IJN 15841 Enhanced Laser Thermal Ablation for in Vitro Liver Cancer de 011311

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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.

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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

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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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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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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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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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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.

References1. Wong RJ, Corley DA. Survival dierences by race/ethnicity and treat-

ment or localized hepatocellular carcinoma within the United States.

Dig Dis Sci. 2009;54(9):20312039.

2. Varela M, Bruix J. Hepatocellular carcinoma in the United States.

Lessons rom a population-based study in Medicare recipients.J Hepatol.

2006;44(1):810.

3. Bosch FX, Ribes J, Daz M, Clries R. Primary liver cancer: worldwide

incidence and trends. Gastroenterology. 2004;127(5 Suppl 1):S5S16.

4. Kiyosawa K, Umemura T, Ichijo T, et al. Hepatocellular carcinoma: recent

trends in Japan. Gastroenterology. 2004;127(5 Suppl 1):S17S26.


12/13


Dovepress

Dovepress

140

Ianu t al

5. Rampone B, Schiavone B, Martino A, Viviano C, Conuorto G.

Current management strategy o hepatocellular carcinoma. World J

Gastroenterol. 2009;15(26):32103216.

6. Lai EC, Fan ST, Lo CM, Chu KM, Liu CL, Wong J. Hepatic resection

or hepatocellular carcinoma. An audit o 343 patients.Ann Surg. 1995;

221(3):291298.

7. Cance WG, Stewart AK, Menck HR. The National Cancer Data Base

Report on treatment patterns or hepatocellular carcinomas: improved

survival o surgically resected patients, 19851996. Cancer. 2000;88(4):

912920.

8. Ferrari M. Cancer nanotechnology: opportunities and challenges.Nat

Rev Cancer. 2005;5(3):161171.

9. Chakravarty P, Marches R, Zimmerman NS, et al. Thermal ablation o

tumor cells with antibody-unctionalized single-walled carbon nano-

tubes.Proc Natl Acad Sci U S A. 2008;105(25):86978702.

10. Liu Z, Tabakman S, Welsher K, Dai H. Carbon nanotubes in biology

and medicine: in vitro and in vivo detection, imaging and drug delivery.

Nano Res. 2009;2(2):85120.

11. Liang P, Wang Y. Microwave ablation o hepatocellular carcinoma.

Oncology. 2007;72 Suppl 1:S124S131.

12. Wang ZY, Song J, Zhang DS. Nanosized As2O

3/Fe

2O

3complexes com-

bined with magnetic fuid hyperthermia selectively target liver cancer

cells. World J Gastroenterol. 2009;15(24):29953002.

13. Cardinal J, Klune JR, Chory E, et al. Noninvasive radiorequency abla-

tion o cancer targeted by gold nanoparticles. Surgery. 2008;144(2):

125132.14. Lapotko D, Lukianova E, Potapnev M, Aleinikova O, Oraevsky A.

Method o laser activated nano-thermolysis or elimination o tumor

cells. Cancer Lett. 2006;239(1):3645.

15. Welsher K, Liu Z, Daranciang D, Dai H. Selective probing and imaging

o cells with single walled carbon nanotubes as near-inrared fuorescent

molecules.Nano Lett. 2008;8(2):586590.

16. Bianco A, Kostarelos K, Partidos CD, Prato M. Biomedical applications

o unctionalised carbon nanotubes. Chem Commun (Camb). 2005;(5):

571577.

17. Srinivasan C. Carbon nanotubes in cancer therapy. Curr Sci. 2008;

94(3):300301.

18. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R.

Nanocarriers as an emerging platorm or cancer therapy. Nat

Nanotechnol. 2007;2(12):751760.

19. Bhirde AA, Patel V, Gavard J, et al. Targeted killing o cancer cellsin vivo and in vitro with EGF-directed carbon nanotube-based drug

delivery.ACS Nano. 2009;3(2):307316.

20. Dumortier H, Lacotte S, Pastorin G, et al. Functionalized carbon

nanotubes are non-cytotoxic and preserve the unctionality o primary

immune cells.Nano Lett. 2006;6(7):15221528.

21. Shi Kam NW, Jessop TC, Wender PA, Dai H. Nanotube molecular

transporters: internalization o carbon nanotube-protein conjugates into

Mammalian cells.J Am Chem Soc. 2004;126(22):68506851.

22. Ghosh S, Dutta S, Gomes E, et al. Increased heating eciency and

selective thermal ablation o malignant tissue with DNA-encased

multiwalled carbon nanotubes.ACS Nano. 2009;3(9):26672673.

23. Schipper ML, Nakayama-Ratchord N, Davis CR, et al. A pilot toxicol-

ogy study o single-walled carbon nanotubes in a small sample o mice.

Nat Nanotechnol. 2008;3(4):216221.

24. Di Steano G, Fiume L, Bolondi L, Lanza M, Pariali M, Chieco P.Enhanced uptake o lactosaminated human albumin by rat hepatocar-

cinomas: implications or an improved chemotherapy o primary liver

tumors.Liver Int. 2005;25(4):854860.

25. Kratz F. Albumin, a versatile carrier in oncology.Int J Clin Pharmacol

Ther. 2010;48(7):453455.

26. Kratz F. Albumin as a drug carrier: design o prodrugs, drug conjugates

and nanoparticles.J Control Release. 2008;132(3):171183.

27. Dennis MS, Jin H, Dugger D, et al. Imaging tumors with an albumin-

binding Fab, a novel tumor-targeting agent. Cancer Res. 2007;67(1):

254261.

28. Tiruppathi C, Finnegan A, Malik AB. Isolation and characterization o

a cell surace albumin-binding protein rom vascular endothelial cells.

Proc Natl Acad Sci U S A. 1996;93(1):250254.

29. Tiruppathi C, Song W, Bergeneldt M, Sass P, Malik AB. Gp60 activation

mediates albumin transcytosis in endothelial cells by tyrosine kinase-

dependent pathway.J Biol Chem. 1997;272(41):2596825975.

30. Heister E, Neves V, Tilmaciu C, et al. Triple unctionalisation o single-

walled carbon nanotubes with doxorubicin, a monoclonal antibody, and

a fuorescent marker or targeted cancer therapy. Carbon. 2009;47(9):

21522160.

31. Raa V, Cioani G, Nitodas S, et al. Can the properties o carbon

nanotubes infuence their internalization by living cells? Carbon. 2008;

46(12):16001610.

32. Suh Y, Aaq F, Khan N, Johnson JJ, Khusro FH, Mukhtar H. Fisetin

induces autophagic cell death through suppression o mTOR signaling

pathway in prostate cancer cells. Carcinogenesis. 2010;31(8):

14241433.

33. Kovtyukhova NI, Mallouk TE, Pan L, Dickey EC. Individual single-

walled nanotubes and hydrogels made by oxidative exoliation o carbon

nanotube ropes.J Am Chem Soc. 2003;125(32):97619769.

34. Socrates G. Infrared and Raman Characteristic Group Frequencies.

Tables and Charts. 3rd ed. Chichester (UK): John Wiley & Sons;

2001.

35. Maeda H, Ishida N, Kawauchi H, Tsujimura K. Reaction o

fuorescein-isothiocyanate with proteins and amino acids. I. Covalent

and non-covalent binding o fuorescein-isothiocyanate and fuoresceinto proteins.J Biochem. 1969;65(5):777783.

36. Azamian BR, Davis JJ, Coleman KS, Bagshaw CB, Green ML.

Bioelectrochemical single-walled carbon nanotubes.J Am Chem Soc.

2002;124(43):1266412665.

37. Feng Y, Feng W, Noda H, et al. Photoinduced anisotropic response o

azobenzene chromophore unctionalized multiwalled carbon nanotubes.

J Appl Phys. 2007;102(5):053102053105.

38. Botos E, Klumperman J, Oorschot V, et al. Caveolin-1 is transported to

multi-vesicular bodies ater albumin-induced endocytosis o caveolae

in HepG2 cells.J Cell Mol Med. 2008;12(5A):16321639.

39. Xiao Y, Gao X, Taratula O, et al. Anti-HER2 IgY antibody-

unctionalized single-walled carbon nanotubes or detection and selec-

tive destruction o breast cancer cells.BMC Cancer. 2009;9:351.

40. Kam NW, OConnell M, Wisdom JA, Dai H. Carbon nanotubes as

multiunctional biological transporters and near-inrared agents orselective cancer cell destruction. Proc Natl Acad Sci U S A. 2005;

102(33):1160011605.

41. Dresselhaus MS, Dai H. Carbon nanotubes: continued innovations and

challenges.MRS Bull. 2004;29(4):237243.

42. Zharov VP, Galitovskaya EN, Johnson C, Kelly T. Synergistic enhance-

ment o selective nanophotothermolysis with gold nanoclusters:

potential or cancer therapy.Lasers Surg Med. 2005;37(3):219226.

43. Cherukuri P, Gannon CJ, Leeuw TK, et al. Mammalian pharmacokinet-

ics o carbon nanotubes using intrinsic near-inrared fuorescence.Proc

Natl Acad Sci U S A. 2006;103(50):1888218886.

44. Chang WJ, Rothberg KG, Kamen BA, Anderson RG. Lowering the

cholesterol content o MA104 cells inhibits receptor-mediated transport

o olate.J Cell Biol. 1992;118(1):6369.

45. Tiruppathi C, Naqvi T, Wu Y, Vogel SM, Minshall RD, Malik AB.

Albumin mediates the transcytosis o myeloperoxidase by means ocaveolae in endothelial cells.Proc Natl Acad Sci U S A . 2004;101(20):

76997704.

46. Dauty E, Remy JS, Zuber G, Behr JP. Intracellular delivery o nano-

metric DNA particles via the olate receptor.Bioconjug Chem. 2002;

13(4):831839.

47. Garber K. Stromal depletion goes on trial in pancreatic cancer.J Natl

Cancer Inst. 2010;102(7):448450.

48. Bathori G, Cervenak L, Karadi I. Caveolae an alternative endocytotic

pathway or targeted drug delivery. Crit Rev Ther Drug Carrier Syst.

2004;21(2):6795.


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49. Bareord LM, Swaan PW. Endocytic mechanisms or targeted drug

delivery.Adv Drug Deliv Rev. 2007;59(8):748758.

50. Fesce R, Meldolesi J. Peeping at the vesicle kiss.Nat Cell Biol. 1999;

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