Functional single-walled carbon nanotubes based on an integrin alpha v beta 3 monoclonal antibody for highly efficient cancer cell targeting.

Abstract

The application of single-walled carbon nanotubes (SWNTs) in the field of biomedicine is becoming an entirely new and exciting topic. In this study, a novel functional SWNT based on an integrin alpha(v)beta(3) monoclonal antibody was developed and was used for cancer cell targeting in vitro. SWNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). The PL-PEG functionalized SWNTs were then conjugated with protein A. A SWNT-integrin alpha(v)beta(3) monoclonal antibody system (SWNT-PEG-mAb) was thus constructed by conjugating protein A with the fluorescein labeled integrin alpha(v)beta(3) monoclonal antibody. In vitro study revealed that SWNT-PEG-mAb presented a high targeting efficiency on integrin alpha(v)beta(3)-positive U87MG cells with low cellular toxicity, while for integrin alpha(v)beta(3)-negative MCF-7 cells, the system had a low targeting efficiency, indicating that the high targeting to U87MG cells was due to the specific integrin targeting of the monoclonal antibody. In conclusion, SWNT-PEG-mAb developed in this research is a potential candidate for cancer imaging and drug delivery in cancer targeting therapy.

Full text

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IOP PUBLISHING
NANOTECHNOLOGY
Nanotechnology 20 (2009) 105102 (7pp)
Functional single-walled carbon
nanotubes based on an integrin αvβ3
monoclonal antibody for highly efficient
cancer cell targeting
doi:10.1088/0957-4484/20/10/105102
Zhongmin Ou1, Baoyan Wu1, Da Xing2, Feifan Zhou,
Huiying Wang and Yonghong Tang
MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science,
South China Normal University, Guangzhou 510631, People’s Republic of China
E-mail: xingda@scnu.edu.cn
Received 12 September 2008, in final form 23 December 2008
Published 16 February 2009
Online at stacks.iop.org/Nano/20/105102
Abstract
The application of single-walled carbon nanotubes (SWNTs) in the field of biomedicine is
becoming an entirely new and exciting topic. In this study, a novel functional SWNT based on
an integrin αvβ3monoclonal antibody was developed and was used for cancer cell targeting in
vitro. SWNTs were first modified by phospholipid-bearing polyethylene glycol (PL–PEG). The
PL–PEG functionalized SWNTs were then conjugated with protein A. A SWNT–integrin αvβ3
monoclonal antibody system (SWNT–PEG–mAb) was thus constructed by conjugating protein
A with the fluorescein labeled integrin αvβ3monoclonal antibody. In vitro study revealed that
SWNT–PEG–mAb presented a high targeting efficiency on integrin αvβ3-positive U87MG cells
with low cellular toxicity, while for integrin αvβ3-negative MCF-7 cells, the system had a low
targeting efficiency, indicating that the high targeting to U87MG cells was due to the specific
integrin targeting of the monoclonal antibody. In conclusion, SWNT–PEG–mAb developed in
this research is a potential candidate for cancer imaging and drug delivery in cancer targeting
therapy.
1. Introduction
Frequent challenges encountered by current cancer treatments
fall into low specificity in cancer cell killing and low
drug delivering efficiency as well as serious side effects on
normal tissues [1, 2]. A rational strategy to couple the
therapeutics to a targeted molecule that recognizes tumor-
associated antigens, namely cancer targeting therapy, could
increase the target selectivity of cancer cells and overcome
the issues of deleterious side effects of traditional cancer
therapy [3, 4]. On the other hand, the high impermeability of
cell membranes to biological cargoes remains another barrier
to the high efficiency of drug delivery. Hence, the pursuit of
efficient and highly target-selective transporters to have better
1Zhongmin Ou and Baoyan Wu contributed equally to this study.
2Author to whom any correspondence should be addressed.
drug delivery and molecular imaging is an active topic in
cancer targeting therapy.
Single-walled carbon nanotubes (SWNTs) have been
consideredpotentialbiomedicalmaterialsbecause oftheirflex-
ible structure and propensity for chemical functionalization;
moreover, cargoes can be attached to the surfaces (inner or
outer) or even packaged within the core of the tubes [5, 6].
The use of SWNTs in biological systems has opened up
an entirely new and exciting field because of the special
structural, electrical and optical properties of SWNTs [7].
Various research has been done focusing on the functional-
ization and immobilization of biomolecules on nanotubes for
characterization, manipulation, purification, separation, and
for device application such as sensors [8–11]. Recent studies
showed that SWNTs can shuttle various molecular cargoes,
including proteins [12, 13] and DNA oligonucleotides [14]
as well as anticancer drugs [6, 15], crossing through the
0957-4484/09/105102+07$30.00
© 2009 IOP Publishing LtdPrinted in the UK
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Nanotechnology 20 (2009) 105102Z Ou et al
cellular membrane without cytotoxicity due to their distinct
architecture, hollow interior and cagelike structures [16]. For
example, the functionalized soluble SWNTs can serve as
‘longboats’tocarry Pt(IV) prodrugsintocellsthroughclathrin-
dependent endocytosis [6]. The unique properties of SWNTs
as molecular transporters have a potential significance in the
field of biomedicine, which needs to be fully developed.
Integrins, which consist of heterodimeric, transmembrane
glycoprotein subunits (α and β), are a large family of cell
surface receptors [17]. One of the most prominent members
of this family is integrin αvβ3, which plays a key role in tumor
proliferation, metastasis and tumor-induced angiogenesis [18].
Integrin αvβ3was found to have high expressions on various
cancer cells (glioma, melanoma and ovarian) and tumor
neovasculature compared to normal tissues [19, 20], which
makes integrin αvβ3 a promising candidate as a cancer cell
targeting marker. Antagonists of integrin αvβ3, such as
monoclonal antibodies (mAbs) and arginine–glycine–aspartic
(RGD) peptide, have been found to inhibittumor angiogenesis,
cancer growth and cancer cell metastasis [21]. Although RGD
peptides are commonly used as binding ligands to integrin
αvβ3due to their small molecular size, drawbacks have also
been revealed: relatively low bindingspecificity was found due
totheir universaltargetingabilitytothe other integrins(α11bβ3,
αvβ6, α5β1) [22, 23]; moreover, some RGD peptide containers
are prone to degrade rapidly in vivo [24, 25]. Comparing with
RGD peptides, integrin αvβ3mAbs exhibit unique specificity
to integrin αvβ3due to their high selectivity to the antigenic
target and relative stability in vivo. Therefore, they might play
a potential role in effective drug delivery and target imaging in
cancer targeting therapy.
A variety of research has been done using RGD as a
targeting molecule; however, so far to our knowledge, no
research has been done to conjugate the integrin αvβ3mAb
with SWNTs as a transporter for the detection of cancer cells.
In this study, integrin αvβ3mAb was conjugated with SWNTs:
the antibodywas used as a specific targetingcomponent, aimed
at integrin αvβ3-positive cancer cells via specific integrin αvβ3
binding; SWNTs were used as a molecular carrier.
2. Materials and methods
2.1. Materials
The major agents used in our research are:
purchased from Chengdu Organic Chemicals Company
Ltd of the Chinese Academy of Sciences; 1,2-distearoyl-
sn-glycero-3-phosphoethanolamine-N-[carboxy (polyethylene
glycol) 2000] (PL-PEG), purchased from Avanti Polar Lipids
Inc.(Alabaster, AL, USA); integrin αvβ3 (23C6) mono-
clonal antibody (integrin αvβ3mAb), purchased from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA, USA); pro-
tein A, purchased from Beijing Biosynthesis Biotechnol-
ogy Co., Ltd (Beijing, China); fluorescein isothiocyanate
(FITC), N-hydroxysulfosuccinimide (NHS) and 1-ethyl-3-(3-
(dimethylamino)-propyl)carbodiimide(EDC), purchased from
Sigma-Aldrich Co. Since all the reagents were of analytical
grade and used without further purification.
SWNTs,
2.2. Preparation of functional SWNTs based on integrin αvβ3
monoclonal antibody
2.2.1. Functionalization of SWNTs by PL–PEG.
were sonicated in an aqueous solution of PL–PEG (1 mg of
SWNTs: 1 mg of PL–PEG: 1 ml of water) for 6 h.
mixture was then centrifugated at 10000 g for 15 min. The
sediment comprising of impurities, aggregates and bundles of
nanotubes at the bottom of the centrifuge tube was discarded,
then the supernatant was collected. Excess phospholipids were
completelyremovedbyrepeated filtrationusing100kDa filters
(Millipore) and rinsing with phosphate-buffered saline (PBS).
Then SWNTs were resuspended in PBS by sonication for 1 h.
Finally, the purified PL–PEG-functionalized SWNTs (SWNT–
PEG) were composed and were resuspended in PBS.
SWNTs
The
2.2.2. Conjugation of protein A and SWNTs.
PEG solution was activated by EDC/NHS to afford SWNT–
PEG–NHS (molar ratio, SWNT–PEG: EDC: NHS = 1:1:1).
SWNT–PEG–protein A was produced by incubating protein A
with SWNT–PEG–NHS (pH 7.4) for 4 h. The solution was
thenfiltrated using100kDafilters(Millipore)toremoveexcess
protein A.
The SWNT–
2.2.3.
antibody.
procedures according to Wisdom [26]. Briefly, the solution of
integrinαvβ3mAbata concentrationof40nMinstandardPBS
was mixed with 50 μl of sodium bicarbonate solution. The
solution was then mixed with FITC (13 mM, 100 μl) dissolved
in DMSO (Aldrich). After incubating the mixture for 1 h at
room temperature, protected from illumination, the conjugated
integrin αvβ3mAb–FITC was filtrated through 100 kDa filters
(Millipore) to remove excess FITC. The resultant fluorescein-
labeled protein solution was then diluted with PBS to a
concentration of 8 nM.
Fluorescent labeling of integrin αvβ3 monoclonal
Integrin αvβ3mAb was labeled with FITC by the
2.2.4. Preparation of SWNT–PEG–protein A–integrin αvβ3
monoclonal antibody–FITC.
of integrin αvβ3 mAb–FITC and the solution of SWNT–
PEG–protein A was incubated at room temperature for 4 h
(protectingfrom illumination)toproduceSWNT–PEG–protein
A–integrin αvβ3monoclonal antibody–FITC (abbreviated as
SWNT–PEG–mAb). Then the mixture was dialyzed against
PBS by using a membrane (molecular weight cutoff
500 kDa, PVDF) to remove unbound integrin αvβ3 mAb–
FITC. To ensure complete removal, the dialysis lasted 3
to 4 days with frequent replacement of the PBS buffer
until no fluorescence was detected in the rinse.
purified SWNT–PEG–mAb was collected. The concentration
of SWNTs in the solution after these processes was 0.3–
0.4 mg ml−1.
The mixture of the solution
=
Finally,
2.3. Cell culture
U87MG human glioblastoma cancer cells and MCF-7 human
breast cancer cells were cultured in Eagle’s minimal essential
medium (EMEM) and Dulbecco’s modified Eagle’s medium
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Nanotechnology 20 (2009) 105102Z Ou et al
(DMEM), respectively. The media were supplemented with
10% fetal bovine serum (FBS) and 1% penicillin–streptomycin
in 5% CO2, 95% air at 37◦C in a humidified incubator.
2.4. In vitro cytotoxicity assay of SWNT–PEG–mAb
A cell cytotoxicityassay of SWNT–PEG–mAb to U87MG was
performed by colorimetric tetrazolium salt-based assay using
Cell Counting Kit-8 (CCK8 Dojin Laboratories, Kumamoto,
Japan). Cell viability was determined by a 96-well plate reader
(INFINITE M200, Tecan, Switzerland) at an absorbance value
of 450 nm (OD450). The viability of cells was calculated
as: cell viability (% of control) = ODTre/ODCon × 100%
(where ODTre is the absorbance value of treated cells; ODCon
is the absorbance value of untreated cells). After culturing
U87MG cells and MCF-7 cells in a chamber slide for 24 h,
SWNT–PEG–mAb was added to each well at a final SWNT
concentration of 3–6 μg ml−1.
2.5. Characterization
2.5.1. Transmission electron microscope (TEM) measurement.
To examine the dispersion of PL–PEG functionalized SWNTs,
the SWNT–PEG sample was observed by a JEM-2010HR
transmission electron microscope (TEM) by dropping 3 μl of
the solution on top of a TEM grid.
2.5.2. Optical spectra measurements.
theintrinsicopticalproperties ofSWNTs, Ramanspectroscopy
was used to directly detect SWNT–PEG–mAb solution
using capillaries without spinning or stirring during the
measurements. An argon ion laser (514.5 nm) was used for
excitation in combination with a 40× objective of an Olympus
BX-41 microscope, a Acton spectro@2300i spectrometer
system (Princeton Acton, USA) and a Pixis 256 CCD detector
(Princeton Acton, USA). After focusing at the center of the
capillary, the Raman spectrum of the sample with a resolution
of 2 cm−1(10 mW power, 20 s collection time) was recorded.
The optical absorbance and fluorescence characteristics
of SWNT–PEG–mAb were investigated by UV–vis absorption
spectra (Lambda-35 UV–vis spectrophotometer,
Elmer, USA) and fluorescence spectra (LS-55 fluorescence
spectrophotometer, Perkin-Elmer, USA) with an excitation of
490 nm.
To take advantage of
Perkin-
2.5.3. Confocal microscopy.
SWNT–PEG–mAb at 37◦C (5% CO2) for 2 h, the cells
were rinsed by fresh culture medium and were imaged by a
commerciallaser scanningmicroscope(LSM 510/ConfoCor2)
combination system (Zeiss, Germany) equipped with a Plan-
Neofluar 40×/1.3 NA oil DIC objective.
After incubating the cells with
3. Results and discussion
3.1. Fabrication of SWNT–PEG–mAb
Figure 1(a) shows the schematic procedures of producing the
SWNT–PEG–mAb. Firstly, SWNTs were solubilized in the
aqueous phase by non-covalent adsorption of phospholipid
molecules with PL–PEG chains and a terminal carboxylic
acid group (PL–PEG–COOH). On the base of this PL–
PEG functionalization, the carboxylic terminal of PL–PEG
immobilized on SWNTs can then be used to conjugate with
a wide range of biological molecules. Secondly, protein A was
coupled to PEG–SWNT after activation with EDC/NHS based
on the reaction between the NHS group of PEG–SWNT. Note
that protein A was bound to the Fc portion of the antibody,
allowing the functional portions (Fv) of the mAb to recognize
integrin αvβ3. On the other hand, FITC was conjugated
to integrin αvβ3 mAb via the primary amines. Finally, the
SWNT–PEG–protein A was conjugated to integrin αvβ3mAb–
FITC to produce SWNT–PEG–mAb.
It is well known that carbon nanotubes are significantly
hydrophobic: therefore, the transporting capability of SWNTs
requires chemical functionalization with water-solubilizing
moieties to increase its solubility. PEG is a well-established,
benign bio-passivating material [10]. PL–PEG can be well
conjugated to SWNTs via van der Waals and hydrophobic
interactions between two PL alkyl chains and the SWNT
sidewall [14], with the PEG chain extending into the
aqueous phase to impart solubility in water.
diameter and length of SWNT–PEG were about 1–4 nm
and 500 nm–1 μm (figure 1(b)).
showed that non-covalently functionalized SWNTs with PEG
have a higher resistance to systemic clearance than that
without PEG functionalization, because the sheath of PEG
can prevent clearance of SWNTs by the reticuloendothelial
system and can prolong their persistence in blood and humor
circulation [10, 27].
The typical
Previous publications
3.2. Characterization of SWNT–PEG–mAb
Non-covalently functionalized SWNTs exhibit a strong
resonance Raman shift at ∼1580 cm−1(G band, characteristic
of graphitic carbon) [28].
composition of SWNT–PEG–mAb, Raman spectroscopy was
used to directly detect SWNT–PEG–mAb in solution.
shown in figure 2, the G band peak area was integrated
∼1580 cm−1.
existence of SWNTs inside the final sample as indicated by
the black color of the final sample (the inset of figure 2).
Moreover, the suspensions of SWNT–PEG–mAb were well
solubilized and stable in physiological media for at least two
weeks at room temperature without any visible aggregation.
The stabilization of SWNT–PEG–mAb revealed in our study
should be attributed to non-covalent interaction between PL–
PEG and SWNTs [27].
The optical absorbance and fluorescence characteristics
of SWNT–PEG–mAb were investigated by UV–vis absorption
spectra and fluorescence spectra.
integrin αvβ3mAb–FITC was removed by dialysis in all of
the final samples, the optical absorbance and fluorescence
emission detected were the result of SWNT–PEG–mAb alone.
Figure 3(a) shows the absorbance peak of SWNT–PEG–mAb
is at 490 nm (thick curve) (on top of the characteristic
SWNT–PEG absorption spectrum (dotted curve)); the peak
To confirm the successful
As
Raman spectroscopy clearly revealed the
Since the unbound
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Nanotechnology 20 (2009) 105102Z Ou et al
(a)
(b)
Figure 1. (a) An illustration of the procedures of producing SWNT–PEG–mAb. (b) A typical TEM image of SWNT–PEG.
of free integrin αvβ3mAb–FITC (thin curve) is quite similar
to that of SWNT–PEG–mAb, confirming a fine conjugation
betweenintegrinαvβ3mAb–FITC andPL–PEG functionalized
SWNTs.With an excitation of 490 nm, the fluorescence
generated from SWNT–PEG–mAb exhibited a much weaker
fluorescence than free integrin αvβ3mAb–FITC at the same
concentration (figure 3(b)). The fluorescence quenching of
FITC was probably due to the energy transfer between FITC
and SWNTs, which is analogous to the previous results of
SWNT-bound pyrene [29, 30]. The fluorescence quenching in
SWNT–PEG–mAb further confirms integrin αvβ3–FITC was
well coupled on PEG-functionalized SWNTs.
3.3. Cytotoxicity of SWNT–PEG–mAb
An important concern of SWNT–PEG–mAb in vivo applica-
tions is its cellular cytotoxicity. To investigate the cytotoxicity
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Nanotechnology 20 (2009) 105102Z Ou et al
Figure 2. Raman spectra of SWNT–PEG–mAb and a photograph of
stable SWNT–PEG–mAb solution (inset).
of SWNT–PEG–mAb, U87MG were cultured in a 96-well
microplate (1×103per well) for 24 h and were then incubated
with SWNT–PEG–mAb at different concentrations (1.5, 3, 6
and 12 μg ml−1) for 12 h. After rinsing with PBS, the cells
were cultured for another 72 h at 37◦C. The viability of cells
was calculated and no obvious toxic effect of SWNT–PEG–
mAb on U87MG cells was observed compared with the non-
addition of SWNT–PEG–mAb on U87MG cells, suggesting
the low toxicity of SWNT–PEG–mAb.
3.4. Cancer cell targeting of SWNT–PEG–mAb
To investigate the targeting efficiency of SWNT–PEG–mAb,
integrin αvβ3-positive cells (U87MG) and integrin αvβ3-
negative cells (MCF-7) were incubated with SWNT–PEG–
mAb, respectively.The fluorescence of fixed cells was
detected by confocal microscopy: much higher fluorescence
signals were observed in U87MG cells (figure 4(a)), whereas
no obvious fluorescence was observed in MCF-7 cells
(figure 4(b)), indicating the specific targeting of integrin αvβ3-
positive U87MG cells was due to the specific recognition
of integrin αvβ3 on the cytomembrane by the integrin
αvβ3 monoclonal antibody.
impermeable to FITC [12], the high fluorescence signals in
figure 4(a) correspond to FITC labeled on the SWNT–PEG–
mAb conjugation taken up by U87MG cells. Because of low
integrinαvβ3expressioninMCF-7cells, almostnotargetingof
SWNT–PEG–mAb at MCF-7cellswas observed. Toutilizethe
potential molecular target, SWNT–PEG–mAb can selectively
recognize integrin αvβ3-positive cancer cells with integrin
αvβ3mAb, and afford no destruction of integrin αvβ3-negative
cancer cells and normal cells.
In order to have further confirmation that the main
uptake mechanism of SWNT–PEG–mAb is due to the
integrin αvβ3 receptor-mediated delivery pathway, U87MG
cells were treated with SWNT–PEG–mAb in the presence
of integrin αvβ3 antibody (5 nM) and characterized by
confocal microscopy. As shown in figure 4(c),
targeting of SWNT–PEG–mAb was significantly blocked
by integrin αvβ3 mAb: the fluorescence level detected in
Since the cytomembrane is
the
(a)
(b)
Figure 3. (a) Absorbance spectra and of SWNT–PEG–mAb (thick
curve), SWNT–PEG (dotted curve) and mAb–FITC (thin curve);
(b) fluorescence emission spectra of SWNT–mAb–FITC (thick
curve) and mAb–FITC (thin curve) in PBS with an excitation of
490 nm.
the control group was significantly lower than that of the
experiment group, indicating that the main delivery pathway
of SWNT–PEG–mAb is via integrin αvβ3acceptor-mediated
endocytosis [31–34]. Moreover, the block result provides
evidence of the existence of integrin αvβ3 mAb on the
surface of SWNTs. Hence, the confocal microscopy results
together with the Raman, absorbance and fluorescence results
above confirmed that SWNT–PEG–mAb conjugation was
successfully prepared, as shown in figure 1. The results above
hintthat the SWNT–PEG–mAb system with an efficient cancer
targeting delivery ability has the potential to function as a
transport matrix for various biological cargoes into cancer
cells.
4. Conclusion
In conclusion, with a combination of integrin αvβ3-mAb (an
ideal targeting molecular) and SWNTs (a molecular carrier),
an effective target delivering molecular transporter, SWNT–
PEG–mAb, was successfully developed. SWNT–PEG–mAb
shows high aqueous dispersibility and stability as well as
5