Journal of Biomedical & Pharmaceutical Engineering 1:1 (2007) 13-16

ISSN: 1793-4532
All Rights Reserved

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Cellular Uptake Enhancement of Polyamidoamine Dendrimer

Modified Single Walled Carbon Nanotubes
B.F. Pan, D.X. Cui, P. Xu, T. Huang, Q. Li, R. He, F. Gao
Department of Bio-nano-Science and Engineering, National Key Laboratory of Nano/Micro Fabrication
Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of
Micro-Nano Science and Technology, Shanghai JiaoTong University, 1954 Huashan Road, Shanghai
200030, Peoples Republic of China
Corresponding Author: Daxiang Cui; Mailing Address: Institute of Micro-Nano Science and Technology, Shanghai Jiao Tong
University, 1954 Huashan Road, Shanghai 200030, Peoples Republic of China; Tel & Fax: +86-21-62933291; Email:
dxcui@sjtu.edu.cn

Abstract
Carbon nanotubes represent a new class of gene transporters potentially useful for future in vitro and in vivo gene
delivery applications. The surfaces of single walled carbon nanotubes (SWCNTs) were modified with
polyamidoamine dendrimer (PAMAM) to reduce cytotoxicity and enhance the cellular uptake of the nanoparticles.
The PAMAM dendrimer coated SWCNTs were prepared and characterized. The effect of cellular uptake of
uncoated and dendrimer coated SWCNT on a human cancer cell line MCF-7 cells were investigated by 3-(4, 5Dimethylthiazol-2-y1)-2, 5-diphenyl tetrazoliumbromide (MTT) and transmittance electronic microscopy (TEM).
MTT analysis showed that the SWCNTs were toxic to MCF-7 cells, conversely, the dendrimer coated SWCNTs
were essentially non-toxic. Transmission electron microscopy analysis indicated that the dendrimer coated
SWCNTs entered the cytoplasm, while uncoated SWCNTs were excluded indicated that the SWCNTs-based
material endocytosis behaviour was dependent on surface characteristics. This phenomenon may find application in
high efficiency gene delivery system for cancer therapy.
Keywords: Polyamidoamine dendrimer, carbon nanotube, cellular uptake
Received 2 February 2007; Accepted 29 May 2007

INTRODUCTION

Surface modification of single walled carbon

nanotubes (SWCNTs) is a crucial factor that not only
determines the biocompatibility of these nanomaterials,
but also plays an important role in cell adhesion on
biomaterials [1-6]. The nature and adhesion capacity of
cells in the presence of SWCNTs [7] as well as the
subsequent cellular events such as endocytosis have not
yet been fully elucidated. Recently, scientists have
uncovered the ability of single-walled carbon nanotubes
(SWCNTs) to penetrate mammalian cells and further
transport various cargos inside cells, including small
peptides [2], the protein streptavidin [4], and nucleic

acids [6, 8]. On the other hand, polycations possessing a

buffering capacity, such as polyamidoamine (PAMAM)
dendrimer, show a high in vitro transfection activity
owing to the so-called proton sponge effect [8-11].
Herein, PAMAM dendrimer, a cationic polymer, was
chosen as a coating material for SWCNT due to its high
water solubility, low toxicity, non-immunogenic, and
non-antigenic properties. The influence of dendrimer
coated SWCNTs on human breast cancer MCF-7 cells
which were analyzed by MTT, TEM and morphological
observation show that coating markedly decreased
cytotoxicity. This phenomenon may be applied to the
rational design of high efficient gene delivery system
for cancer therapy.

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2007 Biomedical & Pharmaceutical Engineering Cluster, Nanyang Technological University

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Cellular Uptake Enhancement of Polyamidoamine Dendrimer Modified Single Walled Carbon Nanotubes

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EXPERIMENTAL SECTION

2.1 Materials

polycarbonate membrane and subsequently washed

with distilled water until the pH of the filtrate was ca. 7,
yielding SWCNT-COOH. 0.2g amine terminated
PAMAM dendrimer was added to the solution, placed
in an ultrasonic bath for 60 min and stirred for 24 h at
50C. The mixture was subsequently filtered and
washed three times with water. The product was
dispersed in water to give a SWCNT-PAMAM aqueous
solution.
2.3 Transmission electron microscopy (TEM) studies
The average particle size, size distribution and
morphology of SWCNTs and dendrimer coated
SWCNTs were examined using a transmission electron
microscope ((Hitachi H-700H) at a voltage of 80 kV.
The aqueous dispersion of the particles was drop-cast
onto a carbon-coated copper grid and the grid was air
dried at room temperature before viewing under the
microscope.

Figure 1: Chemical structures of polyamidoamine

(PAMAM) dendrimers with generation from 1.0 to 4.0
(G1.0, G2.0, G3.0, and G4.0).
SWCNTs were purchased from the Shenzhen
Nanoport Company. Polyamidoamine (PAMAM)
dendrimers were kept in our Lab and their chemical
structures are shown in figure 1. The MCF-7 cells were
obtained from Shanghai Wuli Biotechnology. DMEM
containing 10% fetal calf serum (FCS, Gibco BRL),
penicillin (100 U/ml), streptomycin (100g/mL), Lglutamine 2mM (ICN Biomedicals, Costa Mesa, CA,
U.S.A.), and amphotericin B 2.5g/mL (Sigma-Aldrich)
were obtained from Gibco (BRL, Gaithersburg, MD,
U.S.A.). All the cell cultures were maintained at 37C
in a humidified atmosphere of 5% CO2. 3-(4,5Dimethylthiazol-2-y1)-2,5-diphenyl tetrazoliumbromide
(MTT) was purchased from Sino-American Biotec.

Figure 2: Complexation between carboxylated

SWCNTs and amine terminated PAMAM dendrimer.
2.2 Preparation of dendrimer modified SWCNT
nanocomposite (Figure 2)
As shown in Figure 2, pristine SWCNTs were added
to 98% H2SO4/10M HNO3 (v/v=3:1). The mixture was
placed in an ultrasonic bath for 60 min and then stirred
for 24 h while being boiled under reflux. The product
was vacuum-filtered through a 0.22 m Millipore

2.4 In vitro cell viability/cytotoxicity studies

To determine cytotoxicity of coated and uncoated
SWCNTs, the MCF-7 cells were plated at a density of
1104 cells/well in a 96 well plate at 37C in 5% CO2
atmosphere. After the cells were cultured for 24 h, the
medium in the wells was replaced with fresh medium
containing dendrimer coated SWCNTs in concentration
range 02.0 mg/ml. After 24 h, 20 ml of MTT dye
solution (5 mg/ml in phosphate buffer pH 7.4) was
added to each well. After 4 h of incubation at 37C, the
medium was removed and formazan crystals were
solubilized with 200 ml of dimethylsulphoxide (DMSO)
and the solution was vigorously mixed to dissolve the
reacted dye. After 15 min, the absorbance of each well
was read on a microplate reader (DYNATECH
MR7000 instrument) at 570 nm. The spectrophotometer
was calibrated to zero absorbance, using culture
medium without cells. The relative cell viability (%)
related to control wells containing cell culture medium
without nanoparticles was calculated by:
Cell viability (%) = Atest/Acontrol 100.
Where Atest is the absorbance of the test sample and
Acontrol is the absorbance of control sample.
2.5 TEM observation of MCF-7 cell uptake of
dendrimer modified SWCNTs
The MCF-7 cells were seeded onto 13-mm glass
coverslips in a 24 well plate at a density of 1104 cells
per well in 1 ml of complete medium for 24 h, after
which the growth medium was removed and replaced
with the medium containing dendrimer coated
SWCNTs (0.1 mg/ml). For control experiments,
medium without SWCNTs was used. After 12 and 24 h
of culture, the cells were fixed with 1.5%
glutaraldehyde (Sigma) buffered in 0.1 M sodium
cacodylate (4C, 1 h). The cells were then post-fixed in
1% osmium tetroxide. The cells were subsequently
embedded in Araldite resin, and ultra-thin sections (60
nm) cut with glass knives were stained with lead nitrate
for TEM observation.

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Cellular Uptake Enhancement of Polyamidoamine Dendrimer Modified Single Walled Carbon Nanotubes

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RESULTS AND DISCUSSION

3.1 Characterization of dendrimer coated SWCNTs

The size and morphology of SWCNTs and dendrimer
coated SWCNTs were determined by TEM. Figure 3(a)
show that the SWCNTs were ~8 nm in diameter with
length over 1m. The morphology of the SWCNTs after
dendrimer coating was greatly changed as shown in
Figure 3(b). SWCNTs were shortened after HNO3
treatment from Figure 3(b), the average length of
SWCNTs was about 400nm. A thick layer of PAMAM
dendrimer coated onto the SWCNTs surfaces indicated
the formation of the nanohybrid.

Figure 3: TEM images of (a) SWCNTs and (b) G4.0

PAMAM dendrimer coated SWCNTs.
3.2 In vitro cell viability/cytotoxicity studies

water-soluble dye to a dark blue insoluble formazan

product. Formazan crystals were dissolved in DMSO
and quantified by measuring the absorbance of the
solution at 570 nm, with the resultant value related to
the number of living cells. Figure 4 demonstrates a
dose-dependent reduction in MTT absorbance in cells
treated with coated SWCNTs (concentration range 0
1.0 mg/ml) for 24 h. As shown in figures 4 (b) ~ (e),
dendrimer coated SWCNTs showed lower cytotoxic
effects to MCF-7 cells. G1.0 PAMAM dendrimer
coated SWCNTs were more cytotoxic than G2.0, G3.0,
G4.0 materials. For example, G2.0, G3.0, G4.0
PAMAM dendrimer coated SWCNTs retained cell
viabilities of 99% as compared with the control at
concentration as high as 1.0 mg/ml. No marked cell
death was observed in the case of dendrimer coated
SWCNTs. These results indicated that the PAMAM
dendrimer coated SWCNTs exhibit little toxicity to
MCF-7 cells. However, the SWCNTs caused cell
apoptosis and death after incubation with MCF-7 cells
for 24 h as shown in Figure 4 (a). The degree of cell
death was substantial as evidenced by the large amounts
of cell debris observed. SWCNTs caused a significant
reduction (96% of control) in cell viability even at 0.2
mg/ml concentration, and induced further reductions at
higher concentrations, reaching a plateau around 0.5
mg/ml. At the highest concentration tested (1.0 mg/ml),
it resulted in about 13% loss of cell viability.
3.3 TEM observation of cellular uptake of dendrimer
modified SWCNTs
TEM images show that dendrimer coated SWCNTs
entered the MCF-7 cells after 24 h incubation (Figure
5). Several electron lucent voids containing SWCNTs
could be seen in the cytoplasm of the MCF-7 cells and
form vacuoles. The dendrimer modified SWCNTs
entered the cells via endocytosis. In control
experiments, little SWCNTs were located inside cells
after incubation with uncoated materials (data not
shown), which could not easily traverse cell membranes
into cytoplasm. In our previous studies, it was observed
that uncoated SWCNTs entered HEK293 cell with
difficulty, but could easily enter HL-60 cells [6, 8].

Figure 4: Cytotoxicity profiles of SWCNTs and

dendrimer coated SWCNTs, when incubated with
MCF-7 cells as determined by MTT assay. (a)
SWCNTs, (b) G1.0 PAMAM dendrimer coated
SWCNTs, (c) G2.0 PAMAM dendrimer coated
SWCNTs, (d) G3.0 PAMAM dendrimer coated
SWCNTs, (e) G4.0 PAMAM dendrimer coated
SWCNTs.
To examine the effect of coating on MCF-7 cell
growth and viability, MCF-7 cells were incubated with
coated SWCNTs complex for 24 hours, isolated by
centrifugation, and incubation continued for 72 h for
MTT assay. Viable cells reduced MTT from a yellow

Figure 5: TEM pictures of MCF-7 cells incubated with

G4.0 dendrimer coated SWCNTs showing SWCNTs
internalization after 24 h incubation.

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Cellular Uptake Enhancement of Polyamidoamine Dendrimer Modified Single Walled Carbon Nanotubes

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The colloidal solution of dendrimer coated SWCNTs

show very high stability at neutral pH with no
sedimentation observed even after 2 months of storage
at room temperature. The strong anchoring of the
PAMAM dendrimer molecules on the surface of
SWCNTs sterically stabilized the SWCNTs. Size
distribution studies using TEM measurements show that
the SWCNTs had length less than 400nm with a
SWCNTs core and outer dendrimer coating (corecoating structure). Cell viability studies indicated that
the dendrimer coated SWCNTs reduced cell
cytotoxicity significantly as compared to the cells that
were exposed to the bare SWCNTs. Explanation for this
decrease in cell cytotoxicity may be that PAMAM
dendrimer is hydrophilic and biocompatible. TEM
studies indicated that MCF-7 cells quickly took up the
coated SWCNTs. The results indicated that the
dendrimer coated SWCNTs reduce cell cytotoxicity and
induce cellular uptake behavior distinct from the
SWCNTs. This suggested that SWCNT endocytosis
response was dependent on the SWCNT coating.
PAMAM dendrimer coated SWCNTs may prove useful
for gene and drug delivery. These findings suggest the
possibility to deliver coated SWCNTs into different
tissues with high uptake efficiency.

CONCLUSIONS

In this paper, dendrimer coated SWCNTs of length

about 400nm having a SWCNT core and dendrimer
coating had been prepared and characterized in vitro.
The colloidal solution of dendrimer coated SWCNTs
show high stability. The results of cell culture
experiments show that the dendrimer coated SWCNTs
were non-toxic as compared to uncoated SWCNTs.
While SWCNTs could not directly enter cells in large
amounts, they readily entered MCF-7 cancer cells after
binding with PAMAM dendrimer by endocytosis. The
uniqueness of PAMAM dendrimers as SWCNTs
transporters is emerging as potentially useful. The
biocompatibility, unique physical, electrical, optical,
and mechanical properties of dendrimer coated SWCNT
provide the basis for new classes of materials for drug,
protein, and gene delivery applications. This may allow
the development of a versatile synthetic gene delivery
system that may help realize the potential of non-viral
gene therapy.

ACKNOWLEDGMENTS
This work was supported by the National Natural
Science Foundation of China (No. 30471599), the

National 973 project (2005CB724300-G), the Bio-X

DNA Computer Consortium (03DZ14025), the
Shanghai Development Foundation of Science and
Technology (No. 03ZR14057, No.054119527), and the
2003 Major Basic Research Program of Shanghai (No.
03DJ14002).

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