Adipocytes differentiation in the presence of Pluronic F127–coated
Giuseppe Bardi, PhDa,⁎, Orazio Vittorio, MScb, Margherita Maffei, PhDc,
Tommaso Pizzorusso, PhDd, Mario Costa, PhDa
aInstitute of Neurosciences, National Research Council (CNR), Pisa, Italy
bCentre for Research in Microengineering (CRIM), Scuola Superiore Sant'Anna, Pisa, Italy
cDulbecco Telethon Institute at Department of Endocrinology and Metabolism, University Hospital of Pisa, Pisa, Italy
dDepartment of Psychology, University of Florence, Florence, Italy
Received 17 September 2008; accepted 14 January 2009
Carbon nanotubes are considered important nanodevices in biomedicine, and several applications in drug and gene delivery in different
organs and tissues are presently under investigation. Among them very little attention has been dedicated to white adipose tissue (WAT),
despite its wide distribution and endocrine role, which could potentially be used to release therapeutic agents. An important premise to use
nanotubes in WAT is to determine their potential for toxicity on adipocytes. Here we show that Pluronic F127 (PF127)–coated
multiwalled carbon nanotubes (MWCNTs) can be tolerated by NIH-3T3-L1 pre-adipocytes without affecting their growth. Moreover, the
differentiation process of NIH-3T3-L1 pre-adipocytes to adipocytes is not compromised by the presence of 5 μg/mL MWCNTs in the
medium. These results suggest that reasonable concentrations of PF127-MWCNTs are not toxic for adipocytes and do not interfere with
their differentiation process.
From the Clinical Editor: This study established that Pluronic F127 (PF127)–coated multiwalled carbon nanotubes (MWCNTs) are tolerated
by NIH-3T3-L1 pre-adipocytes. The differentation of these pre-adipocytes is not compromised by the presence of 5 μg/mL MWCNTs,
suggesting that these nanotubes are not toxic for adipocytes.
© 2009 Elsevier Inc. All rights reserved.
Key words: Carbon nanotubes; Adipocytes; Obesity; Biocompatibility; Toxicity
Western industrialized societies are experiencing the draw-
backs of unlimited food resources, and obesity has been
described as the pandemic disorder of these last 2–3 decades.
Ever-growing attention is consequently being dedicated to the
is certainly the most evident feature of this condition. The
it is associated with the secretion of several factors, generally
indicated as adipokines, including hormones, growth factors,
cytokines, and chemokines. The WAT shows remarkable size
plasticity persistent throughout adulthood. This is evident
during weight gain: in fact, the fat mass may increase to up to
70% (in case of morbid obesity) of body mass. Although most
of this increase is due to hypertrophy of adipocytes already in
place, there is a significant portion of de novo differentiation
originating from the bulk of pre-adipocytes present in WAT.2
In this scenario WAT may represent an important target for the
application of nanotechnologies for its wide distribution and
capacity to release factors into the blood.
Carbon nanotubes (CNTs) are good candidates for such
applications, in that they belong to a set of the so called
“nanodevices” intensely studied when interacting with
biological systems.3Single-walled carbon nanotubes
(SWCNTs) consist of a single layer of graphite lattice rolled
into a perfect cylinder, whereas sets of concentric cylindrical
graphite shells form multiwalled carbon nanotubes
(MWCNTs).4CNTs have been proposed as multipurpose
Available online at www.sciencedirect.com
Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 378–381
The work presented in this article has been supported by the NINIVE
(Non-Invasive Nanotransducer for In Vivo Gene Therapy, STRP 033378)
project, co-financed by the 6FP of the European Commission.
⁎Corresponding author. Institute of Neurosciences, CNR, Via Moruzzi
1, 56124 Pisa, Italy.
E-mail address: firstname.lastname@example.org (G. Bardi).
1549-9634/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Please cite this article as: G. Bardi, O. Vittorio, M. Maffei, T. Pizzorusso, M. Costa, Adipocytes differentiation in the presence of Pluronic F127–coated
carbon nanotubes. Nanomedicine: NBM 2009;5:378-381, doi:10.1016/j.nano.2009.01.016
innovative carriers for drug and gene delivery, because they
can be functionalized with different functional groups to
carry different moieties for specific targeting.5
A necessary premise to pursue this strategy is to assess the
effect of CNTs on adipocytes and further, on adipose
conversion. To address this issue we used CNTs on NIH-
3T3-L1 cell line, a model widely accepted to study
adipogenesis.6Our data suggest that CNTs are perfectly
compatible with viability and differentiation potential of
these cells. We provide convincing evidence that Pluronic
F127 (PF127)-MWCNTs could be an excellent nanodevice
for several applications in adipose tissue.
MWCNTs were supplied by Nanothinx (Rio Patras,
Greece). Purity of nanotubes was estimated with a post-
deposition Thermo Gravimetric Analysis (TGA) treatment
by the supplier, which revealed a carbon content of about
99.2% and a minimal amount of carbon soot (b1%).
Cell cultures and differentiation process
Cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 200 U/mL
penicillin, and 50 μg/mL streptomycin (standard medium) to
confluence (hereafter referred to as day 0). For differentiation,
confluent NIH-3T3-L1 cells were stimulated to differentiate
by addition of hormonal cocktail (0.5 mM 1-methyl-3-
isobutylmethyl-xanthine, 0.5 μM dexamethasone, and
170 nM insulin [MDI]) to the standard medium for 2 days
and were maintained thereafter in standard medium with
fully differentiate into adipocytes. Media (all from Sigma-
Aldrich, St. Louis, Missouri) were changed every other day.
Oil Red O staining
Culture dishes were washed in phosphate-buffered saline
prepared by diluting a stock solution (0.5 g of Oil Red O;
Sigma-Aldrich St. Louis, Missouri) in 100 mL of isopropanol
with water (60:40, v/v) followed by filtration. After staining,
plates were washed twice in water and photographed.
WST-1 and MTT assays
20 × 103cells were seeded into each well of a 96-well
plate and then incubated (Heraeus CO2Incubator, Thermo
Fisher Scientific) with the culture media containing different
concentrations of MWCNTs. After 24 hours of incubation
we added 10 μL of WST-1 (tetrazolium salt 2-(4-iodophe-
lium) solution (as described in quick cell proliferation assay
kit, BioVision, USA) and incubated for 2 hours in standard
conditions. The absorbance of treated and untreated samples
was measured using a Versamax microplate reader (Mole-
cular Devices, Sunnyvale, California) at a wavelength of
450 nm with the reference wavelength 650 nm.
20 × 103cells wereseeded intoeachwell ofa 96-well plate
and then incubated with the culture media. After 24 or 72
hours of incubation the culture media were then replaced with
100 μL of medium containing 0.5 mg/mL of MTT (3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide,
M-2128, from Sigma-Aldrich), and the plates were incubated
for 2 hours at 37°C and 5% CO2. Mitochondrial dehydro-
water-insoluble formazan crystals. The MTT-containing
medium is again removed and replaced with 100 μL of
dimethylsulfoxide (Sigma-Aldrich) and left for 10 minutes on
a platform shaker to solubilize the converted formazan on the
culture plates. The absorbance was measured on a Versamax
microplate reader (Molecular Devices) at a wavelength of
570 nm with background subtracted at 690 nm.
Hoechst 33342 (1 μg/mL) staining and Trypan Blue
exclusion was used to evaluate differences between viable
and dead cells after treatments. Phase contrast and fluo-
rescence cell images were acquired by a camera-mounted
Zeiss Axioskop microscope (Carl Zeiss Microimaging,
Thornwood, New York).
To evaluate the statistical significance of all the described
experiments, five microscopic fields per coverslip were
counted, and three coverslips per treatment were used for
each experiment. Three independent experiments were
performed in triplicate. One-way statistical analysis of
variance followed by analysis by Student-Newman-Keuls
method were performed.
Pluronic F127–coated PF127-MWCNTs have shown a
reduced toxicity in previous work with proliferating cell
lines7or postmitotic differentiated neurons.8Because each
cell type shows a different compatibility with CNTs and
several preparations of CNTs are available on the market,
we decided to pre-test a range of MWCNT concentrations
soluble in the NIH-3T3-L1 cell medium. To determine the
effect of CNTs on cell viability we used WST-1 and MTT
cell assays. Figure 1, A shows the results of the WST-1
asssay. MWCNT concentrations ranging between 2.5 and
10 μg/mL do not interfere with NIH-3T3-L1 cell viability.
On the other hand, PF127 surfactant alone at a concentration
of 0.01% or a lower concentration of PF127-MWCNTs
(1 μg/mL) slightly reduce cell metabolism (Figure 1, A) in
379G. Bardi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 378–381
alone. These data have been confirmed by MTT assay
performed at a PF127-MWCNT concentration of 5 μg/mL at
5 μg/mL PF127-MWCNTs (99% pure) for the following
experiments. After 72 hours in the presence of MWCNTs-
Figure 2. PF127-MWCNTs do not interfere with differentiation of
adipocytes. (A) Oil Red O staining of lipid droplets produced by
differentiated adipocytes was performed on day 8 after the induction. (B)
Differentiation of adipocytes was followed for 9 days culturing NIH-3T3-L1
cells in the absence (left-hand panels) or in the presence (right-hand panels)
of 5 μg/mL MWCNTs. Fatty acids vesicles produced by the differentiated
adipocytes(yellowarrowheads) areshowninthe lower panelsintheabsence
(left) or in the presence (right) of MWCNT-supplemented media. Red
arrowheads point at MWCNT aggregates (right panels) found in the
MWCNT-media. (C) Numbers of differentiated NIH-3T3-L1 adipocytes
treated for 48 hours with medium (white bar), 0.01% PF127-medium (gray
bar), or 5 μg/mL MWCNT-supplemented media (hatched bar). The short
black bars represent the number of dead cells present in each treatment, as
measured by Trypan Blue exclusion. DC. Fix part labels: A, B, C. Correx:
Part A 3T3-L1 Controls; 0.01% PF127; PF127-MWCNTs. Parts B & C,
Figure 1. PF127-MWCNTs do not affect viability and growth of NIH-3T3-
L1 pre-adipocytes. (A) AWST-1 assay was performed on NIH-3T3-L1 cells
after a 24-hour treatment with different concentrations of PF127-MWCNTs
(hatched bars) in the medium compared with control cell medium (white
bar) or medium with 0.01% PF127 (gray bar). (B) WST-1 assay after cells
were grown for 72 hours in the presence of control cell medium (white bar),
medium with 0.01% PF127 (gray bar), or 5 μg/mL PF127-MWCNTs
(hatchedbar). Absorbanceof samplesis shownas mean± SEM(six samples
per treatment by three independent experiments). (C) NIH-3T3-L1 pre-
adipocytes were treated once with medium (white turbots), 0.01% PF127
(gray circles), and 5 μg/mL of PF127-MWCNTs (black triangles). Cell
growth was followed for 3 days in vitro. The graph shows the number of
viable cells over 72 hours. (D) The graph represents the percentage of NIH-
3T3-L1 cell death over 72 hours measured by Trypan Blue exclusion. The
mean ± SEM of three independent experiments is shown. DC with A & B
side by side and C & D below them. Correx:Part A, delete “24h”. y-axis, use
decimals not commas. x-axis, 0.01% PF127; PF127-MWCNTs (μg/mL).
Part B, delete “72h”. y-axis, use decimals not commas. x-axis, PF127-
MWCNTs 5 μg/mL. Part C, (key) 5 μg/mL MWCNTs. x-axis, 24 hr, 48 hr,
72 hr. Can't read numbers on y-axis. Part D, x-axis, 24 hr, 48 hr, 72 hr.
380G. Bardi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 378–381
supplemented medium, NIH-3T3-L1 cell metabolism and
proliferation (Figure 1, B and C) did not present statistically
significant differences with respect to controls. Cell death
in each condition over the 72 hours, as shown in Figure 1, D.
NIH-3T3-L1 adipocytes were differentiated in the
presence or in the absence of PF127-MWCNTs. After
8 days the degree of differentiation was assessed by Oil
Red O staining, which, by showing the lipid droplets
accumulated by cells, is a reliable measurement of their
differentiation state. No difference in lipid accumulation was
observed, as shown in Figure 2, A. To further highlight the
differentiation process of the adipocytes in the presence of
PF127-MWCNTs fluorescent nuclei stained with Hoechst
33342 were superimposed on phase contrast pictures to
better localize the single cell in the field (Figure 2, B). The
presence of MWCNTs is indicated by aggregates of
nonsolubilized nanotubes. These results indicate that
PF127-MWCNTs do not interfere with NIH-3T3-L1 capa-
city to undergo adipose conversion.
After differentiation NIH-3T3-L1 adipocytes can survive
for just a days once they are confluent and fatty acids
are released in the medium. We counted the cell number
(Figure 2, C) of untreated, 0.01% PF127, or PF127-
MWCNTs treated. No differences in the viable cell number
were found among the different conditions after 48 hours.
Moreover, we could not find differences in the number of
studies including bioengineering investigations.9New nano-
technology approaches to investigate the possibility of drug
delivery directly in the WAT could become a new field of
investigation. Here, we have focused our attention on the
biocompatibility of PF127-coated MWCNTs with adipo-
cytes. As described by our results for the first time to our
fat environment, do not interfere with growth and differentia-
tion of pre-adipocytes into adipocytes. Pluronic F127 is a
nonionic surfactant also used as a carrier for poorly water-
soluble compounds.10We had noticed in previous work on
primary neurons that 97% pure and 2-μm-long PF127-
MWCNTs could show a certain degree of toxicity after
48 hours at concentrations lower than 5 μg/mL.8However,
used in this study are 99% pure, and their average length is
300 nm (Table 1). It is known that cell uptake of nanotubes is
independent of the cell type,11but toxicity can be dependent
on the length of the CNTs.12in that long MWCNTs could
pierce the cell membrane and induce cell death. In this
relatively short PF127-MWCNTs would not damage the
NIH-3T3-L1 biological mechanisms leading to the prolifera-
tion and the differentiation in adipocytes.
These results disclose novel perspectives for the applica-
tion of nanotechnologies to WAT. Given its endocrine
potential and its wide distribution, this tissue/organ con-
stitutes, in fact, an attractive target for the experimentation
with nanotechnologies. “Ad hoc” nanodevices could be
designed to specifically target the fat cell and open a new line
of investigation for future treatment of obesity and other
diseases involving the adipose tissue.
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Multiwalled carbon nanotubes (MWCNTs) description and properties
Carbon content (purity)
Amorphous carbon & other carbon impurities†
G = 1581 cm–1
D = 1350 cm–1
*After dispersion in water and sonication treatment.
†In the predetermined carbon content.
381G. Bardi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 378–381