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8/2/2019 IJN 15841 Enhanced Laser Thermal Ablation for in Vitro Liver Cancer de 011311
1/13
2011 Ianu t al, publi and lin Dov Mdial P Ltd. Ti i an Opn A atilwi pmit untitd nonommial u, povidd t oiinal wok i poply itd.
Intnational Jounal of Nanomdiin 2011:6 129141
International Journal of Nanomedicine Dovepress
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O r I g I N A L r e s e A r c h
open access to scientifc and medical research
Opn A Full Txt Atil
DOI: 10.2147/IJN.S15841
enand la tmal ablation fo t in vitotreatment of liver cancer by specic delivery of
multiwalld abon nanotub funtionalizd wituman um albumin
conl Ianu1
Luian Moan1
contantin Bl2
Anamaia Ioana Oza2
Flaviu A Tabaan3
conl catoi3
ra stiufiu4
Aiana sti1
citian Mata2
Dana Ianu1
Luia Aoton-colda1
Floin Zaai1
Todoa Moan1
1Dpatmnt of Nanomdiin, Iuliuhatianu Univity of Mdiinand Pamay, Tid su y clini,cluj-Napoa, romania; 2Dpatmntof Biomity, 3Dpatmnt ofPatoloy, Faulty of VtinayMdiin, Univity of Aiultualsin and Vtinay Mdiin,cluj-Napoa, romania; 4Dpatmntof Biopyi, Iuliu hati anuUnivity of Mdiin and Pamay,cluj-Napoa, romania
copondn: conl Ianuand Luian MoanDpatmnt of Nanomdiin,Iuliu hatianu Univity of Mdiinand Pamay, Tid suy clini,19-21 coitoilo stt, 400162cluj-Napoa, romaniaTl +40 264439696Fax +40 264439696email [email protected];[email protected]
Abstract: The main goal o this investigation was to develop and test a new method o treatment
or human hepatocellular carcinoma (HCC). We present a method o carbon nanotube-enhanced
laser thermal ablation o HepG2 cells (human hepatocellular liver carcinoma cell line) based on
a simple multiwalled carbon nanotube (MWCNT) carrier system, such as human serum albumin
(HSA), and demonstrate its selective therapeutic ecacy compared with normal hepatocyte cells.
Both HepG2 cells and hepatocytes were treated with HSAMWCNTs at various concentrations
and at various incubation times and urther irradiated using a 2 W, 808 nm laser beam. Transmis-
sion electron, phase contrast, and conocal microscopy combined with immunochemical staining
were used to demonstrate the selective internalization o HSAMWCNTs via Gp60 receptors
and the caveolin-mediated endocytosis inside HepG2 cells. The postirradiation apoptotic rate
o HepG2 cells treated with HSAMWCNTs ranged rom 88.24% (or 50 mg/L) at 60 sec to
92.34% (or 50 mg/L) at 30 min. Signicantly lower necrotic rates were obtained when human
hepatocytes were treated with HSAMWCNTs in a similar manner. Our results clearly show
that HSAMWCNTs selectively attach on the albondin (aka Gp60) receptor located on the
HepG2 membrane, ollowed by an uptake through a caveolin-dependent endocytosis process.
These unique results may represent a major step in liver cancer treatment using nanolocalized
thermal ablation by laser heating.
Keywords: carbon nanotubes, albumin, HepG2 cells, noncovalent unctionalization, laser
irradiation, Gp60 receptor
IntroductionHepatocellular carcinoma (HCC) represents a leading cause o cancer deaths
worldwide.13 Despite recent discoveries in screening and early detection, HCC exhib-
its a rapid clinical course with an average survival o 6 months and an overall 5-year
survival rate o 5%.4 As chemotherapy and radiotherapy show modest results5 and
surgery is possible in 10%30% o patients,6,7 new therapeutic methods oer hope
or a better outcome.
Most data suggest that nanotechnologies could play a major role in the development
o new anticancer therapies. A thermal approach using nanoparticles, nanoemulsion, pH-
responsive nanoparticles, nanoparticles combined with radiation, and nanovectors or
drug delivery are the most explored nanoparticle-based cancer treatment methods.8
The ability o carbon nanotubes (CNTs) to convert near-inrared (NIR) laser
radiation into heat, due to the photonphonon and electron interactions,9 provides the
Number of times this article has been viewed
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
13 January 2011
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opportunity to create a new generation o immunoconjugates
or cancer phototherapy, with good perormance and e-
cacy in selective cancer thermal ablation, as well as in the
application o nanotechnology in molecular diagnostics
(nanodiagnostics).8,10
Nanotechnology has already shown promising results
in HCC research and treatment. Microwave11 ablation and
radiorequency12 ablation were proposed or the treatment
o HCC. Intratumorally administered CNTs combined with
laser irradiation proved to be ecient in the treatment o HCC
on animal models.13 However, a major challenge in treating
HCC is represented by therapies strictly directed toward
the tumor cells inside the liver parenchyma. Generally, the
use o targeting molecules such as antibodies, olates, and
growth actors has been specically proposed or carrying
nanomaterials to the cancer cells and tumors.1416 However,
100% selective internalization o nanobioconjugates in the
cancer cells remains problematic.17 This can be explained by
the presence o the receptors used or the specic binding o
the targeting molecules on the membranes o the noncancer-
ous cells, although in smaller concentrations compared with
the cancer cells.18
The use o CNTs as bioactive molecules is still at an early
research stage, but their unique physical and chemical proper-
ties hold great hope or cancer treatment.8,14,1618 Nevertheless,
there are many toxicity concerns to be addressed. It has
been stated that a procient method needed to minimize
toxic eects and also to increase the level o therapeutic
response or CNTs is represented by their conjugation to
a carrier molecule.1923 The use o these biological carriers
or the development o specic and sensitive site-targeted
bionanosystems also allows the selective internalization o
CNTs into cancer cells.
Research data have shown that highly prolierative tumors
have the capacity to create albumin deposits.24 The reports
have demonstrated the liver cancer cells overexpression o
specic human serum albumin (HSA) receptors and their
ability to internalize large amounts o albumin through the
mechanism o caveolae-mediated endocytosis.25 The result-
ing amino acids are urther used or the synthesis o various
substrates needed or tumor growth.26,27 Considering all these
data together, we propose a method or the unctionalization
o multiwalled carbon nanotubes (MWCNTs) with HSA or
the selective targeting and laser-mediated necrosis o liver
cancer cells. To our knowledge, this is the rst demonstra-
tion o selective targeting via Gp60 receptors located on the
membrane o malignant liver cancer cells using a conjugate
o HSA and CNTs.
Material and methodsAntibodi and antFor the experiments involving the noncovalent unctionaliza-
tion o CNTs, MWCNTs (.90% carbon basis, OD ID L
1015 nm 26 nm 0.110 m, product number 677248),
HSA, and Sephacryl 100-HR were purchased rom Sigma-Al-
drich (Steinheim, Germany), and all the other chemicals were
purchased rom Merck (Darmstadt, Germany). HepG2 cells
and immortalized hepatocyte epithelial cells (CRL-4020) were
purchased rom ATCC (Rockville, MD, USA), and all the
other reagents needed or cell culture were purchased rom
Sigma-Aldrich. For the experiments involving cell apoptosis,
Cell Death Detection ELISAPLUS was purchased rom Roche
Applied Science (Mannheim, Germany). For immunostaining
procedures, Draq5, 4-6-diamidino-2-phenylindole (DAPI),
and anti-caveolin-1Cy3 antibody (Ab) produced in rabbit
were purchased rom Sigma-Aldrich. Polyclonal Gp60 Ab
was prepared as previously described,
28
and or use as afuorescent probe a cy3 derivative o anti-Gp60 was prepared
according to the existing protocol.29
Nonovalnt funtionalizationof cNT wit hsAA total o 60 mg MWCNTs were dispersed in a 3:1 (v/v)
mixture o concentrated suluric and nitric acid and sonicated
or 3 10 s with a tip sonicator. Subsequently, the mixture was
refuxed at 120C or 30 min. The oxidized MWCNTs treated in
water solution were then centriuged at 8000 rpm to remove any
large unreacted CNTs rom the solution and metallic impurities.
Finally, the oxidized MWCNTs were vacuum ltered through
a 0.2-m polycarbonate lter (Whatman) until the elution was
clear and at neutral pH. The lter cake was dried overnight at
room temperature. Ater ltration, the solution concentration
was re-estimated using UVVisNIR spectroscopy (JASCO
V530, Gross-Umstadt, Germany). A total o 1 mg o fuorescein
isothiocyanate (FITC) (10 mg/mL in dimethyl suloxide) was
mixed with 50 mg HSA in sodium buer (20 mM, pH 8.5),
ollowed by incubation or 2 h in darkness, at room temperature,
with continuous stirring. The HSAFITC conjugate was puri-
ed by gel chromatography using a Sephacryl 100-HR column
eluted with 10 mM phosphate buered saline (PBS).30
Oxidized MWCNTs and HSAFITC were mixed with
deionized water at a concentration o 0.25 and 1.25 mg/mL,
respectively. The mixture was sonicated or 1 h with a
tip sonicator in an ice bath and was then centriuged or
5 min at 12,000 rpm. The solid was settled at the bottom
o the centriuge tube and consisted o unbound nanotubes,
impurities, metals, and bundles o oxidized nanotubes.
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Nanopototmolyi of liv an in an in vito aay uin albumin-onjuatd abon nanotub
The resulting supernatant was collected and subjected to
a second centriugation round. The supernatant collected
contained the desired MWCNTHSA conjugate.
For urther purication, the supernatant was subjected
to a gel chromatography purication process. Sephacryl
100-HR that was presoaked and deaerated using a vacuum
pump was packed up to 15 cm in a 2.5 cm diameter 24 cm
long glass column. The oxidized MWCNTHSA supernatant
recovered ater centriugation was layered on the top o the
gel and eluted using water fowing under gravity. Volume
ractions were collected or periods o 1 min duration and
analyzed or the presence o MWCNTs and HSA by measur-
ing the absorbance at 500 and 280 nm, respectively, using
the spectrophotometer (JASCO V530). Fractions showing
protein content were pooled or urther use.
Ppaation of MWcNTThe nonconjugated highly puried MWCNT control solution
was prepared as previously described.31 The solution was
diluted in minimum essential medium at a 1:10 (v/v) ratio.
caatization of MWcNTbioonjuatThe morphology o MWCNTs unctionalized with HSA was
examined using a WITEC alpha 300 Atomic Force Micro-
scope (Ulm, Germany), operating under ambient conditions.
The images were collected in tapping mode using a silicon
nitride cantilever.
The optical properties o oxidized MWCNTs unction-
alized with HSAFITC were monitored using a UVVis
spectrophotometer (JASCO V570).
Fourier transorm inrared (FTIR) measurements
were perormed with a JASCO 6100 spectrometer in the
4000500 cm1 spectral region, with a resolution o 4 cm1
using the KBr pellet technique.
cll ultuHepG2 and CRL-4020 cells, purchased rom the American
Type Culture Collection (ATCC) (Manassas, VA, USA),
were grown in 25 cm3 Corning plastic plates in minimum
essential medium, supplemented with 10% etal bovine
serum and 1% penicillinstreptomycin. The cells were main-
tained in a humidied 5% CO2
incubator at 37C. The cells
were kept in the logarithmic growth phase by routine passage
every 34 days. When reaching confuence, the cells were
split ater rinsing with PBS and detached with trypsin.
For the experiments, the cells were cultivated to confu-
ence on 60 mm plates. The MWCNTs unctionalized with
HSA were urther administered to the cell cultures by adding
to the culture medium and incubating or various periods o
time (1 min; 30 min; 1 h; 5 h; 24 h) at increased concentra-
tions: 1, 5, 20, 50 mg/L. For each concentration, all the
experiments were perormed in triplicate.
cll aatizationFor the microscopy analysis, the cells were trypsinated and
transerred to 35 mm plates, at a density o 25 104 cells/dish.
Ater administration and irradiation, the cells were thoroughly
washed with 1PBS three times, xed with 10% ormaldehyde
solution or 10 min, washed three times with PBS, and stained
with methyl green dye or 10 min. Cells in culture were exam-
ined using an Olympus CKX 31 (Munich, Germany) inverted
microscope with phase contrast.
cll viabilityThe extent o apoptosis was evaluated using a Cell Death
Detection ELISAPLUS assay kit rom Roche Applied Science.
The assay is a quantitative sandwich enzyme-linked immuno-
sorbent assay (ELISA) that uses the act that, due to cellular
death, nucleosomes are released rom the nucleus into the
cytosol. These nucleosomes can be detected by antihistone
biotin-labeled Abs. The nucleosomeAbs complex will bind
to streptavidin-coated well plate and give a signal at 405 nm
on the addition o substrate. Ater irradiating the cells that were
previously treated with various doses o HSAMWCNTs, the
culture media were removed and briefy centriuged in order to
collect the foating cells. The culture dish was rinsed with PBS,
and then 0.25% trypsin was added to detach the cells. Once
detached, the cell suspension was combined with the cells col-
lected rom the media. The resulting mixture o the cells was
briefy spun to collect the cells. The supernatant was discarded,
and the pelleted cells were resuspended in ice-cold PBS. The
cell suspension was subjected to the nal centriugation, and
the pellet was resuspended in the Roche lysis buer. Ater
30 min incubation at room temperature, the reaction mixture
was centriuged at 200 g (4C) or 10 min. The pellet, which
contains the nucleus, was removed, and the supernatant, which
represents the cytoplasmic raction, was aliquoted into new
tubes and kept rozen at 80C until use.
This supernatant solution would contain the ragmented
nucleosomes i the cells underwent apoptosis. Ater measur-
ing the protein concentration o the resulting supernatant using
bicinchoninic acid (BCA) assay, 20 g o total protein in 20 L
were added to the streptavidin-coated 96-well plate. Twenty
microliters o each incubation buer and DNA histone com-
plex was used as a background control and positive control,
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respectively. Then, 80 L o immunoreagent was added to
each well and incubated or 2 h at room temperature, with
gentle and continuous stirring. Ater the incubation, the
solution in the wells was thoroughly removed using gentle
suction and rinsed three times in incubation buer. Finally,
100 L o 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulonic
acid) (ABTS) substrate solution was added to each well and
incubated until the desired strength o color was achieved,
which took about 7 min.
The multiwell plates were then placed into a Labsystem
Multiskan Plus Spectrophotometer (Helsinki, Finland).
The absorbance was measured at 405 nm, with 495 nm as
the reerence wavelength. The absorbance at 495 nm was
deducted rom the absorbance at 405 nm or all samples
and controls. Then, the OD405OD
495value o the back-
ground control, which is composed o the incubation buer
and ABTS solution, was subtracted rom all OD405OD
495
values o the samples. The intensity o apoptosis can be
expressed as enrichment actor= (mU o the sample)/(mU
o the corresponding negative control), where mU is the
absorbance 103 ater subtracting the reerence absorbance
and the OD405OD
495value o the background control. The
enrichment actor exhibits the specic enrichment o mono-
nucleosomes and oligonucleosomes released into the cyto-
plasm o the cells that are dying and dead due to apoptosis.
Finally, the values were normalized so that the untreated
sample could have an enrichment actor equal to 1.32
La tatmntWe used 2 W o power laser (Apel Laser, Bucharest, Romania)
operating at 808 nm or a 2 minutes irradiation o a monolayer
o cells placed on a glass substrate, ater being incubated with
HSAMWCNTs or various periods o time. The laser diode
was placed 3 cm away rom the surace o the glass, at a vertical
angle, and the beam had a Gaussian distribution with a 1/e2
value o 2 mm.
La onfoal mioopy of llFluorescent images were acquired using a Zeiss LSM
710 conocal laser scanning unit (Oberkochen, Germany)
equipped with argon and an HeNe laser mounted on an Axio
Observer Z1 Inverted Microscope. Hep2G cells or human
hepatocytes rom the suspension were briefy rinsed with
PBS and xed in 4% ormaldehyde (pH 7) or 15 min. Ater
three washing procedures in PBS or 15 min, the slides were
covered or 60 min with a serum-ree blocking buer (Dako
Cytomation, Glostrup, Denmark). The dying procedures were
made in accordance with manuacturers protocols. Specic
visualization o cell structures was perormed using 364,
488, and 568 nm excitation laser lines to detect Draq5 (BP
590650 nm emission), DAPI (BP 385470 nm emission),
FITC (BP505550 emission), and cy3 fuorescence (LP585
emission), respectively.
Tanmiion lton
mioopy analyiThe internalization o the unctionalized nanotubes was
investigated using transmission electron microscopy (TEM)
in conventional electron beam conditions. Live cells were
incubated in an HSAMWCNT solution as described pre-
viously. Ater the nal PBS rinsing, the cells were xed
using 2.5% glutaraldehyde in 0.1 M cacodylate buer
and embedded in agarose. Ater three rinses with sodium
phosphate buer, the monolayers were sectioned into small
pieces, postxed with 1% osmium tetroxide, en bloc stained
with 1% uranyl acetate, dehydrated in graded ethanol series
(30%, 50%, 75%, 100%, 10 min each), and embedded in
EMbed 812 resin. Ultrathin (100 nm) sections were cut on
an LEICA EM UC6 Ultramicrotome (Leica Microsystems,
Wetzlar, Germany), poststained with 4% uranyl acetate and
lead citrate, and viewed using a Jeol JEM 1010 TEM (Jeol,
Tokyo, Japan). The images were captured using a Mega
VIEW III camera (Olympus, Sot Imaging System, Mnster,
Germany).
statitial data analyiAll data were expressed as mean standard error o the mean.
Nonparametric tests were selected due to data nonnormality
(KolmogorovSmirnov test). Between-group comparisons
or the same concentration were tested using the Wilcoxon
test. Alpha error level o,0.05 was selected or all tests.
SPSS Statistics Version 17.0 (Chicago, IL, USA) packages,
as well as the Microsot Oce Excel application, were used
or data analysis.
ResultsFuntionalization of MWcNT wit hsAIn order to obtain a directly targeted delivery o MWCNTs
into the cancer cells and to visualize and detect the localiza-
tion o the nanotubes inside the cell, the FITCHSA system
was preormed and noncovalently labeled on the oxidized
surace o MWCNTs.
To provide clues regarding the success o noncova-
lent HSAMWCNT unctionalization, conocal micros-
copy was proposed or the identication o FITC-labeled
CNTs in solution. As shown in Figure IC, globular green
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Nanopototmolyi of liv an in an in vito aay uin albumin-onjuatd abon nanotub
CNTs corresponding to large molecules o luorescent
albumin were observed.
The oxidation o the nanotubes using a 3:1 (v/v) mixture
o concentrated suluric and nitric acid gave them hydrophi-
licity and stability in aqueous systems due to the ormation
o COOH, OH groups at the end and along the sidewalls
o the tubes.21
FTIR spectra rom Figure 2A conrm successul oxidation.
Comparing the FTIR spectra o pristine MWCNTs (black) with
those o oxidized MWCNTs (red), the characteristic bands o
the oxygen-containing groups appear at 3422 cm1, correspond-
ing to the stretching vibration o OH and water,33 a band at
1721 cm1, corresponding to the carbonyl and carboxyl C=O
stretching vibration, at 1582 and 1380 cm1, corresponding to
the OH deormation vibration, and the band at 1117 cm1,
corresponding to the CO stretching vibration. The band at
620 cm1 corresponds to the CO out-o-plane deormation.34
Further, we conjugated the HSAFITC system noncova-
lently on the surace o oxidized MWCNTs. First, we covalently
labeled HSA with FITC at an increased pH (above pH = 9), as
shown schematically in Figure IA.35 FITC covalently attached
to the protein through the alpha-amino group. Second, HSA
FITC complex was adsorbed on the nanotubes, presumptively,
through electrostatic interactions between the unctional groups
o MWCNTs and the protein-positive domains (Figure IB).
Considering the act that not all the surace o the nanotubes is
oxidized, hydrophobic interactions can also occur.36
UVVis spectroscopy is a simple but ecacious method
that conrms the ormation o the oxidized MWCNTHSA
FITC complex. The nanotubes solutions give an adsorption
band at 295.7 cm1, which corresponds to the +-plasmon
transition o MWCNT.37
The yellowish HSAFITC solution has the characteristic
adsorption band at 489 cm1 and a second adsorption band
at 292 cm1, suggesting the existence o aromatic amino
acids rom HSA. Comparing the aorementioned spectra, the
ormation o the MWCNTsHSAFITC complex becomes
obvious due to the appearance o the oxidized MWNT band
and the HSAFITC band at 475.6 cm1, which is shited and
has low intensity (Figure 2C).
Figure 1 A) Illutation of t ovalnt lablin of hsA wit FITc. B) T fomation of oxidizd MWcNThsAFITc. C) A typical uorescent image of HSAMWCNTs
(100 mg/L): globular uorescent CNTs corresponding to attached large molecules of uorescent albumin are being observed. D) 140 120 nm AFM topoapi ima of
hsA (blak aow) onjuatd wit MWcNT (wit aow). T d aow indiat t pn of an unonjuatd hsA molul. T al ba pnt 20 nm
(bottom-it panl).
Abbreviations: AFM,atomic force microscopy; FITC, uorescein isothiocyanate; HSA, human serum albumin; MWCNTs, multiwalled carbon nanotubes.
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The conjugation o HSAFITC onto the surace o the
nanotubes is also conrmed by FTIR spectroscopy as seen in
Figure 2B. No similarity can be observed when comparing the
spectra o HSAFITC with those o the nanotube-conjugated
HSAFITC. All the corresponding peaks had shited their
position, and some even disappeared. In the higher region,
the stretching vibration band o the NH groups at 3409 cm1
changed their shape in a broad band that included two peaks:
one at 3389 cm1 (NH groups stretching vibration) and the
second at 3303 cm1, which is the pyridine aromatic CH
vibrations band. The aliphatic CH stretching vibration at
2929 and 2873 cm1 moved at 2922 and 2865 cm1, such
that these groups were involved in electrostatic bonds. In
addition, the amide I and II are shited to low requency:
amide I, rom 1656 to 1649 cm1; amide II, rom 1544 to
1532 cm1. The asymmetric and symmetric deormations
o CH3
have changed their bands rom 1459 to 1447 cm1
and 14161389 cm1, respectively. The region in between
has dramatically changed their intensity. This is due to the
spontaneous adsorption o the crystalline HSAFITC com-
plex on the MWCNTs and the ormation o a well-organized
oxidized MWCNTHSAFITC.
To that end, atomic orce microscopy (AFM) analysis o the
HSAMWCNTs solution was perormed. Representative AFM
evidence o the successul attachment o HSA molecules onto
the surace o the nanotubes is shown in Figure ID. By AFM,
analysis at the nanometric scale o the two HSA molecules
(black arrows in Figure ID) attached at the end o the nanotubes
(white arrows) was carried out. A single HSA molecule (red
arrow) has also been observed in the topographic image shown
here. The length o the CNTs was estimated as being,200 nm.
The lateral resolution o an AFM image is determined by the
tip o the object that is imaged. In the presented image, the
width o the nanotube appears to be .2 nm, as we used an
AFM tip with a 15 nm radius o curvature.
hsAMWcNT intnalizationThe ability o an FITC-labeled bioconjugate o HSA
MWCNTs to internalize inside an HepG2 cell was evaluated
by conocal fuorescence microscopy imaging. The results
presented in Figure 3B show that at low concentration and
short exposure time, HSAMWCNT accumulates inside
HepG2 cells. Thus, we provided imaging evidence that
HSA can act as a carrier or MWCNTs, and because we
A
B
C
Absorbance(a.u.)
Absorbance(a.u.)
Absorbance(a.u
.)
0
0.0
0.2
0.4
1
2
4000 3500 2500 2000 1500 1000 500
1.0
0.5
0.0
300 400 500 600
3000
4000 3500 2500 2000 1500 1000 5003000
Wavenumber (1/cm)
Wavenumber (1/cm)
MWCNTs
Oxidized MWCNTs
Oxidized MWCNTs
Oxidized MWCNTsHSAFITC
3422 cm1
3422 cm1
3409 cm1
3389 cm1 3303 cm1
2929 cm1
3060 cm1
2873 cm1
1656 cm1
1649 cm1
1531 cm1
1544 cm1
532 cm1
605 cm1
1582 cm1
1721 cm1
1300 cm1
1117 cm1
620 cm1 HSAFITC
(nm)
HSAFTIC
Oxidized CNTsHSAFTIC
Figure 2 FTIr pta ofA) pitin MWcNT (blak) and oxidizd MWcNT (d); B) hsAFITc (blak) and hsAFITc-oatd oxidizd MWcNT (d); C) UVVi
adoption pta of hsAFITc (blak), oxidizd MWcNT (blu), oxidizd MWcNThsAFITc (d).
Abbreviations: FITc,uorescein isothiocyanate; FTIR, Fourier transform infrared; HSA, human serum albumin; MWCNTs, multiwalled carbon nanotubes.
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Nanopototmolyi of liv an in an in vito aay uin albumin-onjuatd abon nanotub
were unable to identiy any fuorescence in the epithelial
cells in similar conditions (Figure 3A) we reasoned that
HSAMWCNT bioconjugates exhibit specic anity or
liver cancer cells.
Furthermore, phase contrast microscopy was used to
demonstrate the presence o CNTs inside HepG2 cells ol-
lowing HSAMWCNT administration. As seen in Figure 3E
(red arrows), intracellular aggregates o MWCNTs appear as
dark, optically dense signals that associate with a reringent
signal under phase contrast. Once more, we were unable to
identiy any aggregates inside the epithelial cells that have
been similarly treated. (Figure 3D) Moreover, the cellular areas
that appeared to contain MWCNTs were urther subjected to
TEM analysis. When these regions were observed under TEM,
MWCNTs could be clearly identied in the orm o intracel-
lular aggregates, as shown by the red arrows in Figure 3F.
T manim of ltiv
intnalization of hsAMWcNTinid t malinant liv llIn order to shed light on the molecular mechanisms involved
in the specic uptake o HSAMWCNTs in HepG2 cells,
we investigated the possibility that a 60 kDa glycoprotein,
Gp60, which is known to unction in albumin transcytosis
in malignant cells,38 was involved in the selective uptake o
albumin bound to CNTs. To accomplish this, we allowed the
cells treated with 5 mg/L HSAMWCNTs or 1 h to incor-
porate cy3anti-Gp60 Ab or 30 min at 37C. To that end, we
obtained fuorescent images demonstrating the internalized
cy3 fuorescence (Figure 4A, rst panel).
Also, we showed that HepG2 cells internalized with
albumin-bound MWCNTs (fuorescently labeled with FITC)
were distributed into the punctate structure inside the cells
(Figure 4A, 2nd panel). DAPI, which is known to orm fuo-
rescent complexes with natural double-stranded DNA, was
used or nuclei staining. In Figure 4A, ourth panel, nearly
complete colocalization o the FITC fuorescence (green
image) and cy3 fuorescence (red image) was evident by yel-
low in the merged image. This nding suggests that albumin
bound to MWCNTs was incorporated into plasmalemmal
vesicles containing Gp60 as a membrane protein, urther
validating HSAMWCNT specicity or Gp60 receptors.
Importantly, as seen in Figure 4B, no signicant colocaliza-
tion in the hepatocyte cells (CRL-4020) was observed or
A B C
D E F
10 m 10 m 10 m
Figure 3 sltiv nanopototmolyi of hpg2 ll. A) confoal ima of uman patoyt inubatd fo 30 min wit 5 m/L FITchsAMWcNT. (T nulu
wa taind wit DrAQ5-d.) B) confoal dttion of MWcNThsAFITc (n) ltivly intnalizd into hpg2 ll (xpod fo 30 min to 5 m/L of FITchsA
MWcNT). C) hpg2 ll w iadiatd fo 2 min uin a 2-W, 808-nm la bam. Ima of ll lyat and aatd ll aft intnalization of MWcNThsAFITc
and la adiation. D) crL-4020 ll inubatd fo 30 min wit 5 m/L FITchsAMWcNT viualizd by pa ontat mioopy (400 magnication). E) hpg2 ll
inubatd fo 30 min wit 5 m/L FITchsAMWcNT viualizd by pa ontat mioopy (400 magnication). F) Tanmiion lton miopotoap owin
clusters of MWCNTs surrounded by plasmalemmal vesicles, conrming the presence of nanomaterial inside the cell (24,000 magnication).
Abbreviations: FITc,uorescein isothiocyanate; HSA, human serum albumin; MWCNTs, multiwalled carbon nanotubes.
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cy3Gp60 Ab and HSAFITCMWCNTs incubated under
same circumstances.
Thereore, based on these data, we showed that HSA
MWCNTs can act as specic and sensitive site-targeted
nanosystems against Gp60 receptor located on the liver
cancer cell membrane.
Aoiation of avolin-1 witFITchsAMWcNT-ontainin vilMost data indicate that caveolae-mediated endocytosis in
cells is stimulated by the binding o albumin to Gp60, a
receptor located in the caveolae.38
Given these data and the described role o caveolin in
albumin endocytosis, we reasoned that the mechanism o
HSAMWCNT internalization in HepG2 cells was similar. To
test this hypothesis, we immunostained the HepG2 cells with
Cy3anti-caveolin-1 Ab. As shown in Figure 4C, conocal
imaging revealed that the majority o FITCHSAMWCNT-
containing plasmalemmal vesicles stained or caveolin-1
used this fuorescent anti-caveolin-1 monoclonal Ab. Taken
together, all these data demonstrate that HSAMWCNTs
selectively internalize in human hepatocellular cancer cells
via caveolae-mediated endocytosis by the binding o the albu-
min carrier to Gp60, a specic albumin-binding protein.
cytotoxiity indud by laiadiation o by t adminitationof hsAMWcNTBeore testing the in vitro response o HSAMWCNT-treated
cells to laser irradiation, we investigated the possible eect
o cytotoxicity induced by the administration o CNTs in
the cells. HepG2 cells and the epithelial cells were treated
with various concentrations o HSAMWCNT at various
incubation periods. Cell Death Detection ELISAPLUS was
used to evaluate the eect o MWCNT bioconjugates on
cell viability.
Ater 24 h o incubation, HepG2 exposed to 50 mg/L
o HSAMWCNT showed a 5.71% decrease in viability
compared with 1.6% (P, 0.02) (Table I). For human hepa-
tocytes exposed to 50 mg/L o HSAMWCNT, the decrease
in viability was 6.23% compared with the nontreated sample,
in which the percentage o viable cells was 98.7% (P,0.001).
A
B
C
Cy-Gp60 Ab
Cy-Gp60 Ab
Caveolin-1-Cy3 Ab
FITCHSAMWCNTs
FITCHSAMWCNTs
FITCHSAMWCNTs
DAPI
DAPI
DAPI
Merged HepG2
HepG2
CRL-4020Merged
Merged
Figure 4 hsAMWcNT in vito ndoytoi manim in uman liv an ll. A) coloalization of cy-gp60 antibody and FITchsAMWcNT in hpg2 ll.
B) coloalization of cy-gp60 antibody and FITchsAMWcNT in patoyt pitlial ll. C) coloalization of avolin-1-cy antibody and FITchsAMWcNT in
hpg2 ll. rult a pntativ of t xpimnt. sal ba: 20 m in all panl.
Abbreviations: DAPI,4-6-diamidino-2-phenylindole; FITC, uorescein isothiocyanate; HSA, human serum albumin; MWCNTs, multiwalled carbon nanotubes.
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Nanopototmolyi of liv an in an in vito aay uin albumin-onjuatd abon nanotub
The statistical data showed that nanomaterial exposure per se
induced no signicant cytotoxic eects at small and mediumconcentrations (P. 0.05 or all comparisons).
The next step in order to eliminate any potential errors
was represented by a 2 minutes irradiation o a sample o cells
without nanoparticles, using a 2 W, 808 nm laser beam. There
was no lysis among the cells ater irradiation. The process
demonstrates the transparency o HepG2 or NIR beam.
Amnt of llula noiaft la tatmnt andadminitation of hsAMWcNTThe postirradiation lysis rate o HepG2 cells treated with
HSAMWCNTs ranged rom 35.45% (or 1 mg/L) to 88.24%
(or 50 mg/L) at 60 sec (P, 0.001), whereas at 30 min the
necrotic rate increased rom 59.34% (1 mg/L) to 92.34%
(50 mg/L),P value ,0.001. Signicantly lower apoptotic
rates were obtained in irradiated epithelial cells treated or
60 sec and 30 min at concentrations ranging rom 1 mg/L
to 50 mg/L (6.78%64.32% or 60 sec; 9.89%70.78% or
30 min). As can be observed, the optimal apoptotic eect o
malignant cells ater incubation with HSAMWCNT was
obtained at a concentration o 5 mg/L (HepG2/CRL-4020:
65.79%/11.34% at 60 sec, and 75.34%/14.67% at 30 min)
(Figure 5). Ater 60 min o incubation, the dierence among
the apoptotic rates was also statistically signicant among
the two cell lines or low/medium concentrations o HSA
MWCNT (78.92%: 1 mg/L, 88.34%: 5 mg/L, 87.88%:
20 mg/L, or HepG2; 15.56%: 1 mg/L, 21.34%: 5 mg/L,
52.14%: 20 mg/L, or CRL-4020). P values were ,0.001
or comparisons between various orms o nanomaterials.
No signicant dierences (P= 0.143) among the apoptotic
rates o HepG2 and CRL-4020 treated with HSAMWCNT
could be observed (100%: HepG2; 84.13%: CRL-4020) or
a high concentration o nanomaterials (50 mg/L).
Ater 35 h o incubation, a signicant apoptotic rate
o the two cell lines was obtained only when the cells
were treated with low concentrations o nanomaterials
(,20 mg/L). Elevated concentrations recorded a nonsigni-
cant dierence in the cell lysis eect o the two cell lines
(P= 0.25620 mg/L;P= 0.29650 mg/L).
Ater 24 h o incubation, the HepG2 cells treated with
1 mg/L HSAMWCNT were 100% necrotic ater laser irra-
diation, as compared with 52.2% o the CRL-4020 cells simi-
larly treated. For very low concentrations o HSAMWCNTs,
we could observe a dierence among the percentage o
dead cells o the two cell lines. However, the dierence
reached only a marginal signicance (P= 0.07). The lysis
rate o the irradiated cells incubated with more than 5 mg/L
nanomaterials or 24 h was almost similar or the two cell
lines (100% vs 85.94%).
In contrast, no signicant dierences in the percentage
o nonviable cells were obtained between the two cell lines
when the nonunctionalized MWCNT solution was used or
treatment (P. 0.05 or all comparison and each exposure
interval). Moreover, or HepG2 cells, the results showed a
signicant dierence between MWCNTs and MWCNT
HSA-exposed groups or low concentrations (1, 5, and
20 mg/L) and short exposures (60 sec, 30 min, 1 h, 3 h, and
5 h) (Figure 5).
DiscussionThe main goal o this investigation was to develop and test a
new method o treatment o human HCC. Preliminary data
rom literature support the involvement o albumin in tumor
growth. The implication is supported by the act that albumin
enhances tumor expansion, as it is used or synthesis in vari-
ous cellular compartments.38
In order to investigate the toxicity eects o the nano-
conjugates, HepG2 cells and CRL-4020 epithelial cells were
exposed and incubated with HSAMWCNTs at various
concentrations and incubation times. Consistent with other
ndings, we demonstrate that only high concentrations o
MWCNT bioconjugates exhibit cytotoxic eects.31 Never-
theless, the toxicity, which represents a major obstacle in
using CNTs in clinical applications, may be minimized by
administration o low doses o nanoconjugates.9,13,23
Further, we used HSAMWCNTs as heat-inducing
agents under laser radiation during the process o
Table 1 cytotoxi-indud fft on hpg2 and crL-4020
ll by vaiou onntation of bionanomatial at vaiou
inubation tim
HSAMWCNTs
concentration
Cytotoxicity effects at different incubation
intervals (%)
1 min 30 min 1 h 3 h 5 h 24 h
crL-4020 ontol 0 0.2 0.3 0.4 0.9 1.6
hpg2 ontol 0 0.1 0.3 0.5 1.1 1.3
crL-4020 1 m/L 0.3 0.5 0.7 1.9 2.4 3.8
hpg2 1 m/L 0.5 0.8 0.9 2.2 2.5 3.6
crL-4020 5 m/L 0.6 0.9 1 2.4 3.1 4.2
hpg2 5 m/L 0.4 0.7 0.8 2.6 3.2 4.4
crL-4020 20 m/L 0.7 0.8 1.2 3 3.1 4.8
hpg2 20 m/L 0.8 1.2 1.2 2.6 2.8 4.5
crL-4020 50 m/L 0.6 0.8 1.8 2.2 2.8 4.9
hpg2 50 m/L 1.4 1.5 1.6 2.2 2.8 6.2
Abbreviations: hsA, uman um albumin; MWcNT, multiwalld abon
nanotub.
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Ianu t al
nanophotothermolysis. This method is based on the pres-
ence and clustering o HSAMWCNTs inside the cells and
their highly optical absorption capabilities responsible or
inducing thermal eects, especially under NIR irradiation,
where the biological systems have low absorption and high
transparency.10,1922,39,40 The optoelectronic transitions in
the graphitic structures o the MWCNTs clusters generate
thermal energy41 that rapidly diuses into the subcellular
compartments, where the nanoconjugates are present.
Laser-induced thermal ablation o cancer cells labeled
with HSAMWCNTs may be used in two main modes:
pulsed and continuous. The pulsed mode produces localized
(ew micrometers) damage o individual cancer cells by
laser-induced micro- and nanobubbles around overheated
nanoparticles without harmul eects on the surrounding
healthy cells.42 It particularly avors in vivo killing o single
circulating tumor cells using just 1 ns laser pulses. The second
mode is more time consuming (a ew minutes o exposure) and
results in the eects o thermal denaturation and coagulation
as main mechanisms o cell damage. It is more appropriate or
the treatment o primary tumors measuring a ew millimeters
or more.42
1 minute
1 hour 3 hours
Median
deadcellspercent(%)
Median
deadcellspercent(%)
Media
ndeadcellspercent(%)
Mediandeadcellspercent(%)
Median
deadcellspercent(%)
88.24
57.12
60.22
64.373.12
35.45
22.26
22.19
24.67
10.28
14.3711.34
6.03
100
5 hours
100
100
95.1
94.9
91.32
84.32
67.4
37.540.9
68.11
82.35
91.00
90.02
82.42
65.67
36.14
100
94.44
94.5
87.24
86.56
80.78
48.22
19.43
20.72
21.34
15.2614.62
12.87
51.64
52.1
83.24
84.13 87.88
88.34
78.92
8.28
6.78
65.79
87.89
84.12
30 minutes
92.34
68.8664.23
70.78
81.24
59.34
33.72
36.8434.78
10.22
13.19
14.67
8.77
7.98
9.89
75.7
0
20
40
60
80
100
CRL-4020
(MWCNTs)
HepG
2(MWCNTs)
CRL-4020
(HSA
-MWCNTs)
HepG
2(HSA
-MWCNTs)
0
20
40
60
80
100
1mg/L5m
g/L
20mg/L
50mg/L
CRL-4020
(MWCNTs)
HepG
2(MWCNTs)
CRL-4020
(HSA
-MWCNTs)
HepG
2(HSA
-MWCNTs)
0
20
40
60
80
100
1mg/L5m
g/L
20mg/L
50mg/L
CRL-4020
(MWCNTs)
HepG
2(MWCNTs)
CRL-4020
(HSA
-MWCNTs)
HepG2
(HSA
-MWCNTs)
0
20
40
60
80
100
1mg/L5m
g/L
20mg/L
50mg/L
CRL-4020
(MWCNTs)
HepG
2(MWCNTs)
CRL-4020
(HSA
-MWCNTs)
H
epG2(HSA-MWCNTs)
0
20
40
60
80
100
1mg/L5m
g/L
20mg/L50m
g/L
CRL-4020
(MWCNTs)
HepG
2(MWCNTs)
CRL-4020
(HSA
-MWCNTs)
H
epG2
(HSA-MWCNTs)
0
20
40
60
80
100
1mg/L5m
g/L
20mg/L50mg
/L
CRL-4020
(MWCNTs)
HepG
2(MWCNTs)
CRL-4020
(HSA
-MWCNTs)
HepG2
(HSA
-MWCNTs)
1mg/L5m
g/L
20mg/L
50mg/L
Media
ndeadcellspercent(%)
40.65
75.45
90.02 78.8
38.71
39.25
29.11
30.92
27.81
24 hours
100
100
100
100
52.2
85.9
91.01
97.08
90.45
84.69
51.11
49.08
82.51
89.92
95.23
95.34
Figure 5 rult of xpimntal iat xpou to nanomatial (ontol v MWcNThsA) in diffnt onntation, followd by la iadiation. Ba pnt
t ava pnta of dad ll (%).
Abbreviations: hsA,uman um albumin; MWcNT, multiwalld abon nanotub.
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Nanopototmolyi of liv an in an in vito aay uin albumin-onjuatd abon nanotub
The use o continuous laser irradiation proved sig-
nicant dierences in HepG2 postirradiation apoptotic
percentage (P, 0.05) or concentrations o,20 mg/L,
at 60 sec and 30 min, compared with the apoptotic rate
o CRL-4020. This nding may be particularly relevant
or low concentrations o HSAMWCNTs (eg, plasma
levels ater intra-arterial administration).43 It has been
previously stated that the mechanism o HepG2 uptake
or albumin is a caveolae-dependent endocytosis similar
to that or other types o ligands such as cholesterol or
olic acid.44 The mechanism represents a distinct orm o
transport and elicits eatures dierent rom independent
or clathrin-mediated endocytosis. Ater internalization o
caveolae, the biomaterials are accumulated in caveosomes,
a specic type o organelles.45 Folic acid has been intensely
studied or its potential in targeted therapies. Signicant
results were obtained ater binding olate-unctionalized
poly (ethylene glycol)-coated nanoparticles to the targeted
receptor (olate receptor).46 Within the eld o chemo-
therapy, caveolae-mediated transport mechanisms have
been largely used or targeted drug delivery. The pathway
has been preerred as it was demonstrated to be a nondeg-
radative mechanism using pH-dependent chemotherapy
release. For instance, a combination o cytostatic drugs
and albumin called Trexall (Duramed Pharmaceuticals,
New York, NY, USA) is currently prescribed or the treat-
ment o metastatic liver cancer in humans.47 The literature
has already suggested new ideas o targeted therapies that
could elude lysosomal harmul transit and will thereore
oer a higher protection level or drug compounds.48 A
specic endothelin receptor associated with the described
uptake mechanism is the Gp60 receptor (albondin).49 Using
phase contrast, conocal, and TEM, we demonstrated in
this study, without precedent, that the mechanism o HSA
MWCNT uptake in HepG2 cells occurs through caveolae-
dependent endocytosis initiated by the albumin-binding
Gp60 receptor (albondin) (Figure 4).
In the present study, we observed that in the treatment o
HepG2 cells with high concentrations o HSAMWCNTs
or more than 5 h, the percentage o necrotic HepG2 cells
is not signicantly dierent rom that o epithelial cells.
This nding suggests a nonselective, passive intracellular
diusion o nanomaterial inside the cells when the cells are
exposed to high concentrations o nanomaterials or long
periods o time.
In contrast, we obtained a selective lysis o HepG2 cells
treated with HSAMWCNTs or incubation periods shorter
than 30 min, regardless o the concentration. In cellular
systems, the molecular membrane association/dissociation
processes are very short, ranging rom seconds to minutes.50
Thereore, our nding could be o decisive importance when
using HSAMWCNTs or the in vivo targeting o liver
cancer cells.
ConclusionWe have developed a method o unctionalization o CNT
with human albumin or the selective targeting o liver
cancer cells. Moreover, to our knowledge, this is the rst
evidence o improved selective thermal ablation o liver
cancer cells using HSAMWCNTs compared with the
normal epithelial cells. Based on the results presented
here, we believe that HSAMWCNTs selectively attach to
albondin (aka Gp60) receptor located on HepG2 cell mem-
brane, ollowed by uptake through a caveolin-dependent
endocytosis process.
These results may represent a rst step in the process
o complete in vivo elimination o liver cancer cells using
nanolocalized thermal ablation by means o laser heating.
However, urther research is required in order to ully
understand the mechanisms o selective binding o HSA
MWCNTs in malignant cells.
Nevertheless, urther investigations are also required
or the careul assessment o unexpected toxicities and
biological interactions o HSAMWCNTs inside the living
organism.
AcknowledgmentsThe authors acknowledge grant support rom the Romanian
Ministry o Research (CNMP-PNCDI II: NANOPAN 41-009
and NANOHEP 42-115). This research was also supported
by Romanian Society o Nanomedicine.
DisclosureThe authors report no conficts o interest in this work.
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