Article

Journal of
Biomedical
Nanotechnology

Copyright 2013 American Scientic Publishers

All rights reserved
Printed in the United States of America

Vol. 9, 110, 2013

www.aspbs.com/jbn

Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles

Overcome Chemoresistance in Breast Cancer Cells
Yongzhong Wang, Sijia Yi, Leming Sun, Yujian Huang, Scott C. Lenaghan, and Mingjun Zhang
Nano Bio-Systems and Bio-Mimetics Lab, Department of Mechanical, Aerospace and Biomedical Engineering,
The University of Tennessee, Knoxville, TN 37996, USA

The purpose of this study was to design and fabricate a new cyclic peptide-based nanotube (CPNT) and to explore its
potential application in cancer therapy. For such a purpose, the CPNT bundles with a diameter of 10 nm and a length
of 5080 nm, self-assembled in a micro-scaled aggregate, were rst prepared using a glutamic acid and a cysteine
residue-containing cyclic octapeptide. In order to explore the potential application of these supramolecular structures, the
CPNTs were loaded with doxorubicin (DOX), and further modied using polyethylene glycol (PEG). The PEG-modied
DOX-loaded CPNTs, showing high drug encapsulation ratio, were nano-scaled dispersions with a diameter of 50 nm
and a length of 200300 nm. More importantly, compared to free DOX, the PEG-modied DOX-loaded CPNT bundles
demonstrated higher cytotoxicity, increased DOX uptake and altered intracellular distribution of DOX in human breast
cancer MCF-7/ADR cells in vitro. In addition, an enhanced inhibition of P -gp activity was observed in MCF-7/ADR cells by
the PEG-modied DOX-loaded CPNT bundles, which shows their potential to overcome the multidrug resistance in tumor
therapy. These ndings indicate that using cyclic peptide-based supramolecular structures as nanocarriers is a feasible
and a potential solution for drug delivery to resistant tumor cells.

KEYWORDS: Cyclic Peptide, Nanotube, Doxorubicin, Multidrug Resistance, Breast Cancer.

INTRODUCTION
Nanotubes are generally dened as an elongated nanoobject with a denite inner hole,1 which can be fabricated
from a variety of materials, such as inorganic, carbon,
amyloid proteins, synthetic polymers, and other organic
systems.1 Molecular self-assembly is a main bottom-up
approach for the production of this type of supramolecular structures.1 Despite considerable research in different types of nanotubes in recent years, the design and
fabrication of multifunctional nanotubes remains a signicant challenge to the scientic community. Creating
and developing new nanotubes with different supramolecular structures for medicine, diagnostics, drug delivery,
and tissue engineering, has been a fundamental strategy
to meet this challenge.2 In the past several decades, the
most extensively studied nanotube structures are carbon
nanotubes (CNTs). Considering the highly desirable properties of CNTs, they have been designed as nanocarriers

Author to whom correspondence should be addressed.

Email: mjzhang@utk.edu
Received: 29 January 2013
Accepted: 20 May 2013

J. Biomed. Nanotechnol. 2013, Vol. 9, No. xx

for drug and gene delivery, scaffolds for tissue engineering,

and smart devices for disease diagnosis.3 However, numerous concerns exist related to the toxicity, solubility and
biodegradability of CNTs,37 which presents a signicant
obstacle for the widespread use of CNTs in biomedical
applications.
Self-assembled cyclic peptide-based nanotubes (CPNTs),
formed from the stacking of individual cyclic peptides
(CPs),8 has garnered signicant attention since the pioneering work in 1993.9 Extensive studies have been
conducted about the design, self-assembly, and characterization of CPNTs formed from CPs containing alternating D,L-
-amino acids, alternating D,L-
, -amino
acids, -amino acids, -amino acids, and many other
arrangements.815 It has been demonstrated that CP units
with an even number of alternating D,L-
-amino acids
can adopt a at conformation with all the amino acid
side chains pointing outwards and the carbonyl and
amino groups of the amide backbone groups oriented perpendicular to the ring.8 9 Alternating D,L-
-amino acid
residues with conformationally equivalent -type dihedral
angles is crucial to form closed rings by stacking the
CP units through anti-parallel -sheet hydrogen bonding.8

1550-7033/2013/9/001/010

doi:10.1166/jbn.2013.1724

Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells

For example, using the octapeptide cyclo-[(L-Gln-D-AlaL-Glu-D-Ala)2 -], hollow CPNTs, with internal diameters
of 0.75 nm, were formed by stacking the CP units through
their amide backbonebackbone hydrogen bonding.8
In addition, the diameters of CPNTs could be easily tuned
through variation in the number of amino acid residues in
the CP units. For instances, the decapeptide cyclo-[L-Gln(D-Leu-L-Trp)4 -D-Leu-] was reported to self-assemble the
CPNTs with internal diameter of 1.0 nm,1617 whereas the
dodecapeptide cyclo-[(L-Gln-D-Ala-L-Glu-D-Ala)3 -] can
generate the CPNTs with the internal diameter of 1.3 nm.18
In addition to the tunable properties of the CPNTs, due
to the superior biocompatibility, CPNTs are believed to
be a desirable candidate for biomedical applications.8 Further, modication and functionalization of the surface of
CPNTs can be achieved through reactions with the various
side chains of the amino acids present on the CP subunits.2
Therefore, by carefully designing the repeating CP unit
and optimizing the self-assembly conditions, CPNTs could
be tailored to meet needs of specic applications.8
Currently, CPNTs have been proposed for applications in various elds, including biosensing,1921 optics/
electronics,2226 and antimicrobials.2729 However, using
these supramolecular structures as drug delivery systems
for cancer therapy, to the best of the authors knowledge,
has not been done. The purpose of this study is to design
and fabricate a new eight-residue CPNT and to explore
its potential application in cancer therapy. To achieve this
goal, the PEG-modied doxorubicin (DOX)-loaded CPNT
bundles were fabricated using a glutamic acid and a cysteine residue-containing cyclic octapeptide. In previous
reports of the antimicrobial activity of cyclic peptides,
it is notable that some amphipathic D,L-
-CPs have been
reported to exhibit signicant antibacterial activity against
a broad spectrum of bacteria that include methicillinresistant Staphylococcus aureus (MRSA).8 28 Inspired by
these discoveries, we further investigated the cytotoxicity
of the PEG-modied DOX-loaded CPNTs against DOX
sensitive and resistant tumor cell lines to explore the potential of CPNT bundles to function as drug carriers for cancer therapy.

MATERIALS AND METHODS

Chemicals and Cell Lines
The cyclic peptide, cyclo-(-L-Gln-D-Ala-L-Glu-D-Ala-LGln-D-Ala-L-Cys-D-Ala-), was synthesized by Peptide
2.0 Inc (Chantilly, VA). Triuoroacetic acid, acetonitrile,
doxorubicin, and methoxypolyethylene glycol maleimide
(Maleimide-mPEG5000  were purchased from SigmaAldrich (St. Louis, MO). LysoTracker Green DND-26
and Hoechst 33342 were obtained from Invitrogen
Life Technologies (Grand Island, NY). Fetal bovine
serum and RPMI 1640 medium were purchased from
Mediatech (Manassas, VA). Penicillin (10000 units/ml)streptomycin (10000 g/ml) solution was procured from
2

Wang et al.

MP biomedicals (Solon, OH). The human breast tumor

cell line MCF-7 and its resistant cell line MCF-7/ADR
were obtained from the Frederick National Laboratory for
Cancer Research (Frederick, Maryland).
Preparation and Characterization of Cyclic
Peptide Nanotubes (CPNTs)
10 mg of cyclic peptide unit, cyclo-(-L-Gln-D-Ala-LGlu-D-Ala-L-Gln-D-Ala-L-Cys-D-Ala-), was suspended
in 12.1 ml of distilled water. The suspension (1 mM) of
peptide subunit was dissolved by adding NaOH drop-wise
to a pH of 12.0, and the resulting peptide solution was
centrifuged to remove any traces of solid matter. In order
to observe the effect of pH on the formation of CPNTs,
0.5 mM of the cyclic peptide stock solution at pH 12.0 was
titrated with 2% triuoroacetic acid in acetonitrile.9 13 30
The particle sizes and zeta potentials of the resulting nanotubes at different pHs ranging from 12.0 to 2.0 were
detected at 25  C using a Malvern Zetasizer, NANO ZS
(Malvern Instruments Limited, UK), with a HeNe laser
(wavelength of 633 nm) and a detector angle of 173 . All
samples were measured in triplicate.
The morphology of the CPNTs was analyzed using
transmission electron microscopy (TEM). Briey, the
CPNT particles were gradually formed at pH 2.0 as a
white suspension over a period of hours after titration with
2% triuoroacetic acid in acetonitrile. Nanotubes were
then collected by centrifugation and washed repeatedly
with distilled water to remove excess acids and salts.9 13 30
A drop of the CPNT suspension was then dried on a carbon lm-coated copper grid, then negative stained with
0.5% (w/v) phosphotungstic acids, and observed using
TEM (Zeiss LIBRA 200 FE, Germany) at 80 kV.
Preparation and Characterization of DOX-Loaded
CPNT Bundles (CPNT/DOX) and PEG-Modied
CPNT/DOX Bundles
0.5 mM of CP aqueous solution at pH 12.0 was diluted to
0.25 mM by the addition of same volume of HEPES buffer
(20 mM, pH = 7.0). The resulting CP solution was adjusted
using 6 N HCl to a pH of 8.0, 7.0 and 6.0, respectively.
100 l of 3 mM DOX in HEPES buffer was then added
to 900 l of 0.25 mM CP solution with different pH, as
prepared above. The mixtures of CP and DOX in HEPES
buffer (pH 8.0, 7.0 and 6.0) were placed overnight at room
temperature. The nanotube/drug complexes between CPNT
and DOX were centrifuged at 5000 rpm for 10 min, and
the precipitates were washed three times with distilled
water to remove any excess acids and salts. The morphology of the precipitated CPNT/DOX aggregates was
examined using SEM (LEO 1525, high resolution FE-SEM
system, Germany). In order to prepare the PEG-modied
DOX-loaded CPNTs (PEG-modied CPNT/DOX), 14 mg
of maleimide-mPEG5000 was dissolved in 1 ml of distilled water (pH 7.0). The precipitates of the CPNT/DOX,
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Wang et al.

Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells

prepared using the above procedure at pH 7.0, were immediately suspended in 1 ml of maleimide-mPEG5000 aqueous solution, and the maleimide-thiol reaction between
the CP and PEG ([PEG]/[CP] = 12.4, molar ratio) was
performed overnight at room temperature. The resulting
PEG-modied CPNT/DOX was puried by column separation using Sephadex G75 to remove any unreacted
PEG and salts. The puried PEG-modied CPNT/DOX
was then imaged using SEM. The PEG concentration
in the puried PEG-modied CPNT/DOX was quantied using a colorimetric PEG assay based on the formation of PEG-barium iodide complexes31 32 after DOX was
removed from the PEG-modied CPNT/DOX by dialysis
(MWCO = 12 000 Da) against distilled water (pH 5.5) for
three days.
Cytotoxicity of PEG-Modied CPNT/DOX
Bundles on Tumor Cells
The cytotoxicity of the PEG-modied CPNT/DOX was
assessed by the MTT (3-[4, 5-dimethylthiazol-2-yl]-2,
5-diphenyl tetrazolium bromide) assay as previously
reported.33 34 Briey, 1 104 tumor cells (human breast
cancer cell lines, MCF-7 and MCF-7/ADR) were seeded
in 96-well plates in 100 l of RPMI 1640. Serial dilutions
of the PEG-modied CPNT/DOX were added to the plate
and incubated at 37  C in 5% CO2 for 48 hr. 10 l of
the MTT stock solution (5 mg/mL in PBS, pH 7.4) was
then added to the wells, and the plates were incubated at
37  C for another 4 hours. The medium was then completely removed and 100 l of DMSO was added to each
well to solubilize the dye. The absorbance was measured
using a microplate reader (Bio-Tek Quant) at 570 nm,
and the concentration of drug that inhibited cell survival by
50% (IC50  was determined from cell survival plots using
the DoseResp function of OriginPro 8.0.
Cellular Uptake Assay
The cellular uptake of the PEG-modied CPNT/DOX was
quantied according to the reported method.35 Briey,
tumor cells were seeded onto 6-well plates at densities of
2 106 cells/ml and incubated at 37  C until 70% conuence was reached. Free DOX solution or PEG-modied
CPNT/DOX at a DOX concentration of 10 M was then
added and incubated for 4 h at 37  C. The medium was
removed and cell monolayers were washed with cold PBS
three times. After trypsinization, the cell associated uorescence was measured immediately using an Epics XL
Analyzer (Beckman Coulter Inc., Brea, CA) by collecting 20000 events for each sample. Each experiment was
performed in triplicate.
Confocal Laser Scanning Microscopy
Both cells (MCF-7 and MCF-7/ADR) were grown on
cover slips to 50% conuence and incubated with free
DOX solution or PEG-modied CPNT/DOX bundles at
J. Biomed. Nanotechnol. 9, 110, 2013

DOX concentrations of 10 M at 37  C for 4 h. The

cells were then incubated with 100 nM LysoTracker green
DND-26 and 4 M Hoechst 33342 for 30 min prior to
visualization for endolysosome and nuclear labeling.35 The
cell monolayers were then washed three times with PBS,
and immediately imaged with a FluoView FV1000 Confocal Microscope (Olympus, Japan).
P -gp Function Analysis
The intracellular accumulation of Hoechst 33342, a specic substrate of P-gp, was used as an index of Pgp
activity.3638 Both tumor cell lines were grown in 12-well
plates to 80% conuence and treated with free DOX solution or PEG-modied CPNT/DOX bundles at DOX concentrations of 10 M at 37  C for 4 h. 4 M Hoechst
33342 was then added to each well and incubated at 37  C
for another 30 min. After the incubation, the cell monolayers were washed three times with PBS and lysed with
1 mL of 0.1% v/v Triton-X 100 dissolved in 0.3% NaOH.
A 0.5 ml aliquot was then used for the determination of the
Hoechst content, using a Fluorescence Microplate Reader
(BioTek, Winooski, VT). Excitation and emission wavelengths were 370 and 450 nm, respectively. Another 0.5 ml
aliquot was also used to quantify the protein concentration using the BCA assay (Pierce, Rockford, IL) following
the manufacturers protocol. The intracellular uorescence
was standardized to nmol of Hoechst/mg of cell proteins
using a calibration curve.
Statistical Analysis
The data were expressed as mean S.D. Statistical significance was determined using a one-way ANOVA followed
by a Students t test for multiple comparison tests. A p
value of < 005 was considered statistically signicant.

RESULTS AND DISCUSSION

pH-Driven Self-Assembly of Cyclic
Peptide Nanotube (CPNT) Bundles
In this study, a new eight-residue cyclic peptide that contains a cysteine residue and a glutamic acid residue at
the symmetric positions on the peptide unit (Scheme 1(a))
was used to form the CPNTs. According to the previous reports, cyclic peptide units with an even number
of alternating D,L-
-amino acids can self-assemble to a
supramolecular nanotube structures through an extensive
network of hydrogen bonds.8 9 The glutamic acid side
chain functions as a triggering mechanism for initiation
of the self-assembly process, and protonation of the carboxylate groups of glutamic acid in an acidic environment should lead to the formation of the CPNTs. As such,
we predicted that the self-assembly of the CPNTs was a
pH-dependant process (Scheme 1(b)). First, we examined
the pH-dependant prole of self-assembly of the CPNTs.
As shown in Figure 1(a), the initial formation of CPNTs,
3

Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells

Wang et al.

Scheme 1. Structure of the cyclic octapeptide (a) used in this study, pH-driven self-assembly of the blank cyclic peptide nanotube (CPNT) bundles (b), and fabrication of PEG-modied DOX-loaded CPNT bundles (c).

as determined by dynamic light scattering (DLS), was

observed at pH 6.0, with the micro-scaled nanotube aggregates appearing at lower pHs. Due to protonation of the
carboxyl group of the glutamic acid side chain, causing a
hydrophobic property on the surface of the CPNT,8 9 the
nanotube aggregates were formed in acidic pHs, which was
(a)

(b)

(c)

Figure 1. pH-driven self-assembly of cyclic peptide nanotube (CPNT) bundles. (a) Self-assembly prole of the
cyclic octapeptide, cyclo-(-L-Gln-D-Ala-L-Glu-D-Ala-L-Gln-DAla-L-Cys-D-Ala-), over a pH range of 2.012.0; (b)(c) The representative TEM images of the CPNT bundles prepared at pH
2.0 by triuoroacetic acid/acetonitrile method (see Materials
and Methods).

conrmed by the particle size (34 m, Fig. 1(a)). At the

same time, due to the protonation, the zeta potentials of the
nanotube aggregates increased from 20 mV to 0 mV
when the nanotubes spontaneously self-assembled at acidic
pHs ranging from 6.0 to 2.0 (Fig. 1(a)). We also examined
the morphology of the CPNTs prepared at the most acidic
pH 2.0 using TEM. As shown in Figures 1(b)(c), individual nanotubes found in the aggregates had lengths of
5080 nm and diameters of 10 nm. It has been previously reported that the diameter of an un-aggregated CPNT
formed from cyclic octapeptides was demonstrated to be
< 1 nm.8 9 Due to their hydrophobic surface, the CPNTs
can easily aggregate in acidic environments leading to an
increase in the particle size.9 From the TEM analysis,
individual nanotubes with 10 nm in diameter and 50
100 nm in length were the organized bundles of tightly
packed individual CPNTs, which is consistent with previous studies.9 In this study, we designate these individual
nanotubes observed from the TEM images (Figs. 1(b)(c))
as the tightly packed CPNT bundles.
High DOX Loading in CPNT Bundles
and Site-Specic PEGylation of
DOX-Loaded CPNT Bundles
The purpose here is to fabricate a new nanocarrier using
CP for potential biomedical applications. We hypothesized that the individual CPNT bundles with the diameter of 10 nm and the length of 5080 nm prepared
by protonation of the carboxylate group would be effective nanocarriers for drug delivery and cancer therapy if
(1) drugs could be incorporated into the CPNT bundles,
and (2) the nanotube aggregates could be effectively dispersed using a proper hydrophilic polymer in aqueous
solution. First, we utilized the carboxyl group of the glutamic acid side chain of the CP subunit to form ion-pair
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Wang et al.

Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells
(a)

(b)

(c)

(d)

(e)

(f)

(g)

Figure 2. Doxorubicin was loaded into the CPNT bundles and PEG-modied CPNT/DOX bundles. (a)(c) Ion-pair complex formation of CPNT/DOX at different pHs: (1) Cyclic peptide and DOX mixture; (2) DOX solution; (d)(e) SEM images of CPNT/DOX
bundles in aggregates at pH = 7
0; (f)(g) SEM images of PEG-modied CPNT/DOX bundles.

complexes with a cationic antitumor drug, doxorubucin

(DOX) (Scheme 1(c)).33 As shown in Figures 2(a)(c),
when the pH of the mixed solution of DOX and CP was
adjusted to 7.0 and 6.0, the DOX-associated CPNT bundles
were formed. The DOX-containing CPNT bundles rapidly
aggregated, due to hydrophobic surface of the CPNTs,
leading to overnight precipitation. The encapsulation ratio
of DOX within the CPNT bundles of the aggregates was
about 58.3% and 66.7% at pH 7.0 and 6.0, respectively.
However, no precipitates were observed in the mixed solution of DOX and CP at pH 8.0. Presumably, there was a
very small fraction of DOX protonated at pH 8.0 due to
the pKa of DOX ( 8.38.5),39 which resulted in failure to
form the ion-pair complexes between the carboxyl group
and DOX. Such that, the nanotubes cannot be formed and
J. Biomed. Nanotechnol. 9, 110, 2013

no precipitates were observed at this base pH value. We further examined the morphology of the DOX-loaded CPNT
aggregates using SEM. As shown in Figures 2(d)(e), the
CPNT aggregates displayed scaffold-like structures, with
particle sizes of 12 m measured by DLS method (data
not shown); however, individual CPNT bundles within the
aggregates had diameters of 50 nm and lengths of 200
300 nm. Due to their large size, the micro-sized CPNT
aggregates would not be an effective carrier for biomedical
applications; however, we hypothesized that the individual
CPNT bundles may be a potential drug delivery system if
they could be properly dispersed.
In order to disperse the CPNTs in solution, we utilized
the cysteine residue on the CP subunits for site-specic
PEGylation using maleimid-mPEG5000 acting as the
5

Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells

Wang et al.

hydrophilic polymer (Scheme 1(c)). The cysteine residue

was designed so that its position was directly opposite of
the glutamic acid residue (Scheme 1(a)). The thiol-group
in cysteine was used as a specic PEGylation site for a
maleimide-modied PEG.40 We postulated that sulfhydryl
specic PEGylation of the DOX-loaded CPNTs would
facilitate the formation of a dispersed nanotube population. As shown in Figures 2(f)(g), the PEG-modied
CPNT/DOX bundles were well-dispersed, with an average
diameter of 50 nm and length of 200300 nm. After
modication with PEG, the molar ratio of PEG to CP
units in the PEG-modied CPNT bundles was estimated
to be 0.21, and the encapsulation ratio of DOX within
the CPNT bundles slightly decreased from 58.3% of the
unmodied form to 54.2% at pH 7.0.
DOX-Loaded CPNT Bundles Sensitized
Resistant MCF-7/ADR Tumor Cells In Vitro
Due to high DOX loading within the PEG-modied
CPNT/DOX bundles and the nano-scale dimensions of the
well-dispersed nanotubes, we hypothesized that they might
have a potential as a drug delivery system for cancer therapy. To test the hypothesis, we rst analyzed the cytotoxicity of the PEG-modied CPNT/DOX bundles against
the human breast tumor cell line MCF-7. As shown in
Figure 3(a) and Table I, the PEG-modied CPNT/DOX
bundles showed higher cytotoxicity against MCF-7 cells
compared to free DOX (0.53 M vs. 0.16 M), indicating potential application of the PEG-modied CPNT/DOX
bundles as a drug nanocarrier for cancer therapy. Apart
from sensitive MCF-7 tumor cells, we further tested the
cytotoxicity of the PEG-modied CPNT/DOX bundles
against a multidrug resistant cell line, MCF-7/ADR. As
shown in Figure 3(b) and Table I, the PEG-modied
CPNT/DOX bundles showed higher cytotoxicity toward
cultured resistant MCF-7/ADR cells than free DOX as well
(0.84 M vs. 4.21 M). However, compared to the cytotoxicity between the sensitive MCF-7 cells and resistant
MCF-7/ADR cells, the PEG-modied CPNT/DOX bundles had much less effect on drug efcacy in the case
of sensitive MCF-7 cells than resistant MCF-7/ADR cells
(3.3-fold decrease vs. 5.01-fold decrease in IC50 . Interestingly, the cytotoxicity of PEG-modied CPNT/DOX bundles in the resistant cells was decreased to the point where
the sensitive cells responded to free DOX, resulting in the
comparable IC50 values of the same order of magnitude
(0.84 M vs. 0.53 M, Table I). In order to rule out any
possible cytotoxicity from the unloaded carrier, we conducted the MTT assay in both tumor cell lines using the
blank PEG-modied CPNT bundles as a control. As shown
in Figure 3(c), there was no obvious cytotoxicity from
the PEG-modied CPNT bundles at the cyclic octapeptide concentration ranging from 0.01 M to 25 M. The
result is consistent with the previous report that cyclic peptides had no signicant cytotoxicity against rat hepatoma
6

Figure 3. Cytotoxicity of PEG-modied CPNT/DOX bundles

against MCF-7 (a) and MCF-7/ADR cell lines (b). Blank PEGmodied CPNT bundles (c) were used as a control.

cells below 100 M.41 According to a 54.2% of DOX

encapsulation ratio in the PEG-modied CPNT bundles
and the initial feeding amounts of cyclic peptide/DOX
(0.225 mol/0.3 mol, see Section 2.3), 25 M of
cyclic peptide corresponds to 18.1 M of DOX equivalent
concentration in the PEG-modied CPNT/DOX bundles.
From the Table I, 18.1 M of a DOX equivalent concentration is far higher than the IC50 of the PEG-modied
CPNT/DOX bundles against MCF-7 ( 0.16 M) and
MCF-7/ADR cells ( 0.84 M), indicating that there is
no obvious cytotoxicity from the CPNT bundles themselves in the DOX concentration range used for MTT assay
in this study. The cytotoxicity data in both sensitive and
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Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells

Table I. IC50 (mean S.D.) and resistance reversion index

(RRI) of PEG-modied CPNT/DOX bundles against human
breast MCF-7 cells and multidrug resistant MCF-7/ADR cells.
MCF-7
Formulation
Free DOX
PEG-modied
CPNT/DOX bundles

MCF-7/ADR

IC50
M)

IC50
M)

RRI

053 005
016 003

421 065
084 004

5.01

Notes: Resistance reversion index (RRI), i.e., ratio of IC50 of free DOX solution to PEG-modied CPNT/DOX bundles. P < 001, versus free DOX.

resistant cells indicate that the PEG-modied CPNT/DOX

bundles have the potential for DOX delivery to the resistant cells, and are able to sensitize the multidrug resistant cells, exhibiting higher cytotoxicity compared to free
drug.
DOX-Loaded CPNT Bundles Enhanced
Intracellular DOX Uptake and Altered Intracellular
Distribution of DOX in Tumor Cells
It has been reported that the multidrug resistance in
cancer is a major cause for clinical failure of anticancer chemotherapy.34 Therefore, the sensitizing effect
of the PEG-modied CPNT/DOX bundles against multidrug resistant cells is a favorable property for cancer
therapy. It has been demonstrated that increased uptake,
different intracellular localization, inhibition of ABC pump
activity, and enhanced apoptotic effect, play important
roles for overcoming drug resistance in cancer by different drug delivery carriers.42 43 In order to elucidate the
mechanism of the sensitizing effect of the CPNT bundles
toward the resistant MCF-7/ADR cells, we rst tested the
DOX uptake in both sensitive and resistant MCF-7 cells
using ow cytometry by measuring cell-associated DOX
(a)

uorescence intensity. As shown in Figure 4, after a period

of 4 h, the uptake of DOX was signicantly increased by
the PEG-modied CPNT/DOX bundles in both cells compared to free DOX at a DOX concentration of 10 M.
To further determine the sub-cellular distribution of PEGmodied CPNT/DOX bundles, the confocal imaging was
performed, and the nuclei and endolysosome were labeled
with respective nucleus-selective dye (Hoechst 33342,
blue) and acidic endolysosome-selective dye (LysoTracker
green DND-26). As shown in Figure 5, a different intracellular distribution between PEG-modied CPNT/DOX
bundles and free DOX was observed in both cells after a
4-h treatment. MCF-7 cells and MCF-7/ADR cells treated
with PEG-modied CPNT/DOX bundles showed a higher
intracellular accumulation of DOX compared to the treatment with free DOX, which is consistent with the data
from ow cytometry (Fig. 4). However, the majority of
DOX in both cells treated with PEG-modied CPNT/DOX
bundles was predominantly located in the endolysosomal
compartment, whereas most of the free DOX was located
outside endolysosomes. It has been reported that nanoparticles internalized via endocytosis were typically found
mainly within endosomes or lysosomes.44 Therefore, we
presume that the PEG-modied CPNT/DOX bundles were
probably taken up by the endocytic pathway in both tumor
cell lines. In addition, due to a signicant increase of
intracellular DOX in both cells treated with the PEGmodied CPNT/DOX bundles, the nuclear DOX localization accordingly increased compared to the treatment of
free DOX. Enhanced nuclear localization of DOX is a
desirable property in order to maximize DOXs antitumor
activity.45 Overall, the increased uptake and altered intracellular distribution of DOX in both tumor cells probably
is one cause that led to higher cytotoxicity by the PEGmodied CPNT/DOX bundles.
(b)

Figure 4. Flow cytometry analysis for cellular uptake of PEG-modied CPNT/DOX bundles in human breast cancer MCF-7 cells (a)
and the resistant counterpart MCF-7/ADR cells (b). Both cells were treated with the PEG-modied CPNT/DOX bundles or free
DOX at a DOX concentration of 10 M for 4 h, and then the mean DOX uorescence associated with the cells was measured by
collecting 20000 events for each sample.
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Wang et al.

Figure 5. Intracellular distributions of PEG-modied CPNT/DOX bundles and free DOX at DOX concentration of 10 M in human
breast cancer MCF-7 cells (a) and the multidrug resistant cell line MCF-7/ADR (b). The cells were incubated with both samples at
37
C, 5% CO2 for 4 h, and then 100 nM Lysotracker Green DND-26 and 4 M Hoechst 33342 were added for a 30-min incubation
prior to visualization by confocal microscopy. Scale bars represent 20 m.

DOX-Loaded CPNT Bundles Inhibited P -gp

Function in MCF-7/ADR Cell Line
Since P -gp plays an important role in determining and
conferring multidrug resistance in various tumors,46 we
postulated that the increased DOX uptake in the resistant MCF-7/ADR cells was attributable to the inhibition
of P -gp efux activity by the PEG-modied CPNT/DOX
bundles. To validate this hypothesis, we used Hoechst
33342, a commonly used uorescent substrate of P -gp, to
analyze the P-gp activity in multidrug resistant cells after
treatment with the PEG-modied CPNT/DOX bundles.
As shown in Figure 6, in the case of MCF-7/ADR cells
treated with the PEG-modied CPNT/DOX bundles and
8

free DOX for 4 h, both treatments signicantly enhanced

the accumulation of Hoechst 33342 at a DOX concentration of 10 M. Compared to free DOX, more uorescent dye signicantly accumulated in the resistant cells
treated with the PEG-modied CPNT/DOX bundles. However, the treatments with both PEG-modied CPNT/DOX
and free DOX didnt alter the Hoechst accumulation in
the sensitive MCF-7 tumor cells. The quantitative data
of intracellular Hoechest accumulation in sensitive and
resistant cells are consistent with the images from the
confocal analysis in Figure 5. However, considering the
fact that enhanced DOX uptake in the sensitive cells
treated with the PEG-modied CPNT/DOX bundles was
J. Biomed. Nanotechnol. 9, 110, 2013

Wang et al.

Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells

REFERENCES

Figure 6. P -gp activity in the presence of free DOX and PEGmodied CPNT/DOX bundles in the human multidrug resistant
breast tumor MCF-7/ADR cell line. The cells were incubated
for 4 h in fresh medium (ctrl) or with 10 M doxorubicin (DOX)
or PEG-modied CPNT/DOX bundles, and 4 M Hoechst 33342
was added to the culture media for another 30-min incubation
prior to quantitative analysis. The cultures were then washed,
lysed and analyzed uorimetrically for the intracellular content
of the dye. The dye concentration was standardized by the
protein concentration which was measured with BCA assay.
The sensitive breast tumor MCF-7 cells were used as a control. Measurements were performed in triplicate and data are
presented as mean SD (n = 3). p < 0
01, versus MCF-7/ADR
cells ctrl; P < 0
001, versus MCF-7/ADR cells ctrl; P < 0
05,
versus MCF-7/ADR cells treated with free DOX.

also observed (Fig. 4(a)), we propose that the inhibitive

effect of P -gp exerted by the PEG-modied CPNT/DOX
bundles is, at least partially, attributable to the enhanced
DOX uptake in the resistant MCF-7/ADR tumor cells.

CONCLUSION
In this study, cyclic peptide-based nanotube (CPNT) bundles with a diameter of 10 nm and a length of 50
80 nm were prepared using a glutamic acid and cysteine
residue-containing cyclic octapeptide. In order to explore
the potential application of these supramolecular structures as a nanocarrier for cancer therapy, PEG-modied
DOX-loaded CPNT bundles were fabricated, and showed
a high drug encapsulation ratio and good dispersion with
a diameter of 50 nm and a length of 200300 nm.
More importantly, the PEG-modied CPNT/DOX bundles
demonstrated the activity to overcome the multidrug resistance in a human breast cancer cell line in vitro. Further, the PEG-modied CPNT/DOX bundles increased the
uptake of DOX, altered the intracellular DOX distribution,
and inhibited P -gp activity in MCF-7/ADR cells. The ndings of this study highlight that the PEG-modied DOXloaded CPNT bundles may be a useful nanocarrier for drug
delivery to resistant tumor cells.
Acknowledgments: This work was supported in part
by the Ofce of Naval Research Young Investigator Program (award ONR-N00014-11-1-0622). The authors are
grateful for the support.
J. Biomed. Nanotechnol. 9, 110, 2013

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