity was ob-served as the IC50 values of the green, yellow, and redCdTe quantum dots were 3.0, 4.8, and 19.1 mm, respectively,where IC50 corresponds to the concentration causing a 50%reduction in MTT activity. Therefore, they confirmed thefindings presented earlier by Lovric et al. that smaller QDsare significantly more cytotoxic than larger QDs. In addi-

Figure 4. Cytotoxicity results based on intracellular levels of bareQDs (left image, black bars), and 750- (middle image, gray bars) and6000- (right image, light gray bars) Mw PEG-substituted QDs.Reprinted with permission from Ref. [124].



tion, they looked for the effect of CdTe quantum dots in-jected into Sprague-Dawley rats. Few signs of morbid toxici-ty were observed after QD exposure. The group saw noorgan damage, only a small decrease in body weight, and atemporary decrease in locomotion activity occurring justafter injection. This change was attributed to possible effectsof the quantum dots on neural function, which is plausiblesince some nanoparticles can pass the blood–brain barrier.However, the group recognizes that this change could alsobe due to exposure to free cadmium.[134]

The effect of different surface coatings was explored byCho et al. using four cadmium QDs (MPA-, Cys-, or NAC-coated CdTe QDs plus cysteamine-coated CdSe/ZnS QDs)on human breast-cancer (MCF-7) cells. While minimal re-duction in metabolic activity occurred after exposure toCys-coated CdSe/ZnS QDs, exposure to MPA and Cys-coated CdTe QDs caused a significant decrease in cellularmetabolic activity with a less distinct decrease in the NAC-coated CdTe QDs. This finding was confirmed using theTrypan blue cell viability assay, which revealed significantcell death with CdTe QDs but not with CdSe/ZnS QDsafter 24 hours of QD exposure.[130] In addition to using sur-face coatings to reduce cytotoxicity, Liu et al. attempted toalter the synthesis condition to improve biocompatibility ofCdTe QDs. While the dose-dependent reduction in cell via-bility after CdTe QD treatment was confirmed, this wasfound regardless of either air or nitrogen fabrication condi-tions.[135]

Recently, Choi et al. revealed a possible signaling path-way involved in CdTe QD-induced cell death. Activation ofthe Fas receptor results in a signaling cascade that culmi-nates in apoptosis. This study found significant upregulationof Fas expression on the surface of neuroblastoma (SH-SY5Y) cells treated with Cys-coated and NAC-conjugatedQDs in comparison to control cells. However, NAC-cappedQDs had little Fas upregulation, and NAC pretreated cellsexposed to Cys-QDs had no Fas upregulation, suggestingthat oxidative stress caused by QD exposure induces Fas ex-pression.[136]

6. Summary and Outlook

In this paper, cytotoxicity data on carbon-, metal-, andsemiconductor-based nanoparticles have been reviewed. Ingeneral, cells can survive short-term exposure to low con-centrations (<10 mg mL�1) of nanoparticles. However athigh doses, several groups have found cytotoxic effects toemerge in a dose- and time-dependent manner for all of thenanoparticles reviewed here. While the causes for the in-crease in cell death observed at higher concentrations andlonger exposure times are material specific, the generationof reactive oxygen species and the influence of cell internal-ization of nanoparticles are two common findings through-out. While this Review attempts to draw parallels betweenthe research that has currently been conducted and publish-ed on several classes of nanoparticles, there are still gaps inknowledge about the interaction of nanoparticles with thebody. Although several studies have been conducted, many

of the earlier experiments were not designed to isolate thesource of the cytotoxicity, allowing the different physio-chemical properties of the nanoparticles plus experimental-setup factors to influence and confound the findings. In ad-dition, a systematic approach to testing has not been estab-lished.

While much of the function of nanoparticles is due totheir core structure, the surface coating defines much oftheir bioactivity. For many nanoparticles to be useful in bio-logical applications, the addition of some type of surfacecoating is required. In the case of quantum dots, surfacecoatings serve both to contain the cadmium particles fromleeching and to make the particles water soluble. This addi-tion of surface coatings confounds the bioactivity and poten-tial toxicity of the functional groups on the nanoparticle sur-face with the core nanoparticle making it difficult to inter-pret the observed changes. For example, many nanoparticlesare not water soluble and therefore require the addition ofa hydrophilic surface coating. However, as seen in MWNTand QD studies, adding certain hydrophilic molecules re-sults in lower cell viability as the functional groups them-selves were toxic.[11,22,114,118] Surface charge also plays a rolein toxicity with cationic surfaces being more toxic thananionic, and neutral surfaces being most biocompatible.[12]

This may be due to the affinity of cationic particles to thenegatively charged cell membrane. Therefore, adding a coat-ing that makes the nanoparticle more cationic could makethe nanoparticle appear more toxic than it inherently is.

Traditionally, in vitro toxicity testing focuses on whetheror not exposure to a potentially toxic agent results in celldeath. However, although no cell damage or death may beapparent after nanoparticle exposure, changes in cellularfunction may result. Therefore, it is important to verify thatthe end points chosen to signify cytotoxicity are appropriate.For example, if nanoparticle exposure induces cell senes-cence but not cell death, this could be considered a toxiceffect as cell proliferation has been disturbed. Looking overthe cytotoxicity assays commonly used in the studies re-viewed, most either determine membrane damage, metabol-ic irregularities, or inflammatory response, which may notmaterialize with cell senescence. Therefore, sub-lethal cellu-lar changes should also be taken into account and tested forwhen evaluating the effects of nanoparticle exposure oncells. One way to do this, which a few groups have explored,is to conduct genomic and proteomic array tests to explorethe cellular signaling alterations behind the toxicity.

In addition, it is important that the assays used to deter-mine cytotoxicity are valid for the materials being tested.One example, the neutral red test, has come into questionas it relies on the adsorption of the dye to detect living cells.Carbon black has been shown to adsorb neutral red dyemolecules giving false positive results.[9] This suggests thatcarbon nanomaterials could encounter the same interfer-ence, and they have been shown to adsorb a similarly struc-tured chemical, naphthalene.[137,138] The MTT assay has alsocome under scrutiny as groups have found discrepancies be-tween the MTT assay results and those from other assays. Ina study conducted by Pulskamp et al. on SWNTs, the MTTassay was the only test that revealed a dose-dependent de-



crease of cell viability. The WST-assay results did not agreewith the MTT assay as it indicated no significant loss in cellviability except for a small reduction at the highest concen-tration. PI and annexin stains were also used for validation,and they confirmed the WST-assay results.[64] To explain thedifference in results, Worle-Knirsch et al. proposed thatSWNTs interact with the MTT-formazan crystals but notwith WST, XTT, or INT reagents. SWNTs attach to the in-soluble MTT formazan product disrupting the distinguish-ing, colorimetric reaction. This would account for the find-ing that very pure SWNTs reduced cell viability below 50%for MTT assays, but exhibit almost no loss according to theWST, LDH, and MMP assays.[69]

To date, there is a lack of consensus in the published lit-erature on nanoparticle toxicity due to variable methods,materials, and cell lines. Nanotoxicology has emerged re-cently to apply traditional toxicology methodologies to thestudy of nanomaterial toxicity; however, standardization inexperimental set up such as choice of model (cell line,animal species) and exposure conditions (cell confluency,exposure duration, nanoparticle-concentration ranges anddosing increments) is necessary in order for comparisons be-tween studies conducted by different groups to be effec-tive.[139] With respect to model choice, both animal- andhuman-derived cells have been used. Since the potentialtoxicity of nanoparticles in humans is in question, humancells should be used to better predict human toxicity. In ad-dition, the cell types tested in cytotoxicity tests should alsobe consistently studied. Several groups have tested potentiallung or dermal toxicity; however, in the case of oral or in-travenous exposure, many internal organ sites can be ex-posed. While some groups have looked at liver and kidneyexposure, these studies have mainly been conducted usingquantum dots with few to none using fullerenes and goldand iron oxide nanoparticles. Fewer nanoparticle studieshave been conducted using heart, blood, and braincells.[12,49,114,115,136] Detailed recommendations have been out-lined by a Nanomaterial Toxicity Screening WorkingGroup.[140]

Consistency in reporting the physiochemical characteris-tics of nanoparticles would also facilitate re-examinationand cross comparison of nanoparticle toxicity data. Stand-ardization of materials is more challenging as nanoparticlecharacterization can be difficult. Sizing of nanoparticles canbe done using methods such as scanning and transmissionelectron microscopy, dynamic light scattering, and size-ex-clusion chromatography; however, the size values obtainedcan vary between these methods. A standard technique formeasuring and reporting the hydrodynamic sizes of nano-particles would be valuable. Determining the concentrationof nanoparticles in solution is more difficult. Concentrationcan be calculated from the optical density using the Beer–Lambert law given the extinction coefficient of the nanopar-ticle. However, as Yu et al. point out, the extinction-coeffi-cient values published for quantum dots differ betweengroups by an order of magnitude.[141] Cryogenic TEM wasused as an alternative method of determining concentration.This method involves direct counting of particles in a rela-tively fixed volume. As concentration or dose plays a signifi-

cant role in biomedical applications of nanoparticles, havinga standard technique of calculating this value is important.

Another important aspect of toxicology is the burden ofmultiple dosing of nanoparticles. In the case of bioimaging,exposure of nanoparticle contrast agent would not occuronce but repeatedly with each screening or diagnostic scan-ning session. All of the studies reviewed in this paper in-volved only one administration of various concentrations ofnanoparticles with cytotoxicity tests taken at different timepoints. However, this is most likely because most were in vi-tro versus in vivo studies. As toxicity studies move moreinto in vivo nanoparticle evaluation, future experimentsneed to incorporate the effect of multiple exposures tonanoparticles to determine the extent of clearance and bio-accumulation.

Most of the in vitro studies presented in this paperassess dosimetry merely by observing the dose-response re-lationship after external introduction of different concentra-tions of nanoparticles. Yet, as cells are seen to readily inter-nalize nanoparticles, the number of internalized nanoparti-cles correlates to cytotoxicity as Chang et al. revealed.[124]

Future research should measure and record the cellulardose in addition to the administered dose to better charac-terize the extent of nanoparticle exposure. Strategies to de-termine the particokinetics in in vitro systems have beensuggested by Teeguarden et al.[142] In addition, several of thestudies have suggested that after internalization, the groupsobserved a persistence of nanoparticles within cells. This se-questration of nanoparticles could elicit inflammatory re-sponses, cell-cycle irregularities, and gene-expression altera-tions. Unfried et al. have reviewed the mechanisms in whichnanoparticles are taken up and processed by cells; however,knowledge in this area is still limited.[143] Future researchshould focus on understanding how cells internalize nano-particles so that methods to prevent cell opsonization ofnanoparticles can be developed. This potentially could im-prove the in vivo biocompatibility and clearance of nanopar-ticles.

One clear result from this analysis is that there is disa-greement as to what constitutes low toxicity. This may bedue to the lack of a reference nanoparticle system to use asa benchmark for comparison. Given that some standard invitro testing methods will be established, it may be applica-ble to use gold nanoparticles as a reference nanoparticle forlow toxicity. Gold nanoparticles have been reported toinduce little toxicity, around 15% reduction in cell viability,at 200 mg mL�1.[28,29,31,84] While higher concentrations couldelicit a cytotoxic effect, many substances become toxic athigh concentrations. Therefore, it may be reasonable to con-clude that the results from cytotoxicity testing of othernanoparticle types suggest low toxicity if those results aresimilar for gold-nanoparticle solutions containing relativelythe same size particles at the same concentration.

Although nanoparticle-induced cytotoxicity has been re-ported by several groups, it is important to keep in mindthat in vitro results can differ from what is found in vivoand are not necessarily clinically relevant. In addition, therisk of any potentially toxic substance is not only a functionof hazard but also chance of exposure. The nanoparticle



concentrations needed for biomedical applications have notbeen optimized so the levels at which patients may be ex-posed are not certain. At the current stage in nanoparticlesafety research, it would be premature to conclude, basedon the present studies published, that nanoparticles are in-herently dangerous. However, now that a basis has been es-tablished, future research should strive to address the defi-ciencies in current cytotoxicity testing and exploit the find-ings to engineer improved nanoparticles ultimately for clini-cal use.

Acknowledgements

The authors would like to thank Joseph Chang and Ying Hufor their editorial assistance. This work was supported by theCenter for Biological and Environmental Nanotechnology (NSFEEC-0118007 and EEC-0647452) and the Howard HughesMedical Institute.

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Received: July 25, 2007Revised: November 14, 2007Published online on December 28, 2007



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