Nanoparticles can induce changes in the intracellular
metabolism of lipids without compromising cellular
viability
Ewa Przybytkowski, Maik Behrendt, David Dubois and Dusica Maysinger
Department of Pharmacology and Therapeutics, McGill University, Montre
´
al, Canada
Introduction
Quantum dots (QDs) are colloidal semiconductor
nanoparticles (NPs) with unique luminescence charac-
teristics and wide biological and industrial applications
[1,2]. They could become attractive tools for imaging
in basic research and, eventually, in medicine [3]. How-
ever, some QDs can be harmful to cells, particularly if
Keywords
fat oxidation; hypoxia; lipid droplets;
nanoparticles; quantum dots
Correspondence
D. Maysinger, Department of Pharmacology
and Therapeutics, McGill University, 3655
Promenade Sir-William-Osler, Montre
´
al, QC,
Canada, H3G 1Y6
Fax: (514) 398 6690
Tel: (514) 398 1264
E-mail:
(Received 5 June 2009, revised 17 August
2009, accepted 24 August 2009)
doi:10.1111/j.1742-4658.2009.07324.x
There is growing concern about the safety of engineered nanoparticles,

binding protein-1.
6204 FEBS Journal 276 (2009) 6204–6217 ª 2009 The Authors Journal compilation ª 2009 FEBS
their surface is not fully protected or if they degrade
within the biological environment. We have studied
the effects of QDs on living cells and have reported
their internalization, the intracellular production of
reactive oxygen species (ROS) and the damage to mul-
tiple cellular sites induced by various QDs [4–6]. We,
as well as others, have shown that the degree of inter-
nalization of QDs and various other NPs is dependent
on their size, surface charge, concentration in the med-
ium and duration of exposure [4,7–11]. We have also
shown the release of free cadmium from QDs contain-
ing a CdTe core [6]. However, this could not explain
fully their harmful effects. We have postulated that
intracellular ROS formation or interactions with cellu-
lar structures (mitochondria in particular) could con-
tribute to the observed cytotoxicity [4–7]. Although
coating of NPs with ZnS or polyethylene glycol (PEG)
commonly prevents some undesirable effects [6,12], the
long-term stability of these materials in the biological
milieu is not well understood [13].
The purpose of this study was to investigate the
more subtle effects of QDs which could occur without
any evident morphological cellular damage or cell
death. In particular, we explored QD-induced changes
in lipid metabolism. Using well-characterized in vitro
cell model systems [primary mouse hypothalamic
cultures and pheochromocytoma cells (PC12)], we have
provided evidence that poorly fluorescent CdTe NPs,

potential for broad application in the assessment of
the metabolic effect described in this study. Given that
high levels of cytoplasmic LDs present in nonadipose
tissues are considered to be harmful, such assays
would expand the current platform of tests for the
determination of nanomaterial biocompatibility. Excess
fat in nonadipose cells may be involved in several
human pathologies, such as fatty liver, obesity, athero-
sclerosis and type 2 diabetes, and may contribute to
the development of insulin resistance and lipotoxic tis-
sue damage [17]. The accumulation of neutral lipids in
cytoplasmic LDs occurs following exposure to mito-
chondrial toxins [18], during chronic viral infections
[19,20], in response to protease inhibitors [21] and
during hypoxia [22–24].
In this study, we also showed that exposure to
colloidal semiconductor NPs leads to an increased
expression of hypoxia-inducible transcription factor-
1a (HIF-1a). The family of hypoxia-inducible tran-
scription factors (HIFs) regulates the adaptation to
hypoxic conditions, which is critical for cell survival
during decreased availability of oxygen in tissues
[25,26]. Hypoxia and hypoxia-related signaling have
been associated with major pathologies, such as car-
diovascular disease, stroke and cancer [27]. The sig-
naling for hypoxia was of interest in this study
because QDs, which are redox-active NPs, can release
Cd
2+
and induce the intracellular formation of ROS,

brain structure that is not completely protected by the
blood–brain barrier and thus can be accessed by xeno-
biotics and NPs [33]. Various other tissues, such as
liver, kidney, spleen and bone marrow, are known sites
of NP accumulation in vivo [34,35]. The evaluation of
fat oxidation in these tissues could complement current
toxicological assays for the safety screening of NPs
and other nanomaterials.
Results
Short- and long-term effects of uncoated CdTe
QDs on mouse primary hypothalamic cultures
The effects of green, positively charged CdTe QDs with
cysteamine surfaces [4,6,7] were investigated in primary
mouse hypothalamic cultures. Mixed neural cultures
were obtained from 5-day-old animals, and experiments
were initiated at day (in vitro) 8 (DIV 8), when neural
cells were fully differentiated (Fig. 1A). A few neurons
with small cell bodies were visible on top of supporting
glia. In this study, we focused on glial cells.
To examine the short-term effects, the cultures were
exposed to QDs (0–20 lgÆmL
)1
) for a period of 24 h
in serum-free Neurobasal A medium with supplements.
Cells responded to QD treatment by forming multiple
cytoplasmic LDs (Fig. 1B–D). The number of LDs
increased with increasing concentration of QDs
(Fig. 1E). Within this time period, cells exposed to
relatively low concentrations of QDs (5 lgÆmL
)1

)1
(D) of CdTe QDs. (E) The mean number of lipid droplets per microscopic field evaluated as
described in Materials and methods. The data represent the mean and SEM from two independent experiments (n = 10). (F, G) Photomicro-
graphs of cultures stained with Oil Red O at DIV 12. (F) Control untreated cultures. (G) Cultures treated with 5 lgÆmL
)1
of CdTe QDs for
4 days (DIV 8 to DIV 12). The scale bars correspond to 50 l m.
Nanoparticle-induced metabolic changes E. Przybytkowski et al.
6206 FEBS Journal 276 (2009) 6204–6217 ª 2009 The Authors Journal compilation ª 2009 FEBS
LD formation triggered by CdTe QDs in glial cells
depends on the de novo synthesis of lipids and
phosphoinositide 3-kinase (PI3K) signaling
Cytoplasmic LDs contain neutral lipids, including
triacylglycerols, diacylglycerols and cholesterol esters.
To verify whether de novo fat synthesis is involved in
LD formation during treatment with QDs, hypotha-
lamic cultures were exposed to CdTe QDs (5 lgÆmL
)1
)
in the presence of the fatty acid synthase (FAS) inhibi-
tor cerulenin (5 lgÆmL
)1
). FAS is responsible for the
synthesis of FFAs, which can then be esterified to
form components of cell membranes or lipids stored in
LDs. Cerulenin, an antifungal antibiotic isolated from
Cephalosporium caerulens, irreversibly binds to FAS,
thereby inhibiting its activity [36]. Treatment with
5 lgÆmL
)1

E
Fig. 2. Formation of LDs induced by QDs in
glial cells from primary mouse hypothalamic
cultures depends on the de novo synthesis
of lipids. (A–D) Representative photomicro-
graphs of primary mouse hypothalamic
cultures stained with Oil Red O. (A) Control
cultures at DIV 9. Cultures treated for 24 h
with 5 lgÆmL
)1
of cerulenin (B), 5 lgÆmL
)1
of CdTe QDs (C) and both 5 lgÆmL
)1
of
CdTe QDs and 5 lgÆmL
)1
of cerulenin (D).
The scale bars correspond to 50 lm. (E) The
quantification of the lipid droplet number
from (A) to (D). The data represent the
mean and SD from three independent fields.
***P < 0.001.
E. Przybytkowski et al. Nanoparticle-induced metabolic changes
FEBS Journal 276 (2009) 6204–6217 ª 2009 The Authors Journal compilation ª 2009 FEBS 6207
inhibited the formation of LDs in QD-treated cells
(Fig. 3C–E), indicating that the formation of LDs was
dependent on signaling via PI3K. Glial cell survival
was not markedly compromised by the inhibition of
PI3K signaling with LY294002 in a fully supplemented

B
C
D
E
Fig. 3. Formation of LDs induced by CdTe
QDs in glial cells from primary mouse hypo-
thalamic cultures depends on the PI3K
signaling pathway. (A–D) Representative
photomicrographs of primary mouse hypo-
thalamic cultures stained with Oil Red O.
(A) Control cultures at DIV 11. (B) Cultures
treated with 50 l
M LY294002 for 2 days
(DIV 9 to DIV 11). (C) Cultures treated with
5 lgÆmL
)1
of CdTe QDs for 3 days (DIV 8 to
DIV 11). (D) Cultures treated with 5 lgÆmL
)1
of CdTe QDs and 50 lM LY294002 for
3 days (DIV 8 to DIV 11). The scale bars
correspond to 50 lm. (E) The quantification
of the lipid droplet number from (A) to (D).
The data represent the mean and SD from
three independent fields. **P < 0.01.
Nanoparticle-induced metabolic changes E. Przybytkowski et al.
6208 FEBS Journal 276 (2009) 6204–6217 ª 2009 The Authors Journal compilation ª 2009 FEBS
As QDs are redox-active NPs (effective electron
donors and acceptors) which can release Cd
2+

Fig. 4. Uncoated and coated QDs trigger the formation of LDs in PC12 cells in a growth factor-dependent manner without compromising
cellular viability. Pheochromocytoma PC12 cells were treated with QDs in fully supplemented medium (A, C–E, I and K) and in serum-free
medium (B, F–H, J). (A, B) Percentage cell survival relative to control (determined with Alamar blue assay) after exposure to two different
concentrations of QDs in fully supplemented medium (A) and in serum-free medium (B). Data represent the mean and SEM from two inde-
pendent experiments. Representative photomicrographs of cells stained with Oil Red O. (C, F) Control untreated cells. (D, G) Cells treated
with 20 lgÆmL
)1
of CdTe QDs for 24 h. Cells treated with 20 lgÆmL
)1
of CdSe ⁄ ZnS QDs (E, H) and with 100 lM CoCl
2
(I, J) for 24 h. The
scale bars correspond to 50 lm. ***P < 0.001; *P < 0.05. (K) Number of lipid droplets found in PC12 cells treated with two different con-
centrations of QDs in fully supplemented medium. The data represent the mean and SEM from two independent experiments.
E. Przybytkowski et al. Nanoparticle-induced metabolic changes
FEBS Journal 276 (2009) 6204–6217 ª 2009 The Authors Journal compilation ª 2009 FEBS 6209
QDs can induce signaling implicated in the
response to hypoxia and can reduce the rate of
fat oxidation in PC12 cells
We hypothesized that QDs and CoCl
2
induce the accu-
mulation of lipids in cytoplasmic LDs by the activa-
tion of HIF-1a. Transcription factors involved in
signaling for hypoxia are known to stimulate glucose
metabolism [26] and to promote the accumulation of
lipids [23,24]. PC12 cells were incubated with QDs or
CoCl
2
for 24 h, and the expression of HIF-1a protein

and by 20% after treatment with 20 lgÆmL
)1
of
CdSe ⁄ ZnS QDs. In the absence of serum, the effect of
QDs was even more pronounced, as fat oxidation
decreased by 40–50% after treatment with CdTe QDs
and by 19–36% after treatment with CdSe ⁄ ZnS QDs
(Fig. 6A). These results strongly suggest that: (a) QDs
can induce changes in cellular lipid metabolism with-
out affecting cellular viability; and (b) QD-induced
A
B
Fig. 5. Uncoated and coated QDs increase the expression of HIF-
1a in PC12 cells. Pheochromocytoma PC12 cells were treated with
QDs for 24 h in fully supplemented medium (A) and in serum-free
medium (B). After treatment, HIF-1a protein levels were analyzed
by western blot.
A
B
Fig. 6. Uncoated and coated QDs decrease the rate of b-oxidation
of fatty acids in PC12 cells. Pheochromocytoma PC12 cells were
treated with QDs for 24 h in fully supplemented medium (A) and in
serum-free medium (B). After treatment, fatty acid oxidation was
measured using [1-
14
C]palmitate as a substrate. The data represent
the mean and SEM from two independent experiments. *P < 0.05;
**P < 0.01; ***P < 0.001.
Nanoparticle-induced metabolic changes E. Przybytkowski et al.
6210 FEBS Journal 276 (2009) 6204–6217 ª 2009 The Authors Journal compilation ª 2009 FEBS

PI3K ⁄ Akt ensures the supply of substrate for lipid
synthesis and enhances the activities of lipogenic
enzymes, setting the stage for the accumulation of
lipids. Consistent with this, we have observed a small
number of LDs in glial cells and in PC12 cells grown
in a fully supplemented medium (control conditions).
Interestingly, when glial cells from primary mouse
hypothalamic cultures were exposed to QDs, the num-
ber and ⁄ or size of LDs increased markedly. LDs have
been recognized recently as ubiquitous dynamic organ-
elles, which communicate with other cellular compart-
ments and participate in important functions, such as
transport and communication between different vesi-
cles and compartments inside the cell [47]. Some of
these functions are probably dependent on the pres-
ence of specific proteins on the surface of droplets [48].
It has been shown previously that QDs can cause
distortion and ⁄ or damage to cellular membranes,
which are composed mostly of complex lipids [49].
This could result in the release of FFAs, which then
would be available for esterification and the formation
of triacylglycerols (the main components of LDs). We
considered such a possibility; however, the inhibition
of PI3K with LY294002 and the inhibition of FAS
with cerulenin caused the disappearance of droplets
and prevented the formation of new droplets during
exposure to NPs when trophic factors were present in
the medium. These findings indicate that glial cells
(from primary mouse hypothalamic cultures) accumu-
lated newly synthesized lipids when exposed to NPs.

droplets. Activation of the PI3K ⁄ Akt pathway by trophic ⁄ growth
factors creates the metabolic state, in which cells are able to syn-
thesize FFAs. These newly synthesized FFAs are stored in LDs in
the form of triacylgycerols or are oxidized in mitochondria. We
hypothesize that QDs interfere with this processes by down-regu-
lating fat oxidation. As a result, more FFAs become available for
esterification and storage in LDs. The PI3K ⁄ Akt signaling pathway
stimulates lipogenesis via SREBP-1. FAS is the enzyme responsible
for the synthesis of palmitate, the precursor of FFAs. The inhibition
of signaling by trophic factors, removal of trophic factors or inhibi-
tion of fat synthesis result in the down-regulation of lipogenesis
and the disappearance of LDs. QDs can also induce the up-regula-
tion of HIF-1a, most probably via the production of ROS. QDs and
the hypoxia mimetic CoCl
2
down-regulate the oxidation of FFAs
and induce the accumulation of lipids. Further studies are needed
to clarify the relationship between HIF-1a-mediated signaling and
the metabolism of lipids in cells exposed to nanoparticles.
E. Przybytkowski et al. Nanoparticle-induced metabolic changes
FEBS Journal 276 (2009) 6204–6217 ª 2009 The Authors Journal compilation ª 2009 FEBS 6211
caveolae, which consist of small invaginations in
plasma membranes, containing the protein caveolin
and a particular lipid content, could contribute to the
accumulation of lipids. It has been shown recently that
caveolae can act as regulatory sites for the synthesis
and trafficking of triacylglycerols [50]. Moreover, it is
well documented that NGF signaling via the trk recep-
tor in PC12 cells involves caveolin [51], and that cave-
olin associates with LDs [52]. Therefore, NPs may

stearoyl-coenzyme A desaturase-1 (SCD-1) [22]. Hyp-
oxic conditions also enhanced the synthesis of neutral
lipids in human macrophages via the up-regulation of
lipogenesis (increase in SCD-1 activity) and also via
the down-regulation of the b-oxidation of fatty acids
[23]. Hypoxia has also been shown to induce the for-
mation of LDs in various tumors [24]. Both NP and
CoCl
2
treatment induced the up-regulation of HIF-1a
in PC12 cells. However, the up-regulation of HIF-1a
by NP exposure was detectable only in serum-free con-
ditions, whereas LDs were produced mainly in the
presence of serum. These findings suggest that the
up-regulation of HIF-1a may not be necessary for
the accumulation of lipids induced by NPs in trophic
factor-supplemented medium, or that changes in its
levels were too subtle to be detected by western blot-
ting [53]. Further studies are needed to clarify the rela-
tionship between HIF-1a-mediated signaling and the
metabolism of lipids in cells exposed to NPs. HIF-
mediated signaling not only induces the expression of
genes involved in cellular adaptation to low oxygen
[26], but also alters the cell’s response to various stres-
sors [27]. In this regard, it could be predicted that
exposure to NPs could also modify the cellular
responses to various xenobiotics.
The lipid accumulation induced in PC12 cells by two
types of NP was concentration dependent and largely
preceded the manifestation of cell death. QDs are

ment of NP safety are based on viability assays, the
peroxidation of membrane lipids, the depletion of
cellular glutathione or the secretion of inflammatory
mediators [35]. The results from the present study sug-
gest that metabolic measurements, such as the determi-
nation of changes in fat oxidation, could be used as an
additional sensitive test for the evaluation of NP
safety ⁄ biocompatibility. Metabolic measurements,
especially those related to mitochondrial function and
nonhypoxic induction of metabolic effects normally
observed with hypoxia, such as changes in oxygen
Nanoparticle-induced metabolic changes E. Przybytkowski et al.
6212 FEBS Journal 276 (2009) 6204–6217 ª 2009 The Authors Journal compilation ª 2009 FEBS
consumption, are already being considered as impor-
tant criteria for the evaluation of drug-induced toxicity
[62–64].
The accumulation of lipids in cytoplasmic LDs in
glial cells from primary mouse hypothalamic cultures
has not been reported previously. Changes in lipid
metabolism induced by metallic ions and NPs in hypo-
thalamus, liver, kidney, spleen or bone marrow could
compromise organismal homeostasis. Thus, metabolic
measurements, such as the determination of the rate of
fat oxidation, in these organs or tissues, which are also
known sites of NP accumulation in vivo [34,35,65],
could provide useful measures of NP biocompatibility
and safety.
Materials and methods
Materials
The sources of the reagents were as follows: phenol red-free

by Lovric et al. [7]; they contain a CdTe core, their dia-
meter is 2.8 nm and they have an emission maximum at
535 nm. There was no ZnS cap on the CdTe core and
cysteamine was attached directly to the surface [7].
CdSe ⁄ ZnS NPs were prepared as described by Hoshino et al.
[44]; they contain a CdSe core, their diameter is 2.4 nm and
they have an emission maximum of 518 nm. The CdSe core
was capped with one layer of ZnS to which cysteamine was
attached, and thus the particle size, measured by the dynamic
light scattering method, was about 10 nm [44]. QDs were
added to the cellular media in different amounts and for
different lengths of time, as indicated in the figures.
Primary mouse hypothalamic cultures
All experiments were conducted with the approval of the
McGill University Animal Care Committee. Mouse (strain
129T2 ⁄ SV) hypothalamus was obtained by dissection at
postnatal day 5 and freed from the meninges. Tissue pooled
from at least six animals was stored in ice-cold sterile
Ca
2+
⁄ Mg
2+
NaCl ⁄ P
i
. The tissues were dissociated mechan-
ically by gentle pipetting using sterile 1 mL pipette tips,
and digested with 0.25% trypsin ⁄ EDTA at 37 °C for
10 min. Dissociated cells were resuspended in DMEM med-
ium containing 10% fetal bovine serum and PSN cocktail.
Cells in suspension were seeded onto polyornithine- and

Przybytkowski et al. [67]. Briefly, cells were washed with
NaCl ⁄ P
i
and incubated with Oil Red O working solution for
15 min at room temperature. The cells were then washed
once with NaCl ⁄ P
i
, and fixed with 10% formalin for 25 min.
Subsequently, the cells were washed again with NaCl ⁄ P
i
,
stained for 2 min with Harris hematoxylin, washed with
distilled water and mounted on microscopic slides using
Aqueous Mount mounting medium. Photomicrographs were
taken from representative fields using an Olympus BX2
microscope (Olympus America Inc., Center Valley, PA,
E. Przybytkowski et al. Nanoparticle-induced metabolic changes
FEBS Journal 276 (2009) 6204–6217 ª 2009 The Authors Journal compilation ª 2009 FEBS 6213
USA) equipped with a digital camera (Q-Color 5) and
analyzed with Q Capture software. LDs were quantified as
follows. For primary mouse hypothalamic cultures, five
independent fields from each well were photographed (two
wells per condition) with · 400 magnification. LDs were
counted in photomicrographs using Image J software and
the mean number of LDs per field was calculated for each
condition (n = 10). For PC12 cells, at least 10 independent
fields from each well were photographed with · 400 magnifi-
cation. LDs and cells were counted in photomicrographs
and the mean number of LDs per 100 cells was calculated
for each condition.

Rad, Hercules, CA, USA). Membranes were blocked with
5% nonfat milk in NaCl ⁄ Tris containing 0.2% Nonidet
P40, incubated with primary antibodies overnight at 4 °C
and washed in NaCl ⁄ Tris–Tween (0.1% Tween-20). Immu-
noblotting was detected by enhanced chemiluminescence
(ECL Plus; Amersham ⁄ GE Healthcare, Little Chalfont,
UK) and visualized with HyBlot CL autoradiography film
(Danville Scientific Inc., Metuchen, NJ, USA).
Fatty acid oxidation
Fatty acid oxidation was determined as the amount of
14
CO
2
liberated from samples incubated with [1-
14
C]pal-
mitic acid using a modified procedure described in [69].
Briefly, cells were grown and treated in 25 cm
2
flasks. Three
flasks were used for each treatment condition. Cells from
one flask were used for protein determination and western
blotting. Cells from the second and third flasks were
analyzed for fat oxidation, i.e. medium was discarded from
these two flasks and replaced with 0.9 mL of fresh MEM
containing 0.1% BSA. Subsequently, 100 lLof10· reac-
tion mix (prepared freshly 1 h in advance and containing
10 mm carnitine, 1 mm palmitate, 4% BSA and 0.5 lCi per
culture flask of labeled fatty acid) were added to each flask.
The flasks were immediately sealed with rubber serum vial

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