A Toxicologic Review Of Quantum DotsDocument Transcript
Environmental Health Perspectives Volume 114, Number 2, February 2006 Review
A Toxicologic Review of Quantum Dots: Toxicity Depends on Physicochemical
and Environmental Factors
Nicholas School of the Environment and Earth Sciences, Duke University, Durham, North Carolina, USA
matter; not all QDs are alike, and toxicity
As a growing applied science, nanotechnology has considerable global socioeconomic value, and the depends on multiple physicochemical as well
beneﬁts afforded by nanoscale materials and processes are expected to have signiﬁcant impacts on as environmental factors.
almost all industries and all areas of society. A diverse array of engineered nanoscale products and
processes have emerged [e.g., carbon nanotubes, fullerene derivatives, and quantum dots (QDs)], Applications of Quantum Dots
with widespread applications in ﬁelds such as medicine, plastics, energy, electronics, and aerospace. Quantum dots are semiconductor nanocrystals
With the nanotechnology economy estimated to be valued at $1 trillion by 2012, the prevalence of (~ 2–100 nm) with unique optical and electri-
these materials in society will be increasing, as will the likelihood of exposures. Importantly, the cal properties (Bruchez et al. 1998; Dabbousi
vastness and novelty of the nanotechnology frontier leave many areas unexplored, or underexplored, et al. 1997) currently applied in biomedical
such as the potential adverse human health effects resulting from exposure to novel nanomaterials. imaging and electronics industries. One of the
It is within this context that the need for understanding the potentially harmful side effects of these more valuable properties of QDs is their ﬂuo-
materials becomes clear. The reviewed literature suggests several key points: Not all QDs are alike; rescence spectrum, which renders them opti-
engineered QDs cannot be considered a uniform group of substances. QD absorption, distribution, mal fluorophores for biomedical imaging
metabolism, excretion, and toxicity depend on multiple factors derived from both inherent physico- (Alivisatos 2004; Chan et al. 2002). For
chemical properties and environmental conditions; QD size, charge, concentration, outer coating instance, ﬂuorescent QDs can be conjugated
bioactivity (capping material and functional groups), and oxidative, photolytic, and mechanical sta- with bioactive moieties (e.g., antibodies, recep-
bility have each been implicated as determining factors in QD toxicity. Although they offer poten- tor ligands) to target specific biologic events
tially invaluable societal beneﬁts such as drug targeting and in vivo biomedical imaging, QDs may and cellular structures, such as labeling neo-
also pose risks to human health and the environment under certain conditions. Key words: environ- plastic cells (Gao et al. 2004; Wu et al. 2003),
ment, human health, nanomaterials, nanosized particles, nanotechnology, nanotoxicology, quan- peroxisomes (Colton et al. 2004), DNA
tum dots, toxicology. Environ Health Perspect 114:165–172 (2006). doi:10.1289/ehp.8284 (Dubertret et al. 2002), and cell membrane
available via http://dx.doi.org/ [Online 20 September 2005] receptors (Beaurepaire et al. 2004; Lidke et al.
2004). Bioconjugated QDs are also being
explored as tools for site-speciﬁc gene and drug
In 1959 Richard Feynman’s seminal talk on enough material to provide every human delivery (Rudge et al. 2000; Scherer et al.
nanotechnology, “There’s Plenty of Room at worldwide with 300,000 particles each. 2001; Yu and Chow 2005) and are among the
the Bottom,” presented what was theoretically The nascent nature of the nanotechnology most promising candidates for a variety of
possible by manipulating matter at the atomic industry, however, leaves many areas unexplored, information and visual technologies; they are
and molecular scales. Today, nanotechnology or underexplored, such as the potential adverse currently used for the creation of advance ﬂat-
is an applied science, a rapidly growing indus- effects of engineered nanomaterials on human panel LED (light-emitting diode) displays and
try generating a diverse array of nanoscale health and the environment. Currently, the may be employed for ultrahigh-density data
materials and processes (e.g., carbon nano- paucity of toxicologic information and lack of storage and quantum information processing
tubes, fullerene derivatives, quantum dots). standardized testing protocols make assessment (Wu et al. 2004).
Manipulation of materials and processes on a of the adverse effects of engineered nanosized
nanometer scale is opening a world of creative materials on biologic systems difﬁcult (National Quantum Dot Physicochemical
possibilities, and the benefits afforded by Toxicology Program 2005; U.S. Environmental Properties
nanoscale technologies are expected to have Protection Agency 2003). The growing preva- Understanding the potential toxicity of QDs
substantial impacts on almost all industries and lence of nanomaterials in society, in conjunction requires a fundamental grasp of QD physico-
areas of society (e.g., medicine, plastics, energy, with their unique physicochemical properties chemical properties. Although naturally occur-
electronics, aerospace). It is such creative and the risk of unwanted/unanticipated expo- ring biogenic and anthropogenic nanosized
potential that renders nanotechnology of sig- sures, renders them of potential concern to particles abound in nature, engineered QDs
nificant social and economic value. With human health and the environment. It is within differ because of unique physicochemical prop-
approximately $8.6 billion invested in nano- this context that the need for understanding the erties that result from a combination of their
technology research and development world- potentially harmful side effects of these materials crystalline metalloid core structure/composition
wide in 2004 (Nordan et al. 2004), and a is becoming clear (Colvin 2003; Oberdörster and quantum-size conﬁnement, which occurs
projected nanotechnology economy valued at et al. 2005). when metal and semiconductor particles (QD
$1 trillion by 2012 (Service 2004), the preva- Reviewed here are novel nanomaterials cores) are smaller than their Bohr radii
lence of these materials in society is ensured, commonly referred to as quantum dots (QDs). (~ 1–5 nm). Structurally, QDs consist of a
and human exposures, as well as those of Although they offer potentially invaluable soci- metalloid crystalline core and a “cap” or “shell”
wildlife, are likely to increase. Currently, nan- etal beneﬁts such as drug targeting and in vivo
otechnology products are sold by more than biomedical imaging (Alivisatos 2004; Gao Address correspondence to R. Hardman, Duke
200 companies globally; some are widely used et al. 2004; Michalet et al. 2005; Roco 2003), University, Nicholas School of the Environment and
in commercially available products (e.g., elec- they may also, as the reviewed literature sug- Earth Sciences, LSRC A333, Durham, NC 27708
USA. Telephone: (919) 741-0621. Fax: (919) 684-
tronic, cosmetic) (Hood 2004; National gests, pose risks to human health and the envi- 8741. E-mail: email@example.com
Science Foundation 2004). For perspective on ronment under certain conditions. Current The author declares he has no competing ﬁnancial
the size of nanoscale products, consider that literature reveals that assessing QD exposure interests.
2 g of 100 nm-diameter nanoparticles contains routes and potential toxicity is not a simple Received 4 May 2005; accepted 19 September 2005.
Environmental Health Perspectives • VOLUME 114 | NUMBER 2 | February 2006 165
that shields the core and renders the QD for example, would refer to a QD with a CdSe individual QD physicochemical properties and
bioavailable (Figure 1). QD cores consist of a core and ZnS shell, and a CdSe/ZnS QD con- environmental conditions: QD size, charge,
variety of metal complexes such as semi- jugated with sheep serum albumin (SSA) would concentration, outer coating bioactivity (cap-
conductors, noble metals, and magnetic tran- be referred to as CdSe/ZnS–SSA. Controlling ping material, functional groups), and oxida-
sition metals. For instance, group III–V series the physicochemical properties during synthe- tive, photolytic, and mechanical stability have
QDs are composed of indium phosphate sis, which can be done with high precision, each been shown to be determining factors in
(InP), indium arsenate (InAs), gallium arsen- allows tailoring QDs for speciﬁc functions/uses. QD toxicity. For example, some QDs have
ate (GaAs) and gallium nitride (GaN) metal- Herein lies both their strength and weak- been found to be cytotoxic only after oxidative
loid cores, and group II–IV series QDs, of ness: QDs can be given highly speciﬁc bioac- and/or photolytic degradation of their core
zinc sulfide (ZnS), zinc–selenium (ZnSe), tivities by tailoring their coatings, for example, coatings. Last, because QD dosage/exposure
cadmium–selenium (CdSe), and cadmium– for diagnostic (e.g., molecular imaging) and concentrations reported in the literature vary in
tellurium (CdTe) cores (Dabbousi et al. 1997; therapeutic (e.g., drug delivery) purposes. their units of measurement (e.g., milligrams per
Hines and Guyot-Sionnest 1996). Synthetic Their potential weakness is in the very coating milliliter, molarity, milligrams per kilogram
routes to newer heavier structures (e.g., CdTe/ that makes them valuable: Compromise of the body weight, number of QDs per cell), corre-
CdSe, CdSe/ZnTe) and hybrids composed of coating can reveal the metalloid core, which lating dosage across current studies is challeng-
lead–selenium (PbSe) have also been established may be toxic either as a composite core (e.g., ing. Following is a review of in vitro and in vivo
(Kim et al. 2003). CdTe) or upon dissolution of the QD core to studies that describe the characteristics of QDs
Further assignation of biocompatible coat- constituent metals (e.g., Cd). Degradation of that may render them potentially toxic to verte-
ings or functional groups to the QD core–shell the QD coating may also result in reaction of brate systems.
can give QDs a desired bioactivity. Newly syn- the QD in undesirable/unanticipated ways Routes of exposure. Although the potential
thesized QDs are inherently hydrophobic in in vivo. Further, some QD coating materials adverse effects of nanomaterials on the envi-
nature and not biologically useful, given a have themselves been found to be cytotoxic, ronment and human health have recently been
hydrophobic cap formed on the metalloid core such as mercaptoacetic acid (MAA; discussed addressed by research initiatives organized
during their synthesis in organic solvents. To further below). From this, it can be seen that under the National Science Foundation and
render them biologically compatible/active, QD physicochemical properties are fundamen- the U.S. Environmental Protection Agency, no
newly synthesized QDs are “functionalized,” or tal to understanding QD toxicity; it is the sta- factual information is currently available
given secondary coatings, which improves water bility of QD core-coating bioactive complexes regarding routes of QD exposure. QD stabil-
solubility, QD core durability, and suspension that may render QDs potentially harmful, and ity, aerosolization, half-lives, and how they par-
characteristics and assigns a desired bioactivity. because QDs have been found to degrade tition into environmental media are currently
For example, QD cores can be coated with under photolytic and oxidative conditions, poorly understood. However, consideration of
hydrophilic polyethylene glycol (PEG) groups QD stability likely will ﬁgure signiﬁcantly in exposure routes may be extrapolated from what
to render QDs biocompatible and can be fur- commercialization of QD products. is known regarding materials of similar size and
ther conjugated with bioactive moieties to tar- physicochemical properties.
get speciﬁc biologic events or cellular structural Quantum Dot Toxicity Potential routes of QD exposure are envi-
features (described above). Hence, bonding Discussion of QD toxicity can be somewhat ronmental, workplace, and therapeutic or diag-
various molecular entities to the QD core func- confusing because of the diversity QDs being nostic administration. Workplace exposures
tionalizes QDs for speciﬁc diagnostic or thera- synthesized. To make a review of this topic sim- (e.g., engineers, researchers, clinicians) may
peutic purposes. Functionalization may be pler, it should be made clear that not all QDs result from inhalation, dermal contact, or inges-
achieved via electrostatic interactions, adsorp- are alike. Each individual type of QD possesses tion. For inhalation routes, an extensive body of
tion, multivalent chelation, or covalent bond- its own unique physicochemical properties, toxicologic research exists on other nanoscale
ing, important physicochemical features when which in turn determines its potential toxicity particles (e.g., asbestos, ultraﬁne particles) that
considering QD durability/stability and in vivo or lack thereof. In general, there are discrepan- may provide a foundation from which to
reactivity. In the literature, QD physicochemi- cies in the current literature regarding the toxic- approach QD inhalation studies. QDs vary in
cal characteristics are typically referred to as ity of QDs that can be attributed to several size, ranging from approximately 2.5 nm up to
“core–shell-conjugate” or vice versa. CdSe/ZnS, factors: the lack of toxicology-based studies, the 100 nm, depending on coating thickness, and
variety of QD dosage/exposure concentrations vary in their sites of deposition in pulmonary
Quantum dot reported in the literature, and the widely vary- tissues once aerosolized. For instance, QDs
ing physicochemical properties of individual < 2.5 nm may reach the deep lung and interact
QDs. Studies speciﬁcally designed for toxico- with the alveolar epithelium, whereas larger
logic assessment (e.g., dose, duration, frequency aerosolized QDs deposit in bronchial spaces.
of exposure, mechanisms of action) are few. However, under what conditions QDs
Many of the studies from which QD toxicity aerosolize and whether they form aggregates in
information is derived and that have been cited ambient air are not known (a salient review on
ZnS in reference to QD toxicity were performed by nanomaterials and inhalation exposures is given
nanotechnology researchers rather than toxicol- by Oberdorster et al. 2005). Inhalation expo-
ogists or health scientists. Most of the current sures may pose potential risks given that QDs
QD core–shell Bioactive coating
(e.g., CdSe/ZnS) (e.g., protein, peptide) studies reviewed here were designed to ask have been shown to be incorporated via endo-
questions concerning the physicochemical cytosis by a variety of cell types and may reside
5 nm properties of novel QD products such as ﬂuo- in cells for weeks to months. What risks expo-
rescence, detectability, stability, and cell label- sures via dermal absorption and accidental
Figure 1. QDs consist of a metalloid core and a
cap/shell that shields the core and renders the QD
ing efﬁcacy, not QD toxicity per se. ingestion may pose is currently unknown.
bioavailable. The further addition of biocompatible Importantly, and a potential source of con- What will likely be a signiﬁcant concern as
coatings or functional groups can give the QD a fusion in assessing QD toxicity, QD toxicity a route of exposure, given the social and eco-
desired bioactivity. depends on multiple factors derived from both nomic value of therapeutic/diagnostic QD
166 VOLUME 114 | NUMBER 2 | February 2006 • Environmental Health Perspectives
Toxicologic review of quantum dots
products, are exposures resulting from QD characteristics and the environmental media in with the QD capping material mercapto-
administration to humans for medicinal pur- which they partition. As mentioned, certain undecanoic acid (MUA) alone (without QD)
poses. These types of exposures are at present types of QDs have been shown to degrade for 12 hr caused severe cytotoxicity in murine
theoretical, as QD products are not currently under photolytic and oxidation conditions T-cell lymphoma (EL-4) cells at 100 µg/mL.
approved for therapeutic/diagnostic pur- (discussed further below). Treatment with cysteamine alone proved
poses; however, the potential for undesired/ Although little information currently exists weakly genotoxic at 100 µg/mL (12 hr).
unanticipated effects resulting from medicinal/ regarding routes of QD exposure, all routes Hence, in the Hoshino et al. (2004a) study,
diagnostic administration of these materials described are of potential concern given QDs cytotoxicity was attributed to QD capping
likely will ﬁgure prominently in the develop- have been shown to be incorporated into a material rather than the core metalloid com-
ment of medically based QD products. Their variety of cell types via endocytotic mecha- plex itself. It is, however, unlikely that the tox-
potential toxicity via administrative routes of nisms. Current research also suggests that there icity observed by Lovric et al. (2005) can be
exposure is highly dependent on a suite of vari- may be a risk of bioaccumulation of these solely attributed to the QD coatings (MPA
able and poorly understood factors: QD tox- materials (e.g., metals) in organs and tissues, as and cysteamine), as both size and charge and
ico- and pharmacokinetics, toxico- and QDs have been shown to reside in cells for the effects of NAC and BSA suggest other-
pharmacodynamics, and in vivo stability. It weeks to months and potentially may present wise. Brieﬂy, CdTe QD–induced cytotoxicity
may be that once QD kinetics and dynamics problems with body burdens. Common to all in the Lovric et al. study was shown to be
are characterized, the risks posed by these expo- routes of exposure is the issue of QD stability. partly dependent on QD size and may be due
sures may be mitigated through quality control Virtually nothing is known about QD metabo- to QD coating, intracellular reactions of the
mechanisms (e.g., consistency and reliability lism in vertebrate organisms or their routes of surface coatings, or intracellular degradation of
in volume production), as they currently are excretion. Although QDs have been shown to QDs to metalloid ions. QD-induced cytotoxi-
with pharmaceuticals. degrade under photolytic and oxidative condi- city was also observed by Shiohara et al.
Exposures through environmental media tions, degradation products have not been (2004): MUA-coated CdSe/ZnS QDs were
(contamination) are a potential route of con- identiﬁed/deﬁned in vivo except for the release observed to be cytotoxic to HeLa cells and pri-
cern primarily because of QD metalloid core of component core metals such as Cd and Se. mary human hepatocytes at concentrations of
compositions, and to some extent because of Finally, in considering routes of exposure, it is 100 µg/mL (MTT assay).
QD core coatings. Many QD core metals (e.g., important to remember that not all QDs are Several in vitro and in vivo studies have
Cd, Pb, Se) are known to be toxic to vertebrate alike; each individual QD type possesses its been cited in the literature as demonstrating a
systems at relatively low concentrations (parts own unique physicochemical properties that lack of evidence for QD-induced cytotoxicity
per million); however, understanding the risks will dictate its likely route of exposure. (Ballou et al. 2004; Dubertret et al. 2002;
posed by QDs in the environment will prove QD cytotoxicity. In vitro studies suggest Hoshino et al. 2004a; Jaiswal et al. 2003;
complex, as toxicity varies widely with the certain QD types may be cytotoxic. Lovric Larson et al. 2003; Voura et al. 2004).
chemical state of the metals, and environmental et al. (2005) found that CdTe QDs coated However, a few of the above studies do sug-
transformation/degradation and partitioning with mercaptopropionic acid (MPA) and cys- gest that QDs can affect cell growth and via-
will determine the level of the human health teamine were cytotoxic to rat pheochromo- bility. QD micelles, CdSe/ZnS QDs in a
hazard. Currently, nothing is known regarding cytoma cell (PC12) cultures at concentrations hydrophobic core of n-polyethyleneglycol
the stability of QDs in the environment, prod- of 10 µg/mL. Uncoated CdTe QDs were phosphatidylethanolamine (PEG–PE) and
uct lifetimes, or how these materials partition cytotoxic at 1 µg/mL. Cell death was charac- phosphatydilcholine, resulted in cell abnor-
into environmental media. Introduction of terized as chromatin condensation and mem- malities (viability, motility) when injected into
QDs into environmental media may occur via brane blebbing, symptomatic of apoptosis. Xenopus blastomeres at concentrations of 5 ×
waste streams from industries that synthesize or Cytotoxicity was more pronounced with 109 QDs/cell (~ 0.23 pmol/cell), whereas cells
use QDs and via clinical and research settings. smaller positively charged QDs (2.2 ± 0.1 nm) injected with 2 × 109 QDs/cell exhibited a
Consequently, disposal of QD materials and than with larger equally charged QDs (5.2 ± normal phenotype and were said to be statisti-
the risks of leakage and spilling during manu- 0.1 nm) at equal concentrations (cytotoxicity cally similar to uninjected embryos (Dubertret
facturing and transport are potential sources of determined by MTT [3-(4,5-dimethylthiazol- et al. 2002). Hence, QD cytotoxicity was dose
concern. Environmental exposures are a signiﬁ- 2-yl)-2,5-diphenyltetrazolium bromide] assay. dependent. Hoshino et al. (2004a) also found
cant source for several reasons: a) the environ- QD size was also observed to affect subcellular QD-induced cytotoxicity to be dose depen-
mental concentration of anthropogenic distribution, with smaller cationic QDs local- dent. EL-4 cells incubated (10 6 cells/well)
substances increases in direct proportion to izing to the nuclear compartment and larger with concentrations of 0.1, 0.2, and
their use in society, and QDs, given their wide cationic QDs localizing to the cytosol. The 0.4 mg/mL of CdSe/ZnS–SSA QDs exhibited
range of applications, may see substantial pro- mechanisms involved in cell death were not a dose–response relationship (24 hr). Cell via-
duction volumes; b) the half-lives of these known but were considered to be due to the bility decreased at QD concentrations above
materials may be quite long (months to possi- presence of free Cd (QD core degradation), 0.1 mg/mL, and almost all cells incubated
bly years); and c) environmental exposure will free radical formation, or interaction of QDs with 0.4 mg/mL were nonviable beyond 6 hr.
depend on where these materials partition with intracellular components leading to loss Interestingly, approximately 10% of EL-4 cells
(e.g., air, water, soil types). Because of the of function. The effect of QD-induced reac- retained QDs after 10 days of culture. The
diversity of physicochemical properties among tive oxygen species on cell death was assessed ﬂuorescence intensity (QDs) of cells gradually
varied types of QDs, it is likely that elucidating with N-acetylcysteine (NAC; a known decreased and was highly concentrated in
environmental partitioning will be difficult. inhibitor of Cd toxicity), bovine serum albu- endosomes, suggesting intracellular degrada-
These are important considerations given that min (BSA), and Trolox (a water-soluble vita- tion of QDs. Although cytotoxicity was
degradation of these materials in environmen- min E). Both NAC and BSA but not Trolox observed at 0.1 mg/mL in vitro, EL-4 cells
tal media, in the event they reach environmen- signiﬁcantly reduced CdTe QD toxicity, sug- incubated in 0.1 mg/mL SSA-conjugated
tal compartments, will undoubtedly occur, and gesting that QD-induced toxicity may be par- QDs, and subsequently injected into nude
their rates of decay are likely to be highly vari- tially induced by Cd. A similar study by mice (iv), were not observed to be toxic
able, depending on both QD physicochemical Hoshino et al. (2004a) found that treatment in vivo. In a subsequent study, Hoshino et al.
Environmental Health Perspectives • VOLUME 114 | NUMBER 2 | February 2006 167
(2004b) observed reversible DNA damage in potentially toxic “capping” material or intact QD conjugates was an issue during prepara-
WTK1 cells (comet assay). DNA damage was core metalloid complexes or resulting in disso- tion, and QD conjugation procedures were
noted at 2 hr of treatment with 2 µM lution of the core complex to QD core metal performed under little or no light to minimize
QD–COOH (carboxylic acid), but after 12 hr components (e.g., Cd, Se). Primary rat hepato- QD photolysis.
QD-induced DNA damage was efficiently cytes exposed to 62.5 µg/mL MAA–CdSe Intracellular and in vivo degradation.
repaired. QDs exhibited cell death, attributed to photo Given that studies indicate QDs may be sus-
QD-induced cytotoxicity was not observed lysis and oxidation of the QD coating. The ceptible to photolysis and oxidation, the ques-
in several in vivo and in vitro studies. In an hepatotoxicity of MAA–tri-n-octylphosphine tion arises as to their in vivo/intracellular
in vivo study employing mice, Ballou et al. oxide (MAA–TOPO)-capped CdSe QDs was oxidative stability, and a few studies suggest the
(2004) injected (iv) amphiphilic polyacrylic found to be dependent on QD processing con- possibility of intracellular degradation.
acid polymer-coated QDs (amp-QDs), and ditions and QD dose (Derfus 2004). If Although Hoshino et al. (2004a) noted that
amp-QDs conjugated to PEG-amine groups MAA–TOPO-capped CdSe QDs were CdSe/ZnS–SSA QDs could be observed in
(mPEG–QDs), at QD concentrations of exposed to air 30 min before MAA coating, a EL-4 cells for more than a week, with approxi-
20 pmol QD/g animal weight. Necropsy marked dose-dependent decrease in cell viabil- mately 10% of the cells retaining QDs after
showed no signs of necrosis at the sites of tissue ity was observed, from 98 to 21%, at 10 days in culture, the ﬂuorescent intensity of
deposition, and injected mice were viable for 62.5 µg/mL. Likewise, MAA–TOPO-capped cells was observed to gradually decrease and
133 days until the time of necropsy. No obvi- CdSe QDs exposed to ultraviolet (UV) light was highly concentrated in endosomes. QD
ous sign of QD breakdown in vivo was detected (15 mW/cm2) showed a dramatic dose-depen- fluorescence was possibly diminished by low
by electron microscopy (in vivo QD stability dent decrease in cell viability, with longer pH, oxidation of QD surface structures, or
was presumed to be due to the amphiphilic exposure times increasing toxicity (1–8 hr: intracellular factors adsorbed onto QD sur-
polymer coating). In another in vivo study, 91% decrease in cell viability). It was con- faces. Similarly, a substantial loss of QD ﬂuo-
Larson et al. (2003) observed no noticeable ill cluded that prolonged exposure of QDs to rescence over time was noted by Gao et al.
effects in mice injected (iv) with 20 nM and oxidative and photolytic environments can (2004) and Akerman et al. (2002) upon
1 µM solutions of CdSe/ZnS QDs (“ill effects” cause decomposition of MAA–TOPO-capped administration of QDs to live animals.
was not deﬁned). Voura et al. (2004), treating CdSe QD nanocrystals. Relatively high con- Although the exact origin of the loss of QD
B16F10 melanoma cells with dihydroxy- centrations of free Cd were observed in the ﬂuorescence was not clear, Gao et al. (2004)
lipoic acid (DHLA)–capped CdSe/ZnS QDs medium of QD solutions exposed to air stated that recent research in their group sug-
(5 µL/mL), noted no detectable difference in (126 ppm) and UV (82 ppm), with 6 ppm gested that QD surface ligands and coatings
growth between QD-treated and untreated nonoxidized QD core material (CdSe) remain- were slowly degraded in vivo, leading to surface
cells. Similarly, HeLa and Dictyostelium dis- ing in solution. QDs were also observed to defects and fluorescence quenching. They
coideum cells treated with 400–600 nM con- decompose in 1 mM hydrogen peroxide, noted, however, that QDs coated with a high-
centrations of CdSe/ZnS QDs capped with releasing free Cd ions (24 ppm). Derfus (2004) molecular-weight (100 kDa) copolymer and a
DHLA were observed to remain stably labeled concluded that QD toxicity was relative to grafted 8-carbon alkyl side chain demonstrated
for more than a week with no detectable effects environmental conditions; CdSe QD-induced greater in vivo stability than those with simple
on cell morphology or physiology (Jaiswal et al. toxicity was observed only above concentra- polymer and amphiphilic lipid coatings.
2003). Hanaki et al. (2003), exposing Vero tions exceeding 0.25 mg/mL and 1 hr of UV Similarly, Chen and Gerion (2004) attributed
cells to 0.24 mg/mL (2-hr exposure, cells exposure. Adding one or two monolayers of the lack of observable genotoxicity of QDs to a
washed and reincubated) CdSe/ZnS QDs ZnS to the QDs virtually eliminated cytotoxic- silica coating, which successfully prevented the
capped with MUA and coated with SSA, found ity due to oxidation (using the same protocol). interaction of Cd, Se, Zn, and sulfur with pro-
no effect of QDs on cell viability (MTT assay). Although ZnS capping material significantly teins and DNA in the nucleus.
Although it was noted that Vero cells without reduced ambient air oxidation, it did not fully Cytotoxicity of QD capping materials.
MUA–QD granules dominated the population eliminate photooxidation, with high levels of Relative to in vivo degradation, Hoshino et al.
during successive cell divisions, the authors free Cd observed in solution after 8 hr under (2004b) observed that QD surface coatings
stated they could not eliminate the possibility photooxidative conditions. BSA-coated ZnS- such as MUA may be detached under acidic
that MUA-capped QDs affect the cell viability capped QDs were also found to have reduced and oxidative conditions in endosomes and
when MUA-capped QDs are distributed in cytotoxicity compared with non-BSA-coated released into cytoplasm. To assess the toxicity
the cytosol, because they had not investigated ZnS-capped QDs at the same concentration of surface coatings, Hoshino et al. (2004b)
it. Last, Chen and Gerion (2004), using (0.25 mg/mL). Aldana et al. (2001) also assayed three QD coating materials (MUA,
CdSe/ZnS QDs conjugated with the viral observed photochemical instability in thiol- cysteamine, and thioglycerol) and two possi-
SV40 nuclear localization signal peptide, coated CdSe QDs, although not at relevant ble impurities (TOPO and ZnS) for cyto-
observed no cytotoxicity in HeLa cells trans- UV wavelengths (254 nm). It was noted, how- toxicity. Treatment of WTK1 cells with
fected with the peptide-coated QDs. The ever, that the photochemical stability of CdSe MUA alone for 12 hr resulted in cytotoxicity
authors observed that QD concentrations of nanocrystals was closely related to the thickness at doses > 100 µg/mL. DNA damage was
100 pmol/106 cells (~ 100 nM QD concentra- and packing of the ligand monolayer. observed at 50 µg/mL (2 hr of treatment).
tion) had minimal impact on cell survival Last, Staphylococcus aureus cultures exposed Cysteamine was observed to be weakly geno-
(measured by colonigenic assay). to transferrin-conjugated QDs showed marked toxic when cells were treated for 12 hr. The
Photolysis and oxidation: QD stability. increase in ﬂuorescence after 2 weeks of expo- toxicity of thioglycerol was negligible.
Possibly the most important aspect of QD tox- sure, attributed to intracellular oxidation of the Hoshino et al. (2004b), observing TPOP to
icity is their stability, both in vivo and during QDs, with a marked increase in intracellular Se be cytotoxic and genotoxic, stated that
synthesis and storage. Several studies suggest concentration. Kloepfer et al. (2003) observed removal of TPOP from the QD samples is
QD cytotoxicity to be due to photolysis or oxi- the internalization of both free Cd and Se important in reducing toxicity. Their ﬁndings
dation. Under oxidative and photolytic condi- in S. aureus cells but not internalization of provided evidence that QD-induced genotox-
tions, QD core–shell coatings have been found measurable transferrin-conjugated QDs. The icity was not caused by the QD core but by
to be labile, degrading and thus exposing authors also noted that photostability of the hydrophilic QD coatings.
168 VOLUME 114 | NUMBER 2 | February 2006 • Environmental Health Perspectives
Toxicologic review of quantum dots
Summary of QD toxicity. The studies Lovric et al. (2005) observed 5.2 nm cationic Importantly, where endocytic mechanisms
reviewed here suggest that QD toxicity CdTe QDs to localize throughout the cyto- have been observed in a variety of cell types,
depends on multiple factors derived from both plasm of N9 cells (murine microglial cell line) the question of systemic distribution arises.
the inherent physicochemical properties of but not in the nucleus. In contrast, 2.2-nm Although few in vivo studies exist, they suggest
QDs and environmental conditions. QD size, cationic CdTe QDs were observed to localize that QDs may be systemically distributed in
charge, concentration, outer coating bioactivity in the nuclear compartment within the same rodent animal models and accumulate in a
(capping material and functional groups), and time frame. Hence, in this instance, size, not variety of organs and tissues. EL-4 cells con-
oxidative, photolytic, and mechanical stability charge, was a determining factor in subcellular taining CdSe/ZnS–SSA QDs (via endocytosis)
are each factors that, collectively and individu- localization. It was noted, however, that were observed in the kidneys, liver, lung, and
ally, can determine QD toxicity. Of these because relatively unrestrained passage of spleen of mice up to 7 days after injection,
physicochemical characteristics, functional macromolecules up to 9 nm in diameter occurs with spleen and lung having the most accumu-
coating and QD core stability figure promi- through nuclear pores, the size of the QDs (2.2 lation (fluorescence) (Hoshino et al. 2004a).
nently and likely will be signiﬁcant factors in and 5.2 nm) cannot be the only explanation Similarly, Ballou et al. (2004), employing QD
assessing the risk of QD toxicity in real-world for the entry of smaller QDs (2.2 nm) into the coatings of different molecular weights
exposure scenarios. nucleus. Altering the bioactivity of the smaller [MW; methoxy-terminated PEG, MW 750
2.2 nm CdTe QDs by conjugation to BSA was (mPEG-750), carboxy-terminated PEG, MW
Absorption, Distribution, seen to limit its localization to the cytosol. 3,400 (COOH–PEG-3400), and ethoxy-ter-
Metabolism, and Excretion Where nonspeciﬁc endocytic mechanisms minated PEG, MW 5,000 (mPEG-5000)],
of Quantum Dots in Vivo have been shown to be instrumental in QD observed differential tissue and organ deposi-
Several studies have shown QDs may be sys- uptake by cells, receptor-mediated processes tion in mice in a time- and size (MW)-depen-
temically distributed and may accumulate in may also contribute to cellular internalization dent manner. For instance, mPEG-750 QDs
organs and tissues. Absorption, distribution, when QDs carry bioactive moieties specific and COOH–PEG-3400 QDs were cleared
metabolism, and excretion (ADME) charac- for cell receptor types or surface proteins. from circulation by 1 hr after injection,
teristics are, not surprisingly, highly variable Epidermal growth factor (EGF)–conjugated whereas mPEG-5000 QDs remained in circu-
for QDs because of the wide variation in QD CdSe/ZnS QDs proved to be highly speciﬁc lation for at least 3 hr. At 24 hr after injection,
physicochemical properties. QD size, charge, for the EGF receptor (erbB1), demonstrating mPEG-750 QDs were observed in the lymph
concentration, stability, and outer coating rapid internalization into endosomes of nodes, liver, and bone marrow. In contrast, sig-
bioactivity each contribute to not only the Chinese hamster ovary cells. The endocytic niﬁcantly less retention of COOH–PEG-3400
potential toxicity of a given QD but also to vesicles were observed to undergo a directed and mPEG-5000 QDs was observed in lym-
their ADME characteristics. Physicochemical linear motion mediated by microtubule- phatic tissue compared with bone marrow,
properties in conjunction with environmental associated motor proteins and vesicular fusion liver, and spleen. At 133 days, continued ﬂuo-
factors and QD stability (oxidative and photo- (Lidke et al. 2004). QDs coated with anti- rescence of mPEG-750 QDs was observed in
lytic lability) together are a paradigm in which Pgp showed good specificity for live HeLa the lymph nodes and bone marrow. A study by
ADME characteristics of QDs can be highly cells transfected with Pgp-EGFP (EGF pro- Akerman et al. (2002) yielded comparable
variable and difﬁcult to predict. tein), with no apparent nonspeciﬁc cell label- ﬁndings. Lung- and tumor-targeting peptide-
Several in vitro studies have shown QDs to ing (Jaiswal et al. 2003). Other studies yielded coated CdSe/ZnS QDs injected into mice (iv),
be incorporated via endocytic mechanisms by similar results: CdSe/ZnS QDs conjugated to regardless of the peptide used for the coating,
a variety of cell types. Mammalian (HeLa) and peptides speciﬁc for lung, vascular, and lym- accumulated in both the liver and spleen in
Dictyostelium discoideum (AXS) cells were phatic tissues exhibited speciﬁcity for labeling addition to the targeted respiratory tissues.
observed to incorporate avidin and DHLA- cell membranes of their targeted tissue types Interestingly, additionally coating CdSe/ZnS
conjugated CdSe/ZnS QDs via endocytosis (Akerman et al. 2002). Dahan et al. (2003) QDs with PEG (a polymer known to mini-
(Jaiswal et al. 2003), and rat primary hepato- found that QDs conjugated with glycine mize molecular interactions and improve col-
cytes were observed to incorporate receptor (GlyR1) ligands exhibited speciﬁcity loidal solubilities) nearly eliminated the
CdSe–MAA QDs (Derfus 2004). Hoshino for endogenous GlyR1 subunits on cultured nonspeciﬁc uptake of QDs into the liver and
et al. (2004a) observed adherence of spinal neurons. Last, prostate-specific mem- spleen. An in vivo study employing Xenopus
CdSe/ZnS–SSA QDs to the surface of EL-4 brane antigen–conjugated QDs specifically embryos revealed that QDs, once internalized
cells, with subsequent endocytosis and increase targeted prostate tumors in mice (Gao et al. by cells, subsequently may be transferred to
in cytosolic QD concentration in a time- 2004), and QDs complexed with a viral daughter cells on cell division. Dubertret et al.
dependent manner (minutes to hours). Other (SV40) nuclear localization signal peptide (2002), injecting a CdSe/ZnS micelle
studies have shown similar nonspeciﬁc uptake. were observed to readily enter the nuclear (PEG–PE and phosphatidylcholine) conju-
Hanaki et al. (2003), exposing Vero cells to compartment of human HeLa cells (Chen gated with an oligonucleotide into Xenopus
CdSe/ZnS–MUA QDs coated with SSA, and Gerion 2004). embryos, observed QD labeling of all embry-
observed endosomal/lysosomal localization of In invertebrate cell types, Kloepfer et al. onic cell types, including somites, neurons,
the QDs near the perinuclear region 5 days (2003) observed transferrin-conjugated CdSe axonal tracks, ectoderm, neural crest, and
after exposure. Parak et al. (2002) observed QDs to enter S. aureus bacterial cells, which endoderm. The internalized QDs were local-
endocytosis and vesicular storage and trans- do not endocytose but rely on membrane ized to both the cytosol and nuclear envelope
port of CdSe/ZnS silicon dioxide–coated QDs transporters. The transferrin-conjugated QDs and were transferred to daughter cells on cell
to the perinuclear region in human mammary also showed clear internal labeling in the fungi division. The progeny of the QD-injected cells
tumor cells, and an in vivo study by Dubertret Schizosacharomyces pombe and Penicillium were shown to contain ﬂuorescent QDs after
et al. (2002) demonstrated endocytosis and chrysogenum. No internal labeling of nonpath- several days of development.
active transport of QD micelles (phospholipid ogenic staphylococci and micrococci was Metabolic processes and excretory mecha-
block-copolymer) in Xenopus embryos. observed, and it was suggested that transferrin- nisms involved in the elimination of QDs,
In one instance, QD size was shown to be mediated transport processes were involved in as well as in vivo bioactivity, remain poorly
a determining factor in subcellular distribution. cell-speciﬁc uptake. understood and have not been well studied.
Environmental Health Perspectives • VOLUME 114 | NUMBER 2 | February 2006 169
In vivo studies suggest that, regardless of the depending on QD size, QD core–shell compo- systemic distribution, and dependent on QD
speciﬁcity of the QD, vertebrate systems tend nents, and the bioactivity of conjugated or physicochemical properties. Such variables,
to recognize QDs as foreign, with elimination otherwise attached functional groups. Size determined by the unique physicochemical
of the materials through the primary excre- alone can markedly affect distribution kinetics, properties of individual QD types, will prove
tory organs/systems: the liver, spleen, and and QD surface coating can govern serum life- signiﬁcant in developing characterization pro-
lymphatic systems. However, this a rough time and pattern of deposition (Ballou et al. tocols for QD toxicity screening, given the
generalization, and discrepancies in the litera- 2004; Lovric et al. 2005). QDs lacking special- nonuniformity in size, QD functional coatings,
ture exist. For instance, subcutaneous injec- ized functional groups or speciﬁcity have been core–shell complexes, and outer coating photo-
tion of CdSe/ZnS–PEG-coated QDs in mice shown to be incorporated via endocytic mecha- lytic and oxidative stability.
showed clearance of the QDs from the site of nisms by a variety of cell types, both in vivo
injection, with accumulation of QDs in and in vitro. In contrast, QDs bearing natural Correlation of Quantum Dot
lymph nodes. In contrast, Akerman et al. ligands speciﬁc for cell receptors and cell mem- Concentrations and Toxicity
(2002) observed that modification of lung- brane proteins have been shown to be speciﬁc Quantum dot dosage/exposure concentrations
and tumor-targeting peptide-conjugated for given cell membrane proteins/receptor reported in the literature vary widely in units of
CdSe/ZnS QDs with a PEG coating nearly types. Several studies have shown nonspeciﬁc measurement (e.g., micrograms per milliliter,
eliminated nonspecific elimination of QDs QDs to adhere to cell surfaces, possibly molarity, milligrams per kilogram body weight,
via the lymphatic system. through interactions of QD with glycoproteins QDs per cell), and correlating dosage across
The above studies suggest that QDs may and glycoplipids in cell membranes. Although studies is currently challenging. Further, some
see variable systemic distribution dependent on many studies indicate endocytic processes and QDs were found to be cytotoxic only after
individual QD physicochemical properties. intracellular vesicular trafﬁcking and storage of degradation of their core coatings both in vivo
Although studies are limited, QD tissue/organ QDs, the exact mechanisms remain to be elu- and/or in vitro. Nevertheless, reported values of
distribution seems to be multifactorial, cidated. Subcellular localization is variable, like dose–response relationships can be assessed in
Table 1. Review articles summary of QD types, exposure concentrations, experimental conditions, and observed toxicity.
QD Model administration QD concentration Exposure duration Toxicity Reference
CdSe/ZnS–SSA EL-4 cells 1 × 106 cells/well 0.1–0.4 mg/mL 0–24 hr Cytotoxic: 0.1 mg/mL altered cell growth; Hoshino et al.
most cells nonviable at 0.4 mg/mL 2004a
CdSe/ZnS–SSA EL-4 cells 200 µL cell 0.1 mg/mL QDs per 2 hr to 7 days No toxicity in mice in vivo Hoshino et al.
suspension injected 5 × 107 cells 2004a
(iv) into mice
CdSe/ZnS WTK1 cells 5 × 104 cells/mL 1–2 µM 12 hr 2 µM QD–COOH induced DNA damage Hoshino et al.
conjugates: NH2, at 2 hr 2004b
OH, OH/COOH, DNA repair on prolonged incubation
H2/OH, MUA, COOH (12 hr)
CdSe/ZnS–MUA Vero, HeLa, and 100 µL QDs/3 × 104 0–0.4 mg/mL 24 hr Cytotoxic: 0.2 mg/mL, Vero; Shiohara et al.
primary human cells 0.1 mg/mL, HeLa; 2004
hepatocytes 0.1 mg/mL, hepatocytes;
CdTe Rat 1 × 105 cells/cm2 0.01–100 µg/mL 2–24 hr 10 µg/mL cytotoxic Lovric et al.
CdSe–MAA, Primary rat 62.5–1,000 µg/mL 1–8 hr Cytotoxic: 62.5 µg/mL cytotoxic under Derfus 2004
TOPO QDs hepatocytes oxidative/photolytic conditions
No toxicity on addition of ZnS cap
QD micelles: Xenopus 5 × 109 QDs/cell 1.5–3 nL of 2.3 µM QDs Days 5 × 109 QDs/cell: cell abnormalities, Dubertret et al.
CdSe/ZnS QDs in blastomeres (~ 0.23 pmol/cell) injected, ~ 2.1 × 109 to altered viability and motility 2002
(PEG–PE) and 4.2 × 109 injected No toxicity at 2 × 109 QDs/cell
CdSe/ZnS Mice 200-µL tail vein Injections; ~ 180 nM QD, 15 min cell No signs of localized necrosis at the Ballou et al.
amp-QDs, and injection ~ 20 pmol QD/g animal incubations, sites of deposition 2004
mPEG QDs weight 1–133 days
CdSe/ZnS–DHLA Dictyostelium 400–600 nM 45–60 min No effects on cell growth Jaiswal et al.
discoideum and 2003
Avidin-conjugated HeLa cells 0.5–1.0 µM 15 min No effect on cell growth, development Jaiswal et al.
CdSe/ZnS QDs 2003
CdSe/ZnS– Mice Tail vein injection 60 µM QD/g animal weight, Not given Mice showed no noticeable ill effects Larson et al.
amphiphilic micelle 1 µM and 20 nM ﬁnal QD after imaging 2003
CdSe/ZnS–DHLA Mice, B16F10 cells 5 × 104 B16F10 cells 100 µL of B16F10 cells used 4–6 hr cell No toxicity observed in cells or mice Voura et al.
QDs with 10 µL QDs for tail vein injection, incubation, mice 2004
(~ 10 pmol), tail vein ~ 2 × 105 to 4 × 105 cells sacriﬁced at
(iv) injection injected 1–6 hr
CdSe/ZnS–MUA QDs; Vero cells 0.4 mg/mL 0.24 mg/mL 2 hr 0.4 mg/mL MUA/SSA–QD complexes Hanaki et al.
QD–SSA complexes did not affect viability of Vero cells 2003
CdSe/ZnS HeLa cells 1 × 106 cells 10 pmol QDs/1 × 105 cells 10 days (cell 10 nM QD had minimal impact on cell Chen and Gerion
(~ 10 nM) culture) survival 2004
170 VOLUME 114 | NUMBER 2 | February 2006 • Environmental Health Perspectives
Toxicologic review of quantum dots
Table 1. Of note, those studies that observed and in elucidating the mechanisms of action of Derfus A. 2004. Probing the cytotoxicity of semiconductor quan-
no cytotoxicity generally employed protocols QDs, as well as their environmental transport tum dots. Nano Lett 4:11–18.
Dubertret B, Skourides P, Norris DJ, Noireaux V, Brivanlou AH,
that used short-term acute exposures, where and fate. Only with this knowledge can the Libchaber A. 2002. In vivo imaging of quantum dots encapsu-
cells were in contact with QDs for 15 min to biocompatibility of QD technology with the lated in phospholipid micelles. Science 298(5599):1759–1762.
8 hr (e.g., Hanaki et al. 2003; Jaiswal et al. social and ecologic systems in which these Fan TW, Teh SJ, Hinton DE, Higashi RM. 2002. Selenium biotrans-
formations into proteinaceous forms by foodweb organisms
2003; Voura et al. 2004). For instance, studies materials will be applied be achieved, and can of selenium-laden drainage waters in California. Aquat
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