Cytotoxicity Due To Nanomaterials

reviews

R. Drezek et al.

Cytotoxicity DOI: 10.1002/smll.200700595

Cytotoxicity of Nanoparticles Nastassja 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. Semiconductor Nanoparticles............ 39 6. Summary and Outlook .................................. 45

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

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Cytotoxicity of Nanoparticles

Human exposure to nanoparticles is inevitable as nanoparticles become more widely used and, as a result, nanotoxicology research is now gaining attention. However, while the number of nanoparticle types and applications continues to increase, studies to characterize their effects after exposure and to address their potential toxicity are few in comparison. In the medical field in particular, nanoparticles are being utilized in diagnostic and therapeutic tools to better understand, detect, and treat human diseases. Exposure to nanoparticles for medical purposes involves intentional contact or administration; therefore, understanding the properties of nanoparticles and their effect on the body is crucial before clinical use can occur. This Review presents a summary of the in vitro cytotoxicity data currently available on three classes of nanoparticles. With each of these nanoparticles, different data has been published about their cytotoxicity due to varying experimental conditions as well as differing nanoparticle physiochemical properties. For nanoparticles to move into the clinical arena, it is important that nanotoxicology research uncovers and understands how these multiple factors influence the toxicity of nanoparticles so that their undesirable properties can be avoided.

1. Introduction The presence of nanoparticles in commercially available products is becoming more common. Nanoparticles, according to the ASTM standard definition, are particles with lengths that range from 1 to 100 nanometers in two or three dimensions.[1] It is projected that production of nanoparticles will increase from the estimated 2 300 tons produced today to 58 000 tons by 2020.[2,3] With this increase in manufacturing of nanoparticle-containing merchandise along with the constant discovery of new applications of nanoparticles, it is surprising that knowledge on the health effects of nanoparticle exposure is still limited. However, the number of efforts aimed at determining the health risks associated with nanoparticle exposure continues to grow. This is essential as public perception of nanotechnology can be jeopardized by events such as the “nano scare” in 2006 in Germany, involving the aerosol glass protective Magic-Nano,[4] or the sunscreen controversy after the United States Environmental Protection Agency released findings that nanometersized titanium dioxide particles found in sunscreens could cause brain damage in mice.[5] These two incidents exemplify why the need to verify the safety of nanoparticles is increasingly more pertinent. In contrast to nanoparticle exposure through use of consumer products, emerging biomedical applications of nanoparticles as drug-delivery agents, biosensors, or imaging contrast agents involve deliberate, direct ingestion or injection of nanoparticles into the body. For biomedical purposes, especially in vivo applications, toxicity is a critical factor to consider when evaluating their potential. Nanoparticles for imaging and drug delivery are often purposely coated with bioconjugates such as DNA, proteins, and monoclonal antismall 2008, 4, No. 1, 26 – 49

bodies to target specific cells. As these nanoparticles are intentionally engineered to interact with cells, it is important to ensure that these enhancements are not causing any adverse effects. More significant is whether either naked or coated nanoparticles will undergo biodegradation in the cellular environment and what cellular responses degraded nanoparticles induce. For example, biodegraded nanoparticles may accumulate within cells and lead to intracellular changes such as disruption of organelle integrity or gene alterations. While in vitro nanoparticle applications afford less stringent toxicological characterization, in vivo use of nanoparticles requires thorough understanding of the kinetics and toxicology of the particles. In vitro cytotoxicity studies of nanoparticles using different cell lines, incubation times, and colorimetric assays are increasingly being published. However, these studies include a wide range of nanoparticle concentrations and exposure times, making it difficult to determine whether the cytotoxicity observed is physiologically relevant. In addition, different groups choose to use various

[*] N. Lewinski, Prof. R. Drezek Department of Bioengineering MS-142 Rice University PO Box 1892, Houston, TX 77251-1892 (USA) Fax: (+ 1) 713-348-5877 E-mail: [email protected] Prof. V. Colvin Department of Chemistry MS-60 Rice University PO Box 1892, Houston, TX 77251-1892 (USA)

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cell lines as well as culturing conditions, which makes direct comparisons between the available studies difficult. Despite these issues, general trends in the pool of existing data can be extracted. This Review examines the cytotoxicity of several classes of nanoparticles currently being developed for biomedical applications. The nanoparticles included are: 1) Carbon-based nanoparticles, such as ful-

lerenes and single- and multi-walled carbon nanotubes; 2) metal-based nanoparticles, such as gold colloids, nanoshells, nanorods, and superparamagnetic iron oxide nanoparticles, and 3) semiconductor-based nanoparticles such as quantum dots.

2. Cytotoxicity Assays

Nastassja Lewinski graduated from Rice University with a BS in Chemical Engineering in 2006. She is currently a Ph.D. candidate in the Department of Bioengineering at Rice University under the guidance of Prof. Rebekah Drezek. Her research interests include assessing the use of quantum dots as optical contrast agents for enhancing in vivo disease screening and detection, and addressing public policy concerns of nanoparticle safety.

Prof. Vicki Colvin was recruited by Rice University in 1996 to expand its nanotechnology program. Today, she serves as Professor of Chemistry and Chemical Engineering at Rice University as well as Director of the Center for Biological and Environmental Nanotechnology (CBEN). CBEN was one of the nation’s first Nanoscience and Engineering Centers funded by the National Science Foundation. Prof. Colvin has received numerous accolades for her teaching abilities, including Phi 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 20 Scientists to Watch” and received an Alfred P. Sloan Fellowship. Dr. Colvin received her Bachelor’s degree in chemistry and physics from Stanford University, and obtained her Ph.D. in chemistry from the University of California, Berkeley. She is a frequent contributor to Advanced Materials, Physical Review Letters, and other peer-reviewed journals, and holds patents of four inventions. Prof. Rebekah Drezek is currently an Associate Professor in the Departments of Bioengineering and Electrical and Computer Engineering at Rice University. She has been on the faculty at Rice since 2002 where she conducts basic, applied, and translational research at the intersection of medicine, engineering, and nanotechnology towards the development of minimally invasive photonicsbased imaging approaches for detection, diagnosis, and monitoring of cancer. Her research 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 Scientist Award, the MIT TR100’s Top 100 Young Innovators Award, the American Association for Medical Instrumentation Career Achievement Award, and the DOD Era of Hope Scholar Award.

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The first step towards understanding how an agent will react in the body often involves cell-culture studies. Compared to animal studies, cellular testing is less ethically ambiguous, is easier to control and reproduce, and is less expensive. In the case of cytotoxicity, it is important to recognize that cell cultures are sensitive to changes in their environment such as fluctuations in temperature, pH, and nutrient and waste concentrations, in addition to the concentration of the potentially toxic agent being tested. Therefore, controlling the experimental conditions is crucial to ensure that the measured cell death corresponds to the toxicity of the added nanoparticles versus the unstable culturing conditions. In addition, as nanoparticles can adsorb dyes and be redox active, it is important that the cytotoxicity assay is appropriate. Conducting multiple tests is advantageous to ensure valid conclusions are drawn. One simple cytotoxicity test involves visual inspection of the cells with bright-field microscopy for changes in cellular or nuclear morphology. Fiorito et al. used this technique when evaluating the cytotoxicity of single-walled carbon nanotubes (SWNTs).[6] However, the majority of cytotoxicity assays used throughout published nanoparticle studies measure cell death via colorimetric methods. These colorimetric methods can be further categorized into tests that measure plasma membrane integrity and mitochondrial activity. Exposure to certain cytotoxic agents can compromise the cell membrane, which allows cellular contents to leak out. Viability tests based on this include the neutral red and Trypan blue assays. Neutral red, or toluylene red, is a weak cationic dye that can cross the plasma membrane by diffusion. This dye tends to accumulate in lysosomes within the cell. If the cell membrane is altered, the uptake of neutral red is decreased and can leak out, allowing for discernment between live and dead cells. Cytotoxicity can be quantified by taking spectrophotonic measurements of the neutral red uptake under varying exposure conditions.[7] Two studies by Flahaut et al. and Monterio-Riviere et al. exploring the cytotoxicity of carbon nanotubes utilized the neutral red assay.[8,9] Trypan blue, a diazo dye, is only permeable to cells with compromised membranes; therefore, dead cells are stained blue while live cells remain colorless. The amount of cell death can be determined via light microscopy.[10] This assay was used by Bottini et al. and Goodman et al. to determine the cytotoxicity of SWNTs and gold nanoparticles.[11,12] The LIVE/DEAD viability test, which includes two chemicals calcein acetoxymethyl (calcein AM) and ethidium homodimer, is another assay measuring the number of damaged cells. This method has been used to test fullerenes and

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an electron acceptor for enzymes such as NADP and gold nanoshells.[13,14,15] Calcein AM, an electrically neutral, FADH during oxygen consumption.[36] esterfied molecule, can easily enter cells by diffusion. Once within cells, it is converted to calcein, a green fluorescent As not all disruptive effects result in membrane or metamolecule, by intracellular esterases. In contrast, damaged or bolic function defects, more extensive cytotoxicity studies dead cells are stained by ethidium homodimer, a memhave attempted to determine the sub-lethal effects of nanobrane-impermeable molecule, and fluoresce red when the particles. Oxidative stress can be detected using a glutadye binds to nucleic acids. When excited at 495 nm, calcein thione assay. Glutathione (GSH) is a major antioxidant AM and ethidium homodimer emit distinct fluorescence sigcompound that is oxidized to glutathione disulfide (GSSG) natures at 515 nm and 635 nm, respectively.[16] in the presence of reactive oxygen species. In order to sustain its protective role against oxidative stress, a high GSH/ A third cytotoxicity assay used in several carbon-nanoGSSG ratio is required, which is maintained by the enzyme particle studies is lactate dehydrogenase (LDH) release glutathione reductase. The glutathione assay detects levels monitoring.[13,17,18] In this assay, LDH released from damof glutathione using EllmanFs reagent, 5,5’-dithio-bis-2-nitroaged cells oxidizes lactate to pyruvate, which promotes conbenzoic acid (DTNB), which reacts with the sulfhydryl version of tetrazolium salt INT to formazan, a water-soluble group of GSH to produce a yellow-colored product, 5-thiomolecule with absorbance at 490 nm. The amount of LDH 2-nitrobenzoic acid (TNB). Glutathione reductase also recyreleased is proportional to the number of cells damaged or cles GSH from the GSH-TNB complex producing more lysed.[19] TNB. Since the rate of TNB production is directly proporIn addition to distinguishing between live and dead cells tional to the concentration of GSH in the sample, the abby detecting compromised plasma membranes, other colorisorbance of TNB can be measured at 405 or 412 nm to demetric cytotoxicity assays attempt to determine the mechatermine the level of GSH.[37] nism behind the induced cell death. Mitochondrial activity can be tested using tetrazolium salts as mitochondrial dehyLipid peroxidation of the plasma membrane can be dedrogenase enzymes cleave the tetrazolium ring. Only active tected using GSH or a variety of other methods including mitochondria contain these enzymes; therefore, the reaction the widely used thiobarbituric acid (TBA) assay. In the only occurs in living cells.[20] The most widely used test is TBA assay, malondialdehyde (MDA), a toxic byproduct of lipid peroxidation, when heated at acidic pH reacts with 2the MTT viability assay.[8,9,13,21–26] MTT, 3-(4,5-dimethylthiathiobarbituric acid to form a fluorescent pink chromagen, zol-2-yl)-2,5-diphenyl tetrazolium bromide, is pale yellow in which can be measured colorimetrically when excited at solution but produces a dark-blue formazan product within 532 nm. Other methods of lipid peroxidation are listed in an live cells. A variation of this is the Cell Titer 96 Aqueous extensive review by Halliwell et al.[38] One Solution Cell Proliferation Assay distributed by Promega, which has been employed by several groups.[27–29] Here MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and phenazine ethosulfate, is used instead. The number of living cells can be determined similarly by quantifying the production of formazan by measuring the absorbance at 492 nm.[30] Another tetrazolium-based assay used to test the cytotoxicity is the WST assay.[31] WST-1 or WST-8 converts to a yellow–orangecolored formazan product, the concentration of which can be quantified at 450 nm.[32] Resazurin or Alamar blue has also been used to ascertain cytotoxicity.[33–35] This test is also a colorimetric assay where the nonfluorescent alamar blue dye is reduced to a pink fluorescent dye by cell metabolic Figure 1. Structures and human dermal fibroblast Live/Dead cell viability assay results for C60 and derivaactivity, mainly by acting as tives. Reprinted with permission from Ref. [13]. Copyright American Chemical Society, 2004. small 2008, 4, No. 1, 26 – 49

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Inflammation is also a possible adverse effect of nanoparticle exposure. Commonly tested pro-inflammatory cytokines or protein signals of inflammatory response include IL-1b, IL-6, and TNF-a plus the chemokine IL-8.[39,40] These cytokines are detected using enzyme-linked immunosorbant assay (ELISA) and can be quantified by measuring the absorbance from either alkaline phosphatase or strepavidinhorseradish peroxidase labeled antibodies at 405 or 620 nm, respectively.[41] More extensive cytotoxicity studies have attempted to determine the genotoxic potential of nanoparticles by examining the extent of DNA damage using several methods. One test that has been used extensively in studying the effect of carbon-nanoparticle exposure is flow cytometry.[42– 45] This technique utilizes a laser beam that differentiates cells based on their size and density. Using DNA intercalating dyes, the cellular DNA content can also be used to determine the proportion of cells undergoing apoptosis. One such dye is propidium iodide, a membrane-impermeable red dye, which undergoes a fluorescence change proportional to the number of damaged cells. This occurs as binding of the dye to nucleic acids increases with increased membrane permeability.[46] In addition to flow cytometry, the comet assay has also been used to detect DNA damage in individual cells using gel electrophoresis. Cells with damaged DNA appear as “comets” with intact DNA residing in the head portion and broken DNA pieces migrating away, forming the tail. A DNA-specific dye such as propidium iodide is used to read the gel, and the amount of DNA found in the tail is proportional to the amount of DNA damage.[47] More recently, to determine which specific genes are up- or down-

regulated due to nanoparticle exposure, some groups have conducted preliminary DNA microarray studies.[48–51] For nanoparticles, the major biological effects involve interactions with cellular components such as the plasma membrane, organelles, or macromolecules. As different nanoparticles can trigger distinctive biological responses, it is important that cytotoxicity studies are conducted for each nanoparticle type. The following sections review the existing cytotoxicity literature on carbon, metal, and semiconductor based nanoparticles.

3. Carbon Nanoparticles Carbon nanoparticles are materials composed mainly of carbon with one or more dimensions at 100 nm or less. These include, but are not limited to, carbon dots,[52] fullerenes, nanodiamonds,[53] nanofoam,[54] nanohorns,[55] and nano-onions.[46] However, as fullerenes are the most established 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 the most widely used and well developed of the carbon nanoparticles. Their unique physiochemical properties (light weight, high tensile strength, thermal/chemical stability and conductivity) have generated several applications including use in biomedical materials and devices such as tissue scaffolds, drug-delivery agents, and fluorescent-contrast agents.[57–59] In terms of cytotoxicity, a major factor influencing potential toxicity is the carbon nanoparticlesF complexity and variety in size, shape, charge, methods of production,

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

Surface coating

Human dermal fibroCOOH, blasts, HDF; human OH, Na liver carcinoma, HepG2 Guinea pig alveolar pristine macrophages

Human dermal fibroblasts, HDF; human liver carcinoma, HepG2; neuronal human astrocytes, NHA Monocyte-derived macrophages Human monocyte macrophages

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Exposure conditions

NP concentration (average size)

70% confluency

0.24–2400 ppb (d = 100 nm)

2 < 105 cells mL 1 in 24-well plates

8.36 < 104 NP mg 1, 1.41– 226 mg cm 2 0.24–2400 ppb (d = 100 nm)

COOH, OH, Na

70% confluency

pristine

3 < 105 cells mL

pristine

2 < 106 cells well 1 for 24-well plate, 0.5–1 < 106 for 96-well plate 70% confluency, 96-well plate

Human epidermal keratinocytes, HEK

N-BocBaa

Human umbilical vein endothelial cells, HUVEC

C60(OH)24 90% confluency on 6-well plates

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1

30 and 60 mg mL 1 0.16–10 mg mL 1 (d = 60–270 nm)

Test

Exposure duration [h]

Toxicity

MTT, Live/ Dead, LDH MTT

48

LD50 = 20 ppb for bare C60 on HDF; no cytotoxicity observed with C60(OH)24 No significant toxicity up to 226 mg cm 2

Sayes [13]

2004

Jia [21]

2005

MTT, Live/ Dead, LDH

48

Nano-C60 is cytotoxic at 20 ppb level; after 30 h cells begin to have leaky membranes and lipid oxidation

Sayes [49]

2005

Fiorito [6] Porter [62]

2006

Rouse [63]

2006

Nuclear Morph, PI Neutral red

3

Author

1, 24, Did not induce damage or 48 death of macrophages 48 No significant toxicity

24, 48 Cytotoxicity at 0.04 and 0.4 mg mL 1; IL-8, IL-6, and IL-1b levels increased 1–100 mg mL 1 LDH, WST, 24 100 mg mL inhibit cell growth; 10 mg mL 1 inhibit (d = 7.1  2.4 nm) microarray cell attachment 0.00004– 0.4 mg mL

MTT

1

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Year

2006

Yamawaki 2006 [50]

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3.1. C60

chemical compositions, surface chemistry/functionalization, and aggregation tendency. A few reviews have been published that look at the biocompatibility of carbon nanoparticles.[60,61] This section looks at the studies that have been conducted to elucidate the safety of these three major types of carbon nanoparticle. Summaries of the experimental setup and results on C60, SWNTs, and MWNTs are provided in Tables 1–3, respectively.

Several groups have studied the effect of C60 exposure under various experimental conditions with different cell lines and have yielded different results. The most significant factor influencing cytotoxicity in this class of carbon nanoparticles seems to be cell type. The groups that reported noncytotoxic effects studied C60 exposure in macrophage

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

Surface coating

Exposure conditions

3T3 cells

FITC

N/A

Immortalized human epidermal keratinocytes, HaCaT Mouse peritoneal macrophage-like cells, J774.1A Human embryonic kidney, HEK293

Human promyelcytic leukemia cells, HL60; Jurkat T cells Guinea pig alveolar macrophages Human keratinocytes, HaCaT; HeLa cells; Lung carcinoma (A549, H1299) cells Monocyte-derived macrophages Human dermal fibroblasts, HDF

Human epidermal keratinocytes, HEK

Lung epithelial-like cells, A549

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Test

Exposure duration [h]

1–10 mm (d = 1 nm, flow cytome1 l = 300–1000 nm) try (annexin, PI) 80% confluency 0.06, 0.12, or none Alamar blue, 2, 4, 6, (w/30% Fe on 96 well GSH 8, 18 0.24 mg mL 1 plates or 75-cm2 cat) flasks microscopy 4, 8, 12, 0–7.3 mg mL 1 pristine 107 cells (d = 1 nm, l = 1 mm) 18, 24 pristine

24 well plates

COOH, 3 < 106 biotin, fluo- cells mL 1 rescein, streptavidin pristine 2 < 105 cells mL 1 in 24-well plates pristine 5000 cells well 1, 96-well plate

MTT: 0.7812– 200 mg mL 1; others: 25 mg mL (CAS 7782-42-5) 0.05 mg mL 1 (d = 1–5 nm, l = 0.1–1 mm)

3 < 105 cells mL 1

phenyl(SO3H, SO3Na, or (COOH)2), pluronic F108 pristine

70% confluency 0.2–2000 mg mL (d = 1 nm, l = 400 nm)

pristine

pristine

1

1.41–226 mg cm 2 (d = 1.4 nm, l = 1 mm) 0.1, 0.5, 1, 5, 10, 20 mg mL 1

pristine

Rat alveolar macropristine phage cells, NR8383; human alveolar epithelial cells, A549

Mesothelioma cells, MSTO-211H

NP Concentration (average size)

30 and 60 mg mL

5x103 cells well 1

0.8–100 mg mL (d = 2 nm, l = 500 nm)

25 000 cells well 1, 96-well plate 105 cells well 1 in 96-well plates; 2.5 < 104 cells well 1 in 96-well plates (human) 3000 cells well 1 in 24well plates

50 mg mL 1 (d = 1.4 nm) 5–100 mg mL (d = 1–2 nm, l = 100 nm)

1

1

1

MTT

3

MTT, Live/ Dead

72

MTT, WST

1

Author

Year

Pantarot- 2003 to [43]

viability decreased after Shvedova 2003 [33] 4h, 0.24 mg mL 1 ~ 65% viability Cells ingest NT without toxic effects

Cytotoxic effects seen at 0.38 mg cm 2 ; necrosis at 3.06 mg cm 2 Cytotoxic effects seen at 0.5 mg mL 1, NFkB pathway activated by SWNT

nuclear mor- 1, 24, 48 Did not induce damage phology, PI and death of macrophages MTT, Live/ 24, 48 Cytotoxicity decreased Dead w/ decreased C/phenylSO3H ratio; LD50 could not be obtained

MTT, WST, LDH, MMP

7.5, 15, 30 mg mL

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

MTT, western 24–120 Cytotoxicity dose- and blot, flow time-dependent; 43.5% in G1 cell cycle arrest cytometry, microarray after 1 day PI, flow 1 No significant toxicity cytometry for nonstretavidin-modified SWNTs

MTT

1

Toxicity

Cherukuri 2004 [68] Cui [44]

2004

Kam [45]

2004

Jia [21]

2005

Manna [65]

2005

Fiorito [6]

2006

Sayes [66]

2006

24–120 Strongest adverse effect Tian 2006 w/ SWNT; 100 mg mL 1 [23] gave 79%, 50% and 31% viability after 1, 3 & 5 days 24-96 MTT gave different reWorle- 2006 sults from WST, LDH Knirsch and MMP [69] Pulskamp 2007 24–96 Cytotoxicity dose [64] dependent; 100 mg mL 1, 60–80% reduction

MTT

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Cytotoxicity dose dependent; agglomerated worse than well-dispersed

Wick [67]

2007

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toxicity after 24-hour incubation with concentrations of becell lines. Fiorito et al. found non-toxic responses for pristween 1 and 100 mg mL 1.[50] Morphological changes, intine C60 in their studies with murine macrophages.[6] They reported that C60 had low cellular uptake, did not stimulate creases in LDH release, and growth inhibition were reported. In addition, the fullerenes were found to aggregate and nitric oxide release, and did not induce apoptosis in comparinternalize in autophagosomes, suggesting autophagic cell ison to graphite and SWNTs. From this, C60 was considered death. to be fairly noncytotoxic. C60 was also deemed the least toxic of the carbon nanoparticles by Jia et al.[21] The group studied alveolar macrophage response to incubation with 3.2. Single-Walled Carbon Nanotubes C60 and found that, after six hours of exposure to as much Compared to the mixed reports on C60, SWNTs have as 226 mg cm 2 of C60, no significant toxicity resulted. Porter et al. studied the effect of C60 in human monocyte typically been labeled as having cytotoxic effects at high concentrations. In addition, in a few comparison studies macrophages and also found no significant cytotoxicity.[62] SWNTs were reported as more toxic than the other two However, looking at the subcellular level, C60 was found to major types of carbon nanoparticle.[21,23,64] In a study exposaggregate into hexagonal units along the plasma membrane. In addition, they were found to accumulate intracellularly in ing human embryo kidney cells to SWNTs for one to five lysosomes, cytoplasm, along the nuclear membrane and days, Cui et al. found dose- and time-dependent decreases inside the nucleus. In contrast, the groups that studied C60 in cell-adhesion ability, cell proliferation, and increases in induction of apoptosis.[44] In addition, flow cytometry analyexposure in other cell lines found a dose-dependent cytotoxicity relationship. Incubating four different C60 derivatives, sis revealed altered cell-cycle regulation such as G1 phase arrest due to SWNT exposure. Testing four different cell as illustrated in Figure 1, for up to 48 hours with human lines (human keratinocytes, HeLa cells, and two lung carcidermal fibroblast and liver carcinoma cells, Sayes et al. noma lines: A549, H1299), Manna et al. found oxidative found, for all four types, the lowest concentration (0.24 ppb) stress and inhibition of proliferation increased in a dosewas relatively nontoxic while the highest concentration and time-dependent manner.[65] They also studied the NFkB (2400 ppb) was more cytotoxic.[13] pathway, which they found was activated by SWNT expoBetween the four types, the addition of surface chemissure either via MAPK or IKK kinase activation. tries for water solubility decreased the in vitro cytotoxicity, While pristine SWNTs were found to exhibit some cytowith pristine C60 being more cytotoxic while the more hytoxic effects, a few groups found these effects were mitigatdroxylated C60, C60(OH)24, had no apparent cytotoxicity ed by functionalizing the SWNT surface. Kam et al. provide with an LD50 value of > 5 mg mL 1. It has been suggested flow cytometry analysis that revealed no significant toxicity that this difference is due to the generation of reactive due to carboxyl-, biotin-, and fluorescein-coated SWNTs in oxygen species associated with C60. Additionally, particle agHL60 and Jurkat T cells after one hour.[45] Sayes et al. gregation was also determined to cause some of the cytotoxic effects. A more extensive study by Sayes et al. reported looked at the cytotoxicity of water-soluble, SWNT-treated that cell apoptosis due to exposure to C60 was caused by human dermal fibroblasts over a range of concentrations (3–30 mg mL 1) for up to 48 hours.[66] As illustrated in cell-membrane lipid peroxidation from oxygen radicals.[49] Figure 2, cell death was highest in the cultures exposed to This was confirmed when the addition of an antioxidant, Lascorbic acid, prevented membrane damage resulting in cell viability comparable to the control. Rouse et al. studied the effect of amino acidderivatized fullerenes in human epidermal keratinocytes (HEK).[63] The levels of pro-inflammatory cytokine cytotoxicity indicators, IL-8, IL-6, TNF-a, and IL-1b, were measured. After 24 and 48 hours of incubation, a dosedependent decrease in cell viability was found as well as higher phagocytosis of particles observed in cells exposed to concentrations above 0.004 mg mL 1. Yamawaki et al. tested the effect of hydroxyl fullerene, C60(OH)24, on human umbilical vein en- Figure 2. Structures and human dermal fibroblast cytotoxicity data for SWNTs and derivatives. Reprinted dothelial cells and found cyto- with permission from Ref. [66]. Copyright Elsevier, 2007.

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pristine SWNTs while, of the three functionalized SWNTs, the SWNT containing the most functional groups yielded the best cell viability. Several hypotheses have been postulated to explain the cytotoxicity observed with SWNTs. One is related to the mode of production, as the synthesis of SWNTs requires the use of metal catalysts, which can be toxic themselves. Shvedova et al. reported dose- and time-dependent cytotoxicity in human epidermal keratinocytes exposed to SWNTs.[33] At higher concentrations and longer incubation times, increased oxidative stress, reduced glutathione levels, and nuclear and mitochondrial changes were found. They also noted that the addition of a metal chelator reduced cytotoxicity, suggesting that residual iron catalyst in solution may play a role in the cytotoxicity observed. In addition, Jia et al. found more than a 20% growth inhibition at the lowest SWNT dose of 1.41 mg cm 2.[21] This cytotoxicity was also speculated to be due to the 90% purity of the SWNT solution as the presence of metallic catalysts could confound the results. Particle aggregation has also been suggested to be a factor in nanoparticle cytotoxicity. Wick et al. aimed to determine how agglomeration influenced SWNT cytotoxicity and tested four different SWNT solutions: the raw material involved in SWNT production, the SWNT-agglomerates resulting from the synthesis, and the SWNT bundles and the SWNT pellets (devoid of nanotubes) produced from centrifuging the SWNT agglomerate.[67] Aggregation occurred in all SWNT fractions except the well-dispersed SWNT bundles. Correspondingly, the SWNT bundles did not induce adverse cellular effects, and as this was the only solution where agglomerates were not formed, this corroborates the hypothesis that SWNT agglomeration leads to cytotoxic effects. However, an earlier study by Tian et al., testing an unrefined SWNT solution and a SWNT solution with the metal catalysts removed, found lower cytotoxicity with the unrefined SWNTs.[23] The group proposed that the lower cytotoxicity of the unrefined SWNTs was a result of their aggregation into larger, and therefore less toxic, particles. This contradicts the reasoning of Wick et al., who hypothesized that the agglomerated SWNTs were cytotoxic due to the stiffness and larger size, making the nanotubes emulate the effects of asbestos fibers. While the conflicting results may be due to the use of two different cell lines and asbestos-induced lung-cancer cells versus keratinocytes, the effect of SWNT aggregation is still questionable.

3.3. Multi-Walled Carbon Nanotubes Studies on MWNTs have yielded results similar to those of SWNTs. Monteiro-Riviere et al. reported that cells incubated with higher concentrations of MWNTs for longer exposure times contained more MWNTs.[9] The percentage of cells with MWNTs inside increased from 59% at 24 hours to 84% after 48 hours. In addition, a dose- and time-dependent decrease in cell viability was observed, coupled with an increase in release of cytokine IL-8 at the higher MWNT concentrations. While the complimentary study by Shvedova small 2008, 4, No. 1, 26 – 49

et al. on SWNTs proposed that the cytotoxicity may be due to trace amounts of catalyst in the solution, the lack of catalyst particles in these MWNT solutions suggests the MWNTs alone were potentially hazardous. Instead, Monteiro-Riviere et al. hypothesize that the cytotoxicity is due to MWNT attachment to the cell membrane or MWNT internalization, as MWNTs were seen in the cytoplasm and near the nucleus. Sato et al. also found aggregates of MWNT in cytoplasm in their studies.[70] Bottini et al. also saw dose- and time-dependent cytotoxicity in T lymphocyte and Jurkat leukemia cells.[11] In addition, comparing the effects of different surface coatings, hydrophobic MWNTs were less toxic than ones coated with hydroxyl or carboxyl groups. The same conclusions were made by Magrez et al. after studying MWNT in lung carcinoma cells.[22] A dose-dependent decrease in cell viability was also evident after exposing alveolar macrohages to > 95% purified MWNTs, conducted by Jia et al.[21] However, as they tested different diameters of MWNT (10–20 nm) this dose dependence could be due to particle mass, size, or both. Interestingly, while these groups found a dose-dependent trend in cytotoxicity, Flahaut et al. found a decrease in viability in human umbilical vein endothelial cells (HUVEC) with dilution of their MWNT solution.[8] Although they concluded that the MWNTs were nontoxic as metabolic activity was maintained above 75%, HUVEC viability seemed to decrease with exposure to decreasing concentrations of MWNTs with large surface areas. The group suspects this is a result of the aggregation of MWNTs or their enhanced interactions with the cells due to their higher dispersion at lower concentrations. Few groups have studied the inflammatory response to MWNTs. One group, Ding et al., looked at the genetic effects of MWNTs and found that high concentrations induced immune and inflammatory gene overexpression.[48] Witzmann et al. considered the protein-expression changes after human epidermal keratinocyte exposure to MWNTs and noted upregulation of proteins related to irritation and cell apoptosis.[71] Muller et al. incubated peritoneal macrophages for up to 24 hours containing purified MWNTs and ground MWNTs at concentrations of 20–100 mg mL 1.[17] They determined that ground MWNTs had a capacity for inducing dose-dependent cytotoxicity and up-regulating TNF-a expression that is similar to that of asbestos and carbon black. However, the unground MWNT sample exhibited lower effects than the ground sample, which they attributed to the increased agglomeration found in the unground sample preventing cellular uptake. Murr et al. also found that the cytotoxicity of MWNTs was similar to asbestos.[72] Chlopek et al. investigated the viability and stimulation of fibroblasts and osteoblasts exposed to purified MWNTs.[27] The group deemed MWNTs to be biocompatible with the tested cell types, as they found unchanged levels of osteocalcin, cytokine IL-6, and oxygen-free radicals.

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Table 3. Cytotoxicity of MWNTs. Cell Line

Surface coating

Exposure conditions

NP concentration (aver- Test age size)

Human epidermal keratinocytes, HEK

pristine

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

0.1, 0.2, 0.4 mg mL

Human skin fibroblasts, HSF42; human embryonic lung fibroblasts (IMR-90) Guinea pig alveolar macrophages

pristine

70% confluency, 96-well plates

0.06–0.6 mg L

pristine

2 < 105 cells mL 24-well plates

Sprague–Dawley rat peritoneal macrophages Murine alveolar macrophages (RAW267.9) Human acute monocytic leukemia cells (THP-1) Wister male rats T lymphocytes, Jurkat T leukemia cells

pristine

direct lung injection

pristine

5 < 105 cells well in 96-well plates

1

pristine

5 < 105 cells well in 96-well plates

1

Hydroxyl, carboxyl

4.4x104 cells mL

1

Polysulfone (PS)

2 cm3 cells/ 12 well plate

pristine

6000 cells cm 2 in 96-well plates

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

N/A

Human osteoblastic line hFOB 1.19; Human fibroblastic line HS-5 Human umbilical vein endothelial cells, HUVEC

Human lung-tumor cell lines, H596, H446, and Calu-1

Human neonatal HEKs

80% confluency, 6-well plates

1

in 1.41–226 mg cm (d = 10–20 nm, l = 0.5–40 mm) 20–100 mg mL 1 0.5–2 mg rat 1

0.4 mg mL

1

4.1. Gold Nanoparticles Gold colloids, now often referred to as gold nanoparticles, have been used in medical applications during clinical testing of heavy metals to treat rheumatoid arthritis as early as the 1920s.[73] This precedence suggests that current applications of gold nanoparticles should not be limited by their biocompatibility. While gold nanoparticles refer to particles spherical in shape, other geometries, such as gold nanorods, tripods, tetrapods,[74] and nanocages,[75] have been synthesized, with gold nanorods discussed in further detail in a

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2

Neutral red

1, 4, 8, 12, 24, 48

Hoechst 33342, YO-PRO 1, PI, BrdU, microarray MTT

24, 48

LDH

3, 15 days

3

Toxicity

protein array

24, 48

Author

Year

~ 73% viability at Monteiro- 2005 0.4 mg mL 1; IL-8 Riviere [9] increases with MWNT conc. Cytotoxicity doseDing 2005 dependent for puri[48] fied MWNT

Necrosis seen at 3.06 mg cm 2

LDH doubled from 20 to 100 mg mL 1 ground nanotubes 0.005–10 mg mL 1, MTT, ELISA 48 Cytotoxicity begins (d = 5–30 nm, at 2.5 mg mL 1; similar to asbestos l = 0.03–3 mm) 16 TNF-a production 5–500 ng mL 1 0.1 mg HU TNF-a dose dependent, Flexia histolo(d = 20–40 nm, aggregates in sevgy, microscopy l = 0.5–5 mm) eral cell types Trypan blue 24–24 Cell death > 80% 40–400 mg mL 1 (d = 20–40 nm, in oxidized, < 50% l = 1–5 mm) in pristine at 400 mg mL 1 N/A (d = 10–15 nm) Cell titer 96 24, 48, 7 Small viability dedays crease in PS + MWNTs vs. pure PS Max: A: 0.5 mg mL 1 MTT, Neutral 24 None were cytotox(d = 1.1–3.2 nm), red ic but error bars of B: 0.64 mg mL 1 samples A & B (d = 1.1–4.3 nm) below threshold C: 0.9 mg mL 1 (d = 0.7–6.3 nm) 0.002–0.2 mg mL 1 MTT 24–96 Cell viability decreased 33% at (d  20 nm, aspect 0.2 mg mL 1; funcratio = 80–90 nm) tionalized have lower survival

4. Metal Nanoparticles

34

1

1

Exposure duration [h]

Irritation and cell apoptosis proteins upregulated

Jia [21]

2005

Muller [17]

2005

Murr [72]

2005

Sato [70]

2005

Bottini [11]

2006

Chlopek 2006 [27]

Flahaut 2006 [8]

Magrez 2006 [22]

Witzmann 2006 [71]

later section. Gold nanoparticles exhibit an intense color in the visible spectroscopic region and since gold can easily bind functionalizing or targeting ligands, it has great promise as a contrast agent for bioimaging. Due to their small size, gold nanoparticles have been found to easily enter cells. Early studies with cytotoxicity data were focused on utilizing this property for nuclear transfection and targeting. In their work to find nonviral gene-delivery devices, Thomas et al. found polyethylenimine (PEI)-modified gold nanoparticles could transfect monkey kidney (COS-7) cells six times better than PEI alone.[76] Cell viability was recorded after exposure to PEI–gold nanoparticle complexes, and 80% of the cells were still metabolical-

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Cytotoxicity of Nanoparticles

ly active. While PEI–gold nanoparticles with dodecyl–PEI complexes achieved even better transfection, cell viability decreased to 70%. This complex was mainly found inside the cell suggesting that internalization is a factor in cytotoxicity. However, as the gold nanoparticles were conjugated to PEI, whether or not the observed decrease in cell viability was due to the gold nanoparticles is unclear. Another group, Tkachenko et al., looked first at the nuclear targeting ability of gold nanoparticles alone, and then at gold nanoparticles with a full-length peptide containing both the receptor-mediated endocytosis and nuclear localization signal segments from an adenovirus in HepG2 cells.[77] The group found that plain gold nanoparticles were readily taken up into the cytoplasm; however, they did not enter the nucleus. Experiments conducted at 48C indicated that cell entry was energy dependent since a decrease in the number of particles inside the cells was observed. The gold nanoparticles were determined to be able to enter the cell by receptormediated endocytosis but unable to leave the endosomes, hindering nuclear targeting. However, the nanoparticle-peptide complex incorporating both transport signals was found to enter the nucleus. Despite this nuclear exposure, cell viability was greater than 95% after 12 hours of incubation. Tkachenko et al. conducted another study examining four different peptide–BSA–gold nanoparticle conjugates in three cell lines (HeLa, 3T3/NIH, and HepG2).[78] Here they reported differing effects of the nanoparticles between the three cell lines. The four peptide–BSA–gold nanoparticles were able to enter HeLa cells, escape the endosomes, and, except for the particle with the HIV Tat protein, enter the nucleus. In contrast, the four peptide–BSA–gold nanoparticles were found clustered together in endosomes within the 3T3/NIH cells. The HepG2 cells did not seem to uptake the peptide–BSA–gold nanoparticles except for the gold nanoparticle with the integrin-binding domain. The LDH cytotoxicity assay also confirmed these cell-line differences. After three hours of incubation, the peptide–BSA–gold nanoparticles conjugated with the adenovirus fiber protein caused 20% cell death in HeLa cells while only 5% in the 3T3/NIH cells. This suggests that the nuclear delivery of the peptide–BSA–gold nanoparticles influences cell viability due to particle interactions with cellular DNA. Goodman et al. also tested the effect of gold nanoparticle exposure in multiple cell lines.[12] Cationic (ammonium-functionalized) and anionic (carboxylate-functionalized) gold nanoparticles with concentrations of 0.38–3 mm were incubated with COS1 cells, red blood cells, and Escherichia coli cultures for 24 hours. While the cationic nanoparticles were clearly more cytotoxic than the anionic, a small variation was observed in their LC50 values between cell types, showing that different cell types experience similar toxicity. This contradicts the findings of Tkachenko et al. as they found a difference between cell lines; however, this could be due to the use of different surface coatings.[78] In addition, Goodman et al. [12] proposed that the nanoparticles interact with the cells passively rather than by energy-dependent processes, as suggested by Tkachenko et al. [78] since mammalian and bacterial cells exhibited similar nanoparticle uptake. However, reduced-temperature incubation studies were not reported. small 2008, 4, No. 1, 26 – 49

As the results from Tkachenko et al. and Goodman et al. show, the type of surface coating can play an important role in the cytotoxicity of gold nanoparticles. Connor et al. studied the effect of size and different surface modifications on uptake and acute toxicity in human leukemia (K562) cells.[24] The sizes ranged between 4 and 18 nm with surface modifiers including biotin, CTAB, cysteine, citrate, and glucose. After three days of exposure, the largest nanoparticle with citrate and biotin surface modifiers did not appear to be toxic at concentrations up to 250 mm. In contrast, a similar concentration of the gold-salt (AuCl4) solution was found to be over 90% toxic. Glucose and cysteine were found to be less effective in rendering the nanoparticles nontoxic. The gold nanoparticle concentration dropped within the first hour of exposure, suggesting rapid uptake of nanoparticles into cells. The consumed nanoparticles were found clustered in endocytic vesicles and maintained their size after being taken up by the cells. Cytotoxicity may not be the only adverse effect of nanoparticles; nanoparticles may also affect the immunological response of cells. Shukla et al. tested the effect of gold nanoparticles on the proliferation, nitric oxide, and reactive oxygen species production of RAW264.7 macrophage cells.[25] After 48 hours of up to 100 mm gold-nanoparticle treatment, RAW264.7 macrophage cells showed greater than 90% viability with no increase in pro-inflammatory cytokines TNF-a and IL-1b. Cell viability decreased to 85% after 72 hours, which was attributed to depletion of media nutrients since the media was not changed in those 72 hours. The group found that cells take up gold nanoparticles internalizing them in lysosomes, which move in a time-dependent manner toward the nucleus but do not enter the nucleus. They also corroborate the findings of Goodman et al. that gold nanoparticles were not present in cells kept at cold (48C) temperatures.[25] Fu et al. and Shenoy et al. incubated bare gold nanoparticles and gold nanoparticles functionalized with methoxy-PEG-thiol or coumarin-PEG-thiol with breast cancer (MDA-MB-231) cells for 24 hours.[28,29] They found that the functionalized nanoparticles were internalized, by what they suggest is nonspecific endocytosis, within the first hour and localize mainly in the cytoplasm and perinuclear region. This is in agreement with the findings of Shukla et al.[25] Other noncytotoxic effects of nanoparticles, such as the influence of nanoparticle exposure on the proliferation, morphological structure, spreading, migration, and protein synthesis of human dermal fibroblast cells, were examined by Pernodet et al.[79] They found that with increasing concentration of gold nanoparticles, cell area decreased along with cell number and density of actin fibers. Although no cytotoxicity tests were conducted, the decreased number of cells indicates some cytotoxic effects. The number of vacuoles present within the cells increased with time, and the cells were filled with vacuoles by the sixth day. The gold nanoparticles accumulated inside the cells, entering not by endocytosis but rather through diffusion facilitated by their small size (average size ~ 13 nm). The observed cellular changes were both dose- and time-dependent. The group also notes that the gold nanoparticles are not digested in

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Table 4. Cytotoxicity of gold nanoparticles. Cell line

Surface coating

COS-7 cells

3x105 cells/ well BSA, 4 targeting 85% peptides confluency NH3, COOH 80% confluency, 96well plate BSA, 4 targeting 75% conflupeptides ency

Human liver carcinoma, HepG2 COS-1, Red blood cells, E.coli HeLa, 3T3/NIH, HepG2

PEI2

Leukemia cell line, citrate, biotin, LK562 cysteine, glucose, CTAB Human breast coumarin-PEGcarcinoma xenothiol, mPEGgraft cells, thiol (neg. conMDA-MB-231 trol) RAW264.7 lysine, PLL, FITC macrophage cells

Human dermal fibroblasts

Exposure conditions

citrate

104 cells/ well 105 cells/ well in 96-well plates 105 cells/ well in 96-well plates N/A

NP concentration (average size)

Test

Exposure duration

N/A

MTT

6h + 42 h 12 h

10, 25, 50, and MTT 100 mm (d = 3-8 nm)

0-0.8 mg mL 1 (d = 13 + /-1 nm)

4.2. Gold Nanoshells Typically, gold nanoshells are composed of a silica dielectric core coated with an ultrathin metallic gold layer. This core/shell structure allows for the gold nanoshells to be made by either preferentially absorbing or scattering by varying the relative core and shell thicknesses. Because of the “tunability” of their optical properties, nanoshells are being developed for imaging contrast and photothermal therapeutic medical applications.[80,81] A few studies have been published with cytotoxicity results on gold nanoshells. The first to suggest that gold nanoshells are nontoxic was Hirsch et al. While the focus of the study was on the photothermal ablative ability of the nanoshells, they mention that exposure to nanoshells did not cause cell death.[14] In later studies by Loo et al., SKBR2 breast-cancer cells exposed for one hour to 8 mg mL 1 or 3 K 109 nanoshells mL 1 of anti-HER2 bioconjugated nanoshells exhibited no difference in viability compared to control cells.[15,82] A more recent study by James et al. studied the biodistribution of gold nanoshells in female albino mice.[83] A 100 mL nanoshell solution with a 2.4 K 1011 nanoshells mL 1 concentration was injected into the tail veins of 30 mice. Five mice were sacrificed at several time points up to 28 days, and the accumulation of nanoshells in the blood and major organs, such as the liver, kidneys, spleen, lungs, muscle, brain, and bone was measured. The

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Author

70-80% viability after transfection N/A LDH Viability slightly compromised (d = 20-25 nm) (< 5%) 0.38, 0.75, 1.5, or MTT, 1, 2.5, LD50 (Cos-1): anionic ~ 1 mM 3 mm Trypan 6, 24 h and cationic > 7.37 mm; similar blue for other cell types 150 pm LDH 3h Cell viability reduced by 20% (d = 22 nm) in HeLa cells, but only 5% in 3T3/NIH 0-250 mm Au atoms MTT 3 days No apparent toxicity at 250 mm, (d = 4, 12, 18 nm) glucose and cysteine modified not toxic up to 25 mm Cell 24 h Nanoparticles are internalized 50-200 mg mL 1 (d = 10 nm) Titer but essentially non-toxic up to 96 200 mg mL 1

the lysosomes, and even though the vacuoles accumulate near the nucleus, no nuclear penetration was seen. A summary of the experimental setup and results on gold nanoparticles is provided in Table 4.

36

Toxicity

24, 48, 100 mm - after 72 hr cell viability 72 h to decreased to 85%

micros- 2-6 copy days

Dose-dependent decrease in cell area & density; many vacuoles

Year

Thomas 2003 [76] Tkachenko 2003 [77] Goodman 2004 [12] Tkachenko 2004 [78] Connor [24]

2005

Fu/Shenoy 2005 [28/29]

Shukla [25]

2005

Pernodet [79]

2006

nanoshells were found to quickly clear the blood circulation and predominantly accumulated in the liver and spleen. Despite the lack of complete nanoshell clearance from the body after 28 days, the mice are reported to have shown no physiological complications from the residual presence of nanoshells. Cytotoxicity of a gold/copper nanoshell has also been studied by Su et al. Au3Cu nanoshell concentrations between 0.001 and 200 mg mL -1 were incubated with Vero cells for 6 or 24 hours.[84] Using the WST assay, the group found cell damage to be dose dependent with cell viability decreased to 15% at the highest concentration after 24 hours of incubation with the nanoshells. The in vivo effects were also tested in male BALBc mice and, after 30 days, a dose dependence in viability rates was also found with 100% viability in the low-dose mice but 67% viability in the high-dose mice. Urine was collected from the mice three hours after injection, and the amount of gold and copper found suggested the nanoshells were being excreted from the body. The loss of MRI signal after four hours corroborated this finding. A summary of the experimental setup and results on gold nanoshells is provided in Table 5.

4.3. Gold Nanorods The advantage of gold nanorods is that they have both a transverse and longitudinal plasmon. As the optical properties of materials depend both on the type and shape of the metal, the unique properties of these rod-shaped particles can be utilized in several potential applications. While very few groups have published data on the cytotoxicity of gold nanorods, the results are similar to those seen for gold nano-

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Table 5. Cytotoxicity of gold nanoshells. Cell line

Surface coating

Exposure conditions

NP concentration (average size)

Human breast carcinoma SK-BR-3 cells HER2-positive SKBr3 breast adenocarcinoma cells Female albino mice

PEG

N/A

4.4x109 nanoshells mL 1 (core = 55 nm, shell = 10nm) 3x109 nanoshells mL 1 (core = 120 nm, shell = 10nm)

Live/Dead 1 h

No differences in viability

Hirsch 2003 [14]

Live/Dead 1 h

No differences in viability

Loo 2004[15,82] 2005

observation 4 h - 28 days

Limit in mice muscle tissue about 70pg, accumulating in the RES organs, 1–10 ppm levels found in bone, muscle, kidney, lung Cell viability decreased 15% at 200 mg mL 1

James 2007 [83]

Vero cells, BALBc mice

antibody- N/A PEG-thiol

Test

PEG

tail vein injection

2.4x1011 nanoshells mL 1 (core = 110 nm, shell = 10 nm)

PEI/PAA (in mice)

4x103 cells/ well in 96-well plates

0.001-200 mg mL 1 WST (core = 48.9  19.1 nm, shell = 5.8  1.8 nm)

particles. An early study by Salem et al. tested feasibility of gold/nickel nanorods as gene-delivery agents.[31] A concentration of 44 mg mL 1 was used to test transfection of human embryonic kidney (HEK293) cell line with Au/Ni nanorods functionalized with GFP and luciferase reporter genes. The group notes that this is significantly below the LD50 value that was determined by the WST assay to be 750 mg mL 1. Transmission electron microscopy (TEM) revealed that the nanorods localized in vesicles or in the cytoplasm but not the nucleus. While no biodistribution analysis was done during their preliminary in vivo studies, no complications due to skin and muscle exposure to nanorods were reported. While examining the photothermal capabilities of gold nanorods, Takahashi et al. found the viability of cells incubated with nanorods, but without laser irradiation, did not decrease significantly.[85] More recently, other groups have found that the chemicals involved in the synthesis of gold nanorods play a role in their potential cytotoxicity. Niidome et al. looked at the effect of poly(ethylene glycol) (PEG)-modified gold nanorods on HeLa cells after 24 hours of incubation.[86] Strong cytotoxicity was associated with a low concentration of CTAB-stabilized gold nanorods. They proposed that free CTAB in solution was the source of the cytotoxic effect. This was corroborated when removal of excess CTAB from the PEG-modified gold-nanorod solution yielded 90% cell viability at the highest concentration tested (0.5 mm). Takahashi et al., the same group, published another study that tested the cytotoxicity of gold nanorods extracted from CTAB using a phosphatidylcholine (PC)-containing chloroform.[26] Concentrations from 0.09 to 0.72 mm exhibited little cytotoxicity; however, at higher concentrations of 1.45 mm, cell viability was reduced by approximately 20%. This cell death was proposed to be due to nanorod aggregation. In comparison, twice-centrifuged gold-nanorod solutions showed significant cytotoxicity with the lowest tested concentration, 0.09 mm, reducing cell viability by about 15% after 24 hours of incubation. Cytotoxicity was found to be dose dependent with almost 0% cell viability at 1.45 mm. Therefore, the group concluded that extraction process using PC was a better method than twice centrifugation. small 2008, 4, No. 1, 26 – 49

Exposure duration

6, 24 h

Toxicity

Author Year

Su [84]

2007

Huff et al. exposed KB cells to gold nanorods to examine their internalization, whether by endocytosis or by CTAB interaction on cell membranes.[87] This group found that KB cells internalized the majority of CTAB-coated nanorods while mPEG-DTC-coated nanorods were internalized at reduced levels. The CTAB-coated nanorods were found localized near the perinuclear region within the KB cells and, after five days, the cells appeared unaffected by the internalized nanorods as they grew to confluence over that period. This study suggests that CTAB promotes nanorod uptake by cells, which could explain the cytotoxicity observed by Niidome et al. with CTAB stabilized nanorods. A summary of the experimental setup and results on gold nanorods 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 magnetic interaction when placed within a magnetic field. In addition, when encountered by an alternating magnetic field, the particles heat up, allowing for both imaging and therapy applications. Specifically, their utilization as an MRI contrast agent has been extensively studied.[88–92] In terms of cytotoxicity, while bare iron oxide nanoparticles exert some toxic effects, coated SPIONs have been found to be relatively nontoxic. Gupta et al. showed that PEG-coated nanoparticles were biocompatible as exposed cells remained more than 99% viable relative to control at an upper concentration of 1 mg mL 1.[93] On the other hand, bare iron oxide nanoparticles induced a 25–50% loss in fibroblast viability at 250 mg mL 1. In a more extensive study, Gupta et al. found SPION cytotoxicity to be dose dependent. SPIONs caused a 20% reduction in cell viability at the lowest concentration tested (0.05 mg mL 1).[94] Further reductions were seen at higher concentrations, with the highest concentration tested (2.0 mg mL 1) resulting in about 60% loss of cell viability. However, using a different PEGbased coating, Yu et al. found PMAO-PEG-coated SPIONs, illustrated in Figure 3, to be relatively nontoxic, with cell vi-

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Table 6. Cytotoxicity of gold nanorods. Cell line

Surface coating

Human embryonic kidney, HEK293 Hela Cells

AEDP, plasmid, rhodamine, transferrin PEG, CTAB

Hela Cells

KB Cells

Exposure conditions

3x105 cells/ well in 24-well plates 5x103 cells/ well in 96-well plates phosphatidyl5x103 cells/ well in 96-well choline plates CTAB, mPEG-DTC N/A (l = 50 nm)

NP concentration (average size)

Test

Exposure duration

44 mg mL 1 (w = 100 nm, l = 200 nm) 0.01-0.5 mm (w = 11  1 nm, l = 65  5 nm) 0.09-1.45 mm (w = 11  1 nm, l = 65  5 nm) 100 mL

WST

4h

Toxicity

LD50 = 750 mg mL

1

Author Salem [31]

Year 2003

Cell death: ~ 80% at 0.05 mm Niidome 2006 w/CTAB nanorods; only ~ 10% [86] at 0.5 mm w/PEG nanorods MTT 24 h Twice centrifuged more toxic Takahashi 2006 than PC-NRs; 20% cells died at [26] 1.45 mm microscopy 5 days Internalized to perinuclear Huff 2007 region w/ CTAB, little uptake [87] w/mPEG WST

24 h

icity at the concentrations tested.[100] In comparison, MPEG–PAA- and PAAcoated iron oxide nanoparticles significantly reduced cell viability with only 16% of the cell remaining at an iron concentration of 400 mg mL 1. As bare iron oxide nanoparticles adsorbed to the cell surface, MTS analysis was infeasible; Figure 3. Example structure and cell viability data for water-soluble iron oxide nanoparticles. Reprinted however, cell counts after inwith permission from Ref. [95]. cubation indicated that uncoated iron oxide nanoparticles also significantly reduced cell viability. ability decreasing by only 9% at the 400 nm exposure The mechanism for SPION cytotoxicity, when it does level.[95] occur, has been linked to both cellular uptake and ROS proIn addition, other groups testing bare iron oxide nanoduction. Hu et al. found PACHTUNGRE(PEGMA)-immobilized nanoparparticles considered them to be biocompatible. Hussain ticles were relatively nontoxic, as exposed cells had greater et al. had similar findings as the highest dose they tested, than 93% viability.[101] However, pristine iron oxide nano250 mg mL 1, resulted in approximately 30% decrease in cell viability but this was judged as exhibiting little to no particles had a viability reduced to 70% in the first two toxicity.[96] Higher concentrations were tested by Cheng days, increasing to about 90% by day five. The group suggests that this increase in viability is due to the decrease in et al. and at 23.05 mm of nanoparticles the group found no nanoparticle concentration with the increase in cells after significant difference between the exposed cells and the mitosis. This was seen as cell uptake of particles went from control. However, this may be due to the relatively short ex154 pg cell 1 on the first day to 58 pg cell 1 after five days. posure time of four hours.[97] PACHTUNGRE(PEGMA) nanoparticles were taken up at 2 pg cell 1 sugIn addition to PEG, several groups have studied the cytotoxicity of different surface-coated iron oxide particles gesting that their lower toxicity is due to their lack of cell and found little cytotoxicity. Gupta et al. looked at pullulan uptake. Brunner et al. found a cell-specific response to bare (Pn)-coated SPIONs and found no cytotoxic effects, with iron oxide nanoparticle exposure.[102] 3T3 cells remained 1 [94] the cells remaining more than 92% viable at 2.0 mg mL . proliferative with the addition of up to 30 ppm iron oxide; however, human mesothelioma cells exhibited significant reThe group attributed the low toxicity of Pn-SPIONs to the duction in cell viability at only 3.75 ppm iron oxide. The pullulan coating, which prevents the iron oxide core from group attributed the observed toxicity to iron-induced freeinteracting with cells. Petri-Fink et al. observed no cytotoxradical production via the Fenton or Haber–Weiss reactions icity in melanoma after two hours of exposure to aminoin addition to internalization of the iron oxide particles. PiSPION for all polymer/iron ratios tested.[98] After 24 hours, sanic et al. showed anionic dimercaptosuccinic acid cytotoxicity became apparent for high polymer concentra(DMSA)-coated iron oxide nanoparticles are readily endotions. A similarly coated SPION tested by Cengelli et al. cytosed by rat pheochromocytoma cells and are found was found to be nontoxic as N11 microglial cells only took either in the cytoplasm, inside endosomes, or accumulated up aminoPVA-coated SPIONs, and as no nitric oxide was in the perinuclear region within the cells.[103] Most of the cell produced.[99] Wan et al. tested the effects of three surface coatings on iron oxide cytotoxicity and found MPEG–Asp3death occurred during the first 48 hours of exposure with cyNH2-coated iron oxide nanoparticles had almost no cytotoxtotoxicity and cell detachment being dose dependent.

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Changes in cell morphology were observed with nanoparticle exposure with the cells assuming a spherical shape with disruption of the cell cytoskeleton. Muller et al. consistently found a 20–30% decrease in neutral red uptake in monocyte-macrophages with 10 mg mL 1 Ferumoxtran-10 across various incubation times.[104] Similar results were found using the MTT assay. Testing over a longer period showed cell viability remained about the same between Ferumoxtran-10-pretreated cells and control cells after two weeks. The group speculates that the cytotoxicity is due to ROS production via the Fenton reaction, which can result in lipid peroxidation, DNA damage, and protein oxidation. Testing for inflammatory responses, the group found Ferumoxtran10 did not induce increases in cytokines IL-1b, TNF-a, IL-6

or IL-12, or superoxide anion production. This led to the conclusion that the monocyte-macrophages were not activated by the nanoparticles. A summary of the experimental setup and results on superparamagnetic iron oxide nanoparticles is provided in Table 7.

5. Semiconductor Nanoparticles In the case of semiconductor nanocrystals, better known as quantum dots, the concern of their cytotoxicity is not unjustified as several are composed of known toxic elements. However, despite the potential health risks, promising applications of quantum dots include their use in the medical

Table 7. Cytotoxicity of Fe3O4 nanoparticles. Cell line

Surface coating

Exposure conditions

NP concentration (average size)

Test

Exposure duration

COS-7 cells

none

4h

MA-PEG

0.92-23.05 mm (d = 9 nm) 0-1000 mg mL 1 (d = 50nm)

MTT

Human fibroblasts

3x104 cells/well, 24-well plates 10,000 cells mL in 24-well plates

MTT, Live/ Dead MTT

24 h

2h

1

PVA (amino, caroxyl, thiol) Primary human fibro- pullulan blasts, hTERT-BJ1

N/A

0.25 mg mL 1 (core = ~ 9 nm, d = 19-54 nm)

104 cells/well in 96-well plates

0-2 mg mL 1 (d = 40-50 nm)

MTT

24 h

Rat liver cells, BRL 3A

none

confluent in 6 or 24 well plates

0-250 mg mL 1 (d = 30, 47 nm)

24 h

Human mesothelioma MSTO-211H, rodent 3T3 fibroblast cells Rat brain-derived endothelial EC219; murine N9 & N11 microglial cells Mouse macrophages, RAW 264.7

none

N/A

3.75-15 ppm (d = 12-50 nm)

LDH, MTT, GSH MTT

Melanoma cells

Human breast carcinoma SK-BR-3 cells, human dermal fibroblasts Human monocytemacrophages

rat pheochromocytoma cell line PC12M OCTY mouse cells

PVA, ami- 96 or 48-well noPVA, car- plates oxylPVA, ThiolPVA PACHTUNGRE(PEGMA) 105 cells mL

1

PMAO-PEG Confluent

dextran

1–2x106 cells/well in 24-well plates & 0.5-1x106 cells/ well in 48-well plates DMSA 20,000 cells mL 1 in 6 or 12 well plates 104 cells/well in MPEG– Asp3-NH2, 96-well plates MPEG-PAA, PAA

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3, 6 days

2.5 mL NPs mL 1, MTT 48 h 11.3 mg iron mL 1 (core = 8-12 nm, d = 30 nm) 0.2 mg mL 1 ratio of 1, 4 days (d = 6.2  0.7 nm) treated/ control cells 10-400 nm Live/ 1, 24, (9.6 nm) Dead 48 h

0.0001-10 mg mL 1 (d = 30 nm)

15, 1.5 mm and 150 mm (d = 512 nm) 0-400 mg mL 1 (d = 14 nm)

MTT, 24, 48, Neutral 72 h, 4 Red days + 14 day grow Live/ 2, 4, 6 Dead days Cell 72 h Titer 96

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Toxicity

Author Year

No significant difference between control and exposed 250 mg mL 1: 25-50% viability decrease for bare; 1 mg mL 1: 99% viable for PEG-coated No cytotoxicity after 2 h for all polymer/iron ratios; after 24 hr, cytotoxicity at high polymer conc. Plain SPION showed significant decrease in viability, PnSPION showed no cytotoxicity w/ > 92% viability EC50 > 250 mg mL 1

Cheng 2004 [97] Gupta 2004 [93] Petri- 2004 Fink [98] Gupta 2005 [94]

Hussain 2005 [96]

3T3 cells viable w/ up to Brunner 2006 30 ppm; MSTO cell viability [102] decrease at 3.75 ppm, free radicals via Fenton rxn Only aminoPVA-SPION upaken Cengelli 2006 by N11 [99]

Cytotoxicity dose dependent; decrease w/time attributed to cell division

Hu 2006 [101]

91% viability at 400 nm after 48 h in HDF cells

Yu [95]

1 mg mL 1: not toxic after 72 h; 10 mg mL 1: mildly toxic; viability similar over 2 wks

2006

Muller 2007 [104]

SPION exposure reduced PC12 Pisanic 2007 ability to respond to nerve [103] growth factors Wan 2007 MPEG–Asp3-NH2 almost no [100] cytotoxicity; MPEG–PAA- and PAA-coated decrease cell viability

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field as new drug-delivery and biomedical-imaging agents. This section explores the current research that has been conducted on quantum-dot toxicity. Quantum dots (QDs) are nanoscale particles ranging from 2 to 100 nm in diameter depending on the types of surface coating or functional group added. For biological applications, 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) and groups III–V (e.g., GaAs, GaN, InP, and InAs) on the periodic table.[105] Many of these core metals are known to be toxic at low concentrations; examples include cadmium, selenium, lead, and arsenic. Therefore, if these QDs are exposed to conditions promoting degradation, such as an oxidative environment, toxicity related to the release of free metal ions is expected. Thus the crucial factor in QD toxicity is stability. The cytotoxicity of QDs is reduced when their

cores are protected from degradation given that the added coatings are biocompatible. To prevent core degradation, an additional shell layer is added, making the QD more biocompatible. Additional functionalities or bioconjugates can be added to the surface to improve bioavailability or introduce bioactivity. Since CdSe/ZnS quantum dots are believed to be the most versatile for biological applications, most of the published toxicity studies focus on this type.[106,107] Summaries of the experimental setup and results on CdSe and CdTe QDs are provided in Tables 8 and 9, respectively.

5.1. Cadmium Selenide Quantum Dots Historically, QDs were used in animals well before extensive cytotoxicity studies. These results highlighted key issues, such as biodistribution and coating integrity, now of

Table 8. Cytotoxicity studies on CdSe quantum dots. Cell line

Surface coating

BALB/c nu/nu mice

3 peptides (GFE, F3, tail vein LyP-1), PEG injection

Xenopus embryos Vero cells

PEG-PE, N/A phosphatydilcholine MUA, SSA N/A

Hela cells, Dictyostelium discoideum Mice

DHLA

N/A

Micelle

BALB/c mice

tail vein injection tail vein injection

poly(acrylic acid) polymer or mPEG750 QDs, mPEG5000 QDs, COOHPEG-3400 QDs silane, biotin, STV, 300 cells/ peptides, NLS 100 mm

Hela cells

Rat primary hepatocytes

MAA, BSA/EDAC, EGF

Human MUA, cysteamine lymphoblastoid, (NH2), WTK1 thioglycerol (OH) Vero cells, Hela MUA, SSA cells, primary human hepatocytes B16F10 melano- TOPO, DHLA ma cells

DNA

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biotin

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Exposure conditions

NP concentration (average size)

Test

Exposure duration

100-200 mg QDs in 0.1-0.2 mL solution 2.3 mm

histology

5 or 20 min

0.24 mg mL 1 400-600 nm

20 nM

1

microscopy At least 4 days microscopy 2 h microscopy 4560 min

observaN/A tion 50-500 pmol histology/ 1-3 h @ QDs in 50microscopy 1 min 200 mL saline interval mm

1, 10, 100 nm (d = 8-10 nm) 5x105 cells 0.0625, on 35 mm 0.25, 1 mg mL 1 wells 4 5x10 0-2 mm cells/well (d = 9-48 nm) on 96-well plates 3x104 0-4 mg mL 1 cells/well on 96-well plates 5x104 10 mL cells/well on 6-well plates N/A N/A

colonigenic 2 h assay MTT, 24 h microscopy Comet, 12 h flow cytometry, MTT MTT, flow cytometry

24 h

microscopy 4-6 hr

Plasma nicking assay

Toxicity

Author

Year

Specific tissue targeting achievable, Akerman 2002 accumulate in liver and spleen [109]

At > 5x109 QDs/cell abnormalities became apparent 0.4 mg mL 1: no difference in viability of MUA-QD/SSA complexes No differences in viability

No abnormal behavior observed QDs in endosomes in liver, spleen and bone; fluorescence at 1 mo. similar to 24 h signal, mPEG-5000 QDs have longer circ. time & lower accumulation On average > 90% cells survived

Dubertret 2002 [108] Hanaki 2003 [123] Jaiswal 2003 [122] Larson [110] Ballou [111]

2003

Chen [126]

2004

Derfus Coating eliminates air oxidation; [113] however, high conc. with 8 h UV exposure still have 95% cell death Crude QDs exhibited decreased cell Hoshino activity, QD fluorescence lost in low [118] pH oxidation, TOPO is cytotoxic

2004

2004

2004

Damaged cells increased sharply at Shiohara 2004 0.2 mg mL 1 but slowly at [116] 0.1 mg mL 1 No detectable toxicity

0-60 min DNA damage occurs in both in dark environ, UV exposure incr damage or UV

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Voura [129]

2004

Green [127]

2005

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Table 8. (Continued) Cell line

Surface coating

Exposure conditions

NP concentration (average size)

NRK fibroblasts; MDA-MB-435S breast cancer cells; Chinese hampster ovary, CHO; RBL cells COS-7 cells, NIH 3T3 cells, Human liver carcinoma, HepG2 Human breast carcinoma SK-BR-3 cells

MPA, mercaptocarbonic acid

7x104 cells mL

2-10 mm ratio of 18-48 h (core d = 2.4- pre/post 4.5 nm) adherent cells, Live/ Dead

SiO2, MAA, PA

2x104 cells 0-0.6 mm mL 1 in 96- (core well plates d = 5 nm, d = 25 nm)

Alamar blue

PEG (750-, 6000-Mw)

Live/Dead

Human bone marrow mesenchymal stem cells (hBMSC) Sprague– Dawley rats

HIV-derived Tat peptide

22,500 10-150 nM cells/well in 96-well plates 7x105 cells 1.625 mg in 10 cm dishes

1

LM, BSA

jugular vein injection

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

silica, thiol, PEG

N/A

Human breast cancer cells, MCF-7

NAC, Cys, MPA

Hela cells

PEG-g-PEI, polycarboxylate

105 cells cm 2 in 24-well plates 60% confluency

HepG2 cells, Wister mice

PLA, F-68, SDS, CTAB

ctDNA

MAA

primary HEKs

PEG, PEG-amine, polyacrylic acid

5 nmol in 0.2 mL solution (d = 25, 80 nm) 2-10 nm Brca, 880 nm lung, skin (d = 810 nm) 10 mg mL 1

1 nm (core d = 6.5 nm, PEI coated d = 15.3 nm) 6x104 cells 10-400 ppm on 96-well (d = 159plates 266 nm) 5 mg mL 1 3.6x10 7 ctDNA mol L 1

1.5–2x104 cells cm 2, 40-60% confluency

0.2-20 nm (d = 4.6 nm & w = 6 nm, h = 12 nm)

Test

48 h (cos-7 & hepG2), 72 h (3T3) 4h

WST, flow 24 h cytometry, Rt-PCR, microscopy histology 90 min

Toxicity

Author

NRK cells: poisoning occurs around Kirchner 0.65 mm for MPA-CdSe, 5.9 mm for [114] CdSe/ZnS; other 3 cells: only larger particles inside cell, polymer better than MPA

Year

2005

SiO2 coating better than PA, ZnS coating decreases toxicity

Selvan [35]

2005

150 nm - 0%, 70%, 80% cell viability for bare, 750- & 6000-Mw PEG coated QDs

Chang [124]

2006

Growth curve and cell cycle distribution not affected by QDs

Hsieh [121]

2006

QDs uptake in Kupffer cells, majority uptake in liver with some in spleen, lungs, kidneys, lymph nodes, bone marrow No adverse effects in lung cells, skin cells showed < 50 gene expression changes

Fischer [112]

2006

Zhang [128]

2006

microarray

48 h

MTT

24 h

MPA capped QDs more toxic than Cys capped, cells exhibit reactions of oxidative stress

Cho [130]

2007

MTT

2h

PEI-coated dots very toxic, toxicity reduced w/ more PEG added

Duan [119]

2007

MTT

12-72 h

> 80% cell viability up to 400 ppm

Guo [131]

2007

Nucleic Liang 2007 110 min 1.7x10 5 mol L 1 Cd2 + released, acid probe 70% DNA damaged by QDs [132] (RuACHTUNGRE(bipy)2ACHTUNGRE(dppx)2 + ) MTT 24, 48 h Dose–response significant for PEG- Ryman- 2007 amine and carboxylic acid but not Rasmussen [120] PEG alone

concern in cytotoxicity studies. Dubertret et al. injected micelle-encapsulated CdSe/ZnS QDs into Xenopus embryos. At 2 K 109 QDs cell 1, the quantum dot injected and control embryos displayed similar growth patterns with the QDs remaining in the injected cells and their progeny. However, at 5 K 109 QDs cell 1, adverse effects were found. The group speculated that the abnormalities resulted from the QDs affecting the osmotic equilibrium of the cell.[108] The majority of published in vivo studies were conducted in mouse or rat models. Akerman et al. injected three different (GFE, F3, small 2008, 4, No. 1, 26 – 49

Exposure duration

LyP-1) peptide-coated CdSe/ZnS QDs into normal BALBc mice and studied the tissue distribution after 5 or 20 minutes of circulation. Each of the peptide-coated QDs were found to accumulate in the liver and spleen in addition to the targeted tissue; yet, this nonspecific accumulation was reduced by adding PEG to the QD surface. The group did not observe any acute toxicity caused by the QDs after 24 hours of circulation.[109] Similarly, Larson et al. observed no adverse effects after imaging the mice used in their experiment and hypothesized that CdSe/ZnS QDs clear from the

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Table 9. Cytotoxicity studies on CdTe quantum dots. Cell line

Surface coat- Exposure NP concentraing conditions tion (average size)

anionic, catRat pheochromocytoma cells, ionic, BSA PC12 Human hepato- none ma HepG2 cells

Human breast cancer cells, MCF-7

NAC, Cys, MPA

Human neuroblastoma cells, SH-SY5Y

cysteamine, N-acetylcysteine (NAC)

Human hepato- none ma cells, HepG2; Sprague-Dawley rats

105 cells cm 2 on 24-well plates 2x105 cells mL 1 in 96-well plates 105 cells cm 2 in 24-well plates 105 cells cm 2 in 24-well plates 2x105 cells mL 1 in 96-well plates

Test

0.01-100 mg MTT mL 1 (d = 2.25.7 nm w/o coating) 0, 10 8, 10 7, MTT 10 6, 10 5 m

1

10 mg mL

5 mg mL

1

MTT

MTT 0-100 mm; 2 mm,1 mL kg 1 in rats (d = 26nm)

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Toxicity

Author Year

24 h

50% decrease in metabolic activity: 50 mg mL 1 (green) 100 mg mL 1 (red)

Lovric 2005 [133]

24 h

~ 50% viability reduction at 10 5 m; similar cytotoxicity found w/ air and N2 exposed QDs

Liu [135]

2006

24 h

MPA capped QDs more toxic than Cys capped; cys-CdTe QDs more cytotoxic than cys-CdSe/ZnS QDs; cells exhibit oxidative stress 52% viability w/ cyc-QDs, 85% viability if pretreated w/NAC

Cho [130]

2007

Choi [136]

2007

MTT, flow 24 h cytometry

body before the protective coating can breakdown. However, the group did not report a time line for which the animals were kept, only mentioning that the animals were maintained for a long-term toxicity study, which diminishes their findings.[110] More recently, the role of the particle surface on distribution and toxicity has been studied. Ballou et al. also observed QD deposition in the liver, spleen, and bone marrow of 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 QDs were found to have the longest circulation time in addition to reduced nonspecific accumulation. All QDs were found localized internally in endosomes, primarily within perifollicular cells for the spleen, and no visible signs of breakdown were seen via electron microscopy. A long-term stability study conducted with mPEG-750 QDs found the QDs remained in the liver, lymph nodes, and bone marrow for a month. Although the fluorescence decreased after one month, the fluorescence distribution was close to what the group observed 24 hours after injection.[111] More recently, Fischer et al. injected mercaptoundecanoic acid (MUA), lysine, and BSA-coated CdSe/ZnS QDs in Sprague–Dawley rats. A difference in biodistribution was also found as the liver took up 40% of the lysine-QDs and 99% of BSA-QDs after 90 minutes. QDs endocytosed by Kupffer cells, similar to RES processing, were sequestered not excreted. Small amounts of both QDs appeared in the spleen, kidney, and bone marrow, but no QDs were detected in the feces or urine even after ten days. The group also measured the size of the quantum dots within the vesicles and found the QDs retained their size, suggesting no degradation after 90 minutes of exposure.[112] These studies conclude that ZnS capped CdSe QDs are relatively nontoxic as the animals were not killed, nor did they exhibit abnormal behavior

42

Exposure duration

48 h; 24 h (0, 0.5, 1, 2, 4 h) - in rats

IC50: 19.1 mm (red), 4.8 mm (yellow), 3 mm Zhang 2007 (green); rats: few signs of toxicity, but [134] changes in locomotor activity were observed

after QD injection. However, as several of the group found, the QDs are internalized and seem to be retained inside the cells. As clearance from the body is an important aspect of safety, this suggests possible toxicity could result from the bioaccumulation. All of the previously mentioned studies involved ZnS coated CdSe quantum dots; the ZnS coating provides a well-terminated surface with few defects and high quantum yields. 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 the release of free cadmium ions. Cadmium is a known toxic agent that induces cell death via mitochondrial damage and oxidative stress. When QD surface oxidation was prevented with surface coatings, the cadmium atoms remained bound to selenium atoms and the surface-coating molecules rendering them relatively nontoxic. This was demonstrated with the addition of a ZnS shell; the oxidative degradation of the CdSe core due to exposure to air was significantly reduced resulting in lower cytotoxicity.[113] Several groups have confirmed the effectiveness of the ZnS shell in reducing the cytotoxicity of CdSe quantum dots.[34,35,114,115] Chan et al. proposed a mechanism for the bare CdSe QD-induced cell death. In addition to determining that apoptosis, not necrosis, occurred in CdSe exposed cells, the group suggested that QDs induced apoptosis by activating Jun N-terminal kinase (JNK) in a dose-dependent manner. In addition, mitochondrial-dependent apoptotic processes, involving activation of caspase 9 and 3, increases in Bax protein and decreases in Bcl-2, were also observed. Mitochondrial-membrane potential was reduced with exposure to bare CdSe QDs resulting in an increase in cytochrome c release.[115] Researchers have also examined the effect of additional surface coatings on the cytotoxicity of quantum dots. QDs must be appropriately encapsulated to prevent cadmium re-

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lease and subsequent cytotoxicity. At issue for many researchers is the best way to accomplish this. One simple approach is to replace the original organic coat with watersoluble ligands. These ligand-exchange reactions yield QDs with smaller hydrodynamic sizes but generally do not provide biocompatible materials. Shiohara et al. studied the cytotoxicity of three MUA-coated CdSe/ZnS QDs (520-, 570and 640-nm emission) in three different cell lines (Vero cells, Hela cells, and human primary hepatocytes). After incubating the cells with quantum-dot concentrations ranging from 0 to 0.4 mg mL 1 over 24 hours, a concentration dependence of cytotoxicity was found.[116] It is important to recognize that, in this case, the toxicity is due to the surface coating rather than the quantum dots. MUA is a compound that in previous studies was found to water solubilize CdSe/ ZnS quantum dots.[117] This is important in biological applications as hydrophobic compounds have poor bioavailability. This study reveals that the MUA coating is not appropriate for this purpose as it increases the toxicity of the quantum dots. Hoshino et al. did a larger study incorporating more surface coatings (MUA, cystamine, thioglycerol) on CdSe/ZnS QDs. MUA-coated quantum dots were more toxic than ones without. Of the three coatings, thioglycerol was found to induce the least genotoxicity and therefore cytotoxicity. These results indicate that some hydrophilic surface coatings contribute to the cytotoxicity of QDs. Because TOPO was also found to be a cytotoxic compound, the complete removal of TOPO from the QD samples is important in reducing toxicity.[118] Selvan et al. looked at SiO2, mercaptoacetic acid (MAA), polyanhydride (PA) surface-coated CdSe/ZnS quantum dots in three different cell lines (human liver carcinoma (HepG2), NIH T3T cells, COS-7 cells) and obtained dose-dependent results for all surface coatings in each cell line. SiO2/CdSe QDs were found to be much less cytotoxic than MAA or PA coated QDs. SiO2/ZnS-CdSe QDs were less cytotoxic than SiO2/CdSe QDs, suggesting that the combination of ZnS capping and SiO2 coating provided for the optimal protection against CdSe dissolution.[35] A better option for biocompatible QDs is to use amphiphilic polymers to encapsulate the inorganic/organic system. Most commercial sources of QDs prepare their systems in this way. While larger polymeric coatings increase the hydrodynamic size, they yield very bright and stable materials. Kirchner et al. found PEG to lower cellular uptake of silicacoated QDs resulting in lower cytotoxicity.[114] Duan et al. tested PEI-coated QDs in HeLa cells and found they are endocytosed or macropinocytosed after one to two hours of incubation. However, since PEI-coated dots are toxic to cells, PEG was added to reduce this toxicity. The two different forms (PEI grafted with two PEG and PEI grafted with four PEG) of coated QDs exhibited different distribution patterns inside the cells and different cytotoxicities. The PEI-g-PEG4 QDs accumulated in the perinuclear region, which yielded better cell viability while the PEI-g-PEG2 QDs were distributed in the cytoplasm and had significant cytotoxic effects. The group believes the cytotoxicity is due to the PEI polymer and not the presence of cadmium ions.[119] Ryman-Rasmussen et al. tested two different QDs (565- and 655-nm emission) with three different surface small 2008, 4, No. 1, 26 – 49

coatings (PEG, PEG-amine and carboxylic acids). All QDs were localized intracellularly by 24 hours with the PEGcoated QDs found in the cytoplasm, perinuclear region, and for QD 565 within the nucleus. After 24 hours, no cytotoxicity was observed, but by 48 hours toxicity became apparent at the largest concentration of 20 nm, indicating time-dependent cytotoxicity. Surface coating had an observable effect on IL-1b, IL-6, and IL-8 pro-inflammatory cytokine release. Cytokine levels increased after carboxylic acid coated QD exposure, while there was no increase in cytokine release with PEG-coated QDs.[120] In addition to their coatings, size and concentration can influence the toxicity of quantum dots with smaller sizes and higher concentrations being more cytotoxic. The advantage of quantum dots in imaging applications is their tunability. By changing their size or core diameter, the fluorescence emission peak can be shifted to a wavelength of choice within a fairly broad range. This is particularly useful in biological applications as cells contain endogenous fluorophores, which can mask the signal emitted from contrast agents with similar emission peaks. However, several groups have found cytotoxicity to be size dependent with smaller QDs exhibiting larger reductions in cell viability. Kirchner et al. tested the exposure of CdSe/ZnS QDs in several cell lines (NRK fibroblasts, MDA-MB-435S breast cancer cells, CHO cells, RBL cells). After 18 hours exposed to the same concentration, cytotoxic effects were higher for smaller QDs, which they suggest could be due to the higher surfaceto-volume ratio of smaller particles.[114] Interestingly, Hsieh et al. found size-independent internalization of QDs.[121] In addition, while high concentrations of QDs can be toxic to cells, the group found that concentration influenced QDs delivery into cells with a low concentration (15 nm) effectively labeling cells while a 10-fold increase in QD concentration, resulting in poor cellular uptake. However, this was not the typical finding. As with many chemicals, cytotoxicity of QDs was also found by many research groups to be dose dependent with higher concentrations, resulting in significantly higher cell death.[113,114,116,120] In vitro cytotoxicity studies report findings similar to in vivo studies that QDs are taken up and sequestered intracellularly. Jaiswal et al. demonstrated that targeted CdSe/ ZnS QDs could be internalized by HeLa cells and tracked in live cells for more than 10 days with no morphological signs of toxicity.[122] Hanaki et al. studied how long MUAcoated QDs could stay in Vero cells. QD-containing vecontaining vesicles.[123] The number of vesicle-containing cells reduced to half after three days and about 10% of the cells contained QD vesicles after five days. These findings correspond to introducing a QD concentration of 0.4 mg mL 1, which was found to have no cytotoxicity. A more long-term study was conducted by Seleverstov et al., who found that QD-labeled cells retained their fluorescent signal for 52 days in both continuous culture or after cell passaging.[34] The QDs were internalized and observed mainly within endosomes near the perinuclear region with no nuclear involvement. In addition, QD aggregates were found localized around the mitochondria and after 72 hours morphological effects included swollen mitochondria and

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enlarged Golgi cisterns. Hsieh et al. confirmed the perinuclear localization of QDs and found that cells retained QDs for at least three weeks.[121] While these studies have shown that cells can survive long after internalizing QDs, Chang et al. found with three different QDs (bare, two PEG coated) that cytotoxicity is similar between the differently coated QDs when their intracellular concentration is the same, as seen in Figure 4. Therefore, while extracellular concentrations of different QDs suggest dose-dependent cytotoxicity, cytotoxicity is dependent on the intracellular QD content, and biocompatibility can be improved by minimizing QD uptake.[124,125]

was also inhibited. These effects could be reproduced using QDs of various sizes.[121] A DNA microarray study was recently published by Zhang et al., examining the impact of treating human lung and skin epithelial cells with two doses of PEG-silane QDs. No adverse effects were found in lung epithelial cells; however, the skin epithelial cells exhibited cell-cycle regulator gene repression. Overall, fewer than 50 genes showed significant expression changes after PEGsilane QD treatment. However, the group did not find involvement of the genes that are associated with heavy metal exposure. In addition, no pronounced difference in phenotypic response was found between low or high QD doses. Higher QD doses led to more particle uptake made apparent from the stronger measured fluorescent signal.[128]

5.2. Cadmium Telluride Quantum Dots

Figure 4. Cytotoxicity results based on intracellular levels of bare QDs (left image, black bars), and 750- (middle image, gray bars) and 6000- (right image, light gray bars) Mw PEG-substituted QDs. Reprinted with permission from Ref. [124].

The ability of QDs to be internalized by cells has led some groups to pursue using CdSe/ZnS QDs for nuclear targeting. Chen et al. revealed that silane-coated CdSe/ZnS QDs conjugated with the SV40 nuclear localization signal (NLS) protein only entered the nucleus of 15% of the cells.[126] Perinuclear accumulation was still observed for the majority of the NLS-QDs. However, QDs conjugated to a random peptide did not enter the nucleus and only localized randomly within the cells. Testing for cytotoxicity on HeLa cells, the group found most of the transfected cells survived in all the experiments thus implying negligible toxicity even with nuclear exposure to QDs. Since QDs are capable of entering the nucleus, several groups have suggested QD interaction with nuclear DNA or proteins to be a factor in their cytotoxicity. Green et al. reported data corroborating this theory as they found biotincoated CdSe/ZnS QDs were able to nick DNA in an in vitro, cell-free assay.[127] Hsieh et al. showed that QDs can alter gene expression in human bone-marrow mesenchymal stem cells. While flow cytometry analysis showed that the internalized QDs did not change the cell-cycle distribution of hBMSCs compared to the control, an inhibited response of hBMSCs to osteogenesis was found as ALP activity was significantly suppressed, and mRNA expression of osteopontin and osteocalcin, two osteogenesis specific markers,

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Besides CdSe/ZnS QDs, one group has published several studies on CdTe QDs, which also have potential in biomedical applications such as bioimaging. The initial study presented by Lovric et al. tested cell exposure to both red (  5.2 nm) and green (  2.2 nm) CdTe QDs coated with mercaptopropionic acid (MPA) and cysteamine (Cys).[133] A range of concentrations (0.01–100 mg mL 1) was used to test the effect of exposure on metabolic activity. For both types of QD, a decrease in metabolic activity was found at concentrations of 10 mg mL 1 or more. Looking at cell morphology, 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 condensation and membrane fragmentation were observed, indicative of apoptosis. In addition, cytotoxicity was more noticeable with the smaller QDs than with the larger QDs at the same concentrations. To explain this difference, the group noted that the red QDs were found primarily in the cell cytoplasm with none entering the nucleus, while the green QDs were mainly found in the cell nucleus. This suggests that since the smaller QDs could access the nucleus they could cause damage to DNA and induce apoptosis or cell death. As QD cytotoxicity is believed to be due to free-radical formation caused by the presence of free Cd2 + from the degradation of the QD core, the effect of free-radical scavengers N-acetylcysteine (NAC) and Trolox, as well as adding another protective coating, bovine serum albumin (BSA), was tested. Both NAC and BSA but not Trolox significantly reduced CdTe QD toxicity, suggesting that Cd2 + is a factor in QD-induced toxicity.[133] Zhang et al. had similar findings after testing green( 2 nm), yellow- (  4 nm), and red- (  6 nm) light emitting, uncoated CdTe quantum dots in human hepatoma cells (HepG2). A size-dependent difference in toxicity was observed as the IC50 values of the green, yellow, and red CdTe 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 the findings presented earlier by Lovric et al. that smaller QDs are significantly more cytotoxic than larger QDs. In addi-

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tion, they looked for the effect of CdTe quantum dots injected into Sprague-Dawley rats. Few signs of morbid toxicity were observed after QD exposure. The group saw no organ damage, only a small decrease in body weight, and a temporary decrease in locomotion activity occurring just after injection. This change was attributed to possible effects of the quantum dots on neural function, which is plausible since some nanoparticles can pass the blood–brain barrier. However, the group recognizes that this change could also be due to exposure to free cadmium.[134] The effect of different surface coatings was explored by Cho et al. using four cadmium QDs (MPA-, Cys-, or NACcoated CdTe QDs plus cysteamine-coated CdSe/ZnS QDs) on human breast-cancer (MCF-7) cells. While minimal reduction in metabolic activity occurred after exposure to Cys-coated CdSe/ZnS QDs, exposure to MPA and Cyscoated CdTe QDs caused a significant decrease in cellular metabolic activity with a less distinct decrease in the NACcoated CdTe QDs. This finding was confirmed using the Trypan blue cell viability assay, which revealed significant cell death with CdTe QDs but not with CdSe/ZnS QDs after 24 hours of QD exposure.[130] In addition to using surface coatings to reduce cytotoxicity, Liu et al. attempted to alter the synthesis condition to improve biocompatibility of CdTe QDs. While the dose-dependent reduction in cell viability after CdTe QD treatment was confirmed, this was found regardless of either air or nitrogen fabrication conditions.[135] Recently, Choi et al. revealed a possible signaling pathway involved in CdTe QD-induced cell death. Activation of the Fas receptor results in a signaling cascade that culminates in apoptosis. This study found significant upregulation of Fas expression on the surface of neuroblastoma (SHSY5Y) cells treated with Cys-coated and NAC-conjugated QDs in comparison to control cells. However, NAC-capped QDs had little Fas upregulation, and NAC pretreated cells exposed to Cys-QDs had no Fas upregulation, suggesting that oxidative stress caused by QD exposure induces Fas expression.[136]

6. Summary and Outlook In this paper, cytotoxicity data on carbon-, metal-, and semiconductor-based nanoparticles have been reviewed. In general, cells can survive short-term exposure to low concentrations (< 10 mg mL 1) of nanoparticles. However at high doses, several groups have found cytotoxic effects to emerge in a dose- and time-dependent manner for all of the nanoparticles reviewed here. While the causes for the increase in cell death observed at higher concentrations and longer exposure times are material specific, the generation of reactive oxygen species and the influence of cell internalization of nanoparticles are two common findings throughout. While this Review attempts to draw parallels between the research that has currently been conducted and published on several classes of nanoparticles, there are still gaps in knowledge about the interaction of nanoparticles with the body. Although several studies have been conducted, many small 2008, 4, No. 1, 26 – 49

of the earlier experiments were not designed to isolate the source of the cytotoxicity, allowing the different physiochemical properties of the nanoparticles plus experimentalsetup factors to influence and confound the findings. In addition, a systematic approach to testing has not been established. While much of the function of nanoparticles is due to their core structure, the surface coating defines much of their bioactivity. For many nanoparticles to be useful in biological applications, the addition of some type of surface coating is required. In the case of quantum dots, surface coatings serve both to contain the cadmium particles from leeching and to make the particles water soluble. This addition of surface coatings confounds the bioactivity and potential toxicity of the functional groups on the nanoparticle surface with the core nanoparticle making it difficult to interpret the observed changes. For example, many nanoparticles are not water soluble and therefore require the addition of a hydrophilic surface coating. However, as seen in MWNT and QD studies, adding certain hydrophilic molecules results in lower cell viability as the functional groups themselves were toxic.[11,22,114,118] Surface charge also plays a role in toxicity with cationic surfaces being more toxic than anionic, and neutral surfaces being most biocompatible.[12] This may be due to the affinity of cationic particles to the negatively charged cell membrane. Therefore, adding a coating that makes the nanoparticle more cationic could make the nanoparticle appear more toxic than it inherently is. Traditionally, in vitro toxicity testing focuses on whether or not exposure to a potentially toxic agent results in cell death. However, although no cell damage or death may be apparent after nanoparticle exposure, changes in cellular function may result. Therefore, it is important to verify that the end points chosen to signify cytotoxicity are appropriate. For example, if nanoparticle exposure induces cell senescence but not cell death, this could be considered a toxic effect as cell proliferation has been disturbed. Looking over the cytotoxicity assays commonly used in the studies reviewed, most either determine membrane damage, metabolic irregularities, or inflammatory response, which may not materialize with cell senescence. Therefore, sub-lethal cellular changes should also be taken into account and tested for when evaluating the effects of nanoparticle exposure on cells. One way to do this, which a few groups have explored, is to conduct genomic and proteomic array tests to explore the cellular signaling alterations behind the toxicity. In addition, it is important that the assays used to determine cytotoxicity are valid for the materials being tested. One example, the neutral red test, has come into question as it relies on the adsorption of the dye to detect living cells. Carbon black has been shown to adsorb neutral red dye molecules giving false positive results.[9] This suggests that carbon nanomaterials could encounter the same interference, and they have been shown to adsorb a similarly structured chemical, naphthalene.[137,138] The MTT assay has also come under scrutiny as groups have found discrepancies between the MTT assay results and those from other assays. In a study conducted by Pulskamp et al. on SWNTs, the MTT assay was the only test that revealed a dose-dependent de-

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crease of cell viability. The WST-assay results did not agree with the MTT assay as it indicated no significant loss in cell viability except for a small reduction at the highest concentration. PI and annexin stains were also used for validation, and they confirmed the WST-assay results.[64] To explain the difference in results, Worle-Knirsch et al. proposed that SWNTs interact with the MTT-formazan crystals but not with WST, XTT, or INT reagents. SWNTs attach to the insoluble MTT formazan product disrupting the distinguishing, colorimetric reaction. This would account for the finding that very pure SWNTs reduced cell viability below 50% for MTT assays, but exhibit almost no loss according to the WST, LDH, and MMP assays.[69] To date, there is a lack of consensus in the published literature on nanoparticle toxicity due to variable methods, materials, and cell lines. Nanotoxicology has emerged recently to apply traditional toxicology methodologies to the study of nanomaterial toxicity; however, standardization in experimental set up such as choice of model (cell line, animal species) and exposure conditions (cell confluency, exposure duration, nanoparticle-concentration ranges and dosing increments) is necessary in order for comparisons between studies conducted by different groups to be effective.[139] With respect to model choice, both animal- and human-derived cells have been used. Since the potential toxicity of nanoparticles in humans is in question, human cells should be used to better predict human toxicity. In addition, the cell types tested in cytotoxicity tests should also be consistently studied. Several groups have tested potential lung or dermal toxicity; however, in the case of oral or intravenous exposure, many internal organ sites can be exposed. While some groups have looked at liver and kidney exposure, these studies have mainly been conducted using quantum dots with few to none using fullerenes and gold and iron oxide nanoparticles. Fewer nanoparticle studies have been conducted using heart, blood, and brain cells.[12,49,114,115,136] Detailed recommendations have been outlined by a Nanomaterial Toxicity Screening Working Group.[140] Consistency in reporting the physiochemical characteristics of nanoparticles would also facilitate re-examination and cross comparison of nanoparticle toxicity data. Standardization of materials is more challenging as nanoparticle characterization can be difficult. Sizing of nanoparticles can be done using methods such as scanning and transmission electron microscopy, dynamic light scattering, and size-exclusion chromatography; however, the size values obtained can vary between these methods. A standard technique for measuring and reporting the hydrodynamic sizes of nanoparticles would be valuable. Determining the concentration of nanoparticles in solution is more difficult. Concentration can be calculated from the optical density using the Beer– Lambert law given the extinction coefficient of the nanoparticle. However, as Yu et al. point out, the extinction-coefficient values published for quantum dots differ between groups by an order of magnitude.[141] Cryogenic TEM was used as an alternative method of determining concentration. This method involves direct counting of particles in a relatively fixed volume. As concentration or dose plays a signifi-

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cant role in biomedical applications of nanoparticles, having a standard technique of calculating this value is important. Another important aspect of toxicology is the burden of multiple dosing of nanoparticles. In the case of bioimaging, exposure of nanoparticle contrast agent would not occur once but repeatedly with each screening or diagnostic scanning session. All of the studies reviewed in this paper involved only one administration of various concentrations of nanoparticles with cytotoxicity tests taken at different time points. However, this is most likely because most were in vitro versus in vivo studies. As toxicity studies move more into in vivo nanoparticle evaluation, future experiments need to incorporate the effect of multiple exposures to nanoparticles to determine the extent of clearance and bioaccumulation. Most of the in vitro studies presented in this paper assess dosimetry merely by observing the dose-response relationship after external introduction of different concentrations of nanoparticles. Yet, as cells are seen to readily internalize nanoparticles, the number of internalized nanoparticles correlates to cytotoxicity as Chang et al. revealed.[124] Future research should measure and record the cellular dose in addition to the administered dose to better characterize the extent of nanoparticle exposure. Strategies to determine the particokinetics in in vitro systems have been suggested by Teeguarden et al.[142] In addition, several of the studies have suggested that after internalization, the groups observed a persistence of nanoparticles within cells. This sequestration of nanoparticles could elicit inflammatory responses, cell-cycle irregularities, and gene-expression alterations. Unfried et al. have reviewed the mechanisms in which nanoparticles are taken up and processed by cells; however, knowledge in this area is still limited.[143] Future research should focus on understanding how cells internalize nanoparticles so that methods to prevent cell opsonization of nanoparticles can be developed. This potentially could improve the in vivo biocompatibility and clearance of nanoparticles. One clear result from this analysis is that there is disagreement as to what constitutes low toxicity. This may be due to the lack of a reference nanoparticle system to use as a benchmark for comparison. Given that some standard in vitro testing methods will be established, it may be applicable to use gold nanoparticles as a reference nanoparticle for low toxicity. Gold nanoparticles have been reported to induce little toxicity, around 15% reduction in cell viability, at 200 mg mL 1.[28,29,31,84] While higher concentrations could elicit a cytotoxic effect, many substances become toxic at high concentrations. Therefore, it may be reasonable to conclude that the results from cytotoxicity testing of other nanoparticle types suggest low toxicity if those results are similar for gold-nanoparticle solutions containing relatively the same size particles at the same concentration. Although nanoparticle-induced cytotoxicity has been reported by several groups, it is important to keep in mind that in vitro results can differ from what is found in vivo and are not necessarily clinically relevant. In addition, the risk of any potentially toxic substance is not only a function of hazard but also chance of exposure. The nanoparticle

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concentrations needed for biomedical applications have not been optimized so the levels at which patients may be exposed are not certain. At the current stage in nanoparticle safety research, it would be premature to conclude, based on the present studies published, that nanoparticles are inherently dangerous. However, now that a basis has been established, future research should strive to address the deficiencies in current cytotoxicity testing and exploit the findings to engineer improved nanoparticles ultimately for clinical use.

Acknowledgements The authors would like to thank Joseph Chang and Ying Hu for their editorial assistance. This work was supported by the Center for Biological and Environmental Nanotechnology (NSF EEC-0118007 and EEC-0647452) and the Howard Hughes Medical Institute.

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