Biological evaluation of a novel doxorubicin-peptide conjugate for targeted delivery to EGF receptor-overexpressing tumor cells

Article abstract of DOI:10.1021/mp100243j

Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial malignancies and thus can be used for EGFR-targeted therapy to improve antitumor efficacy. Therefore we synthesized a novel conjugate of doxorubicin (DOX) with an EGFR-binding peptide (NH₂-CMYIEALDKYAC- COOH; EBP) via an ester bond at position 14 of DOX through a glutarate spacer. To confirm that the DOX-EBP conjugate is capable of targeting tumor cells overexpressing EGFR, we compared the cellular accumulation, intracellular distribution and in vitro cytotoxicity of DOX-EBP and free DOX. After treating with equimolar concentration of DOX-EBP or free DOX, the conjugate accumulated at significantly higher levels in EGFR-overexpressing cells than in non-EGFR-overexpressing cells, while the intracellular accumulation of free DOX was almost the same in all the cells. However, the intracellular accumulation of DOX-EBP was significantly reduced in EGFR-overexpressing cells preincubated with inhibitory anti-EGFR monoclonal antibody, demonstrating the involvement of EGFR pathway in the transport of the conjugate. Confocal fluorescence microscopy reveals that the conjugate was distributed in cytoplasmic and perinuclear areas during the first 30 min, whereas the free DOX was accumulated in both cytoplasm and nuclei. After 24 h, however, the DOX signal in the cells treated with DOX-EBP was also distributed in the nuclei, suggesting the release of DOX from the conjugate and entry into the nuclei. Biodistribution and in vivo antitumor experiments, together with in vitro cytotoxicity, indicate that the therapeutic competence of DOX-EBP was due to its increased accumulation in EGFR-expressing tumor cells. Furthermore, the survival of tumor-bearing mice treated with DOX-EBP was significantly higher than that with free DOX. These data demonstrate the enhanced anticancer efficacy and reduced systemic toxicity of DOX-EBP conjugate with targeting ability to EGFR-overexpressing tumor cells.

Full text of DOI:10.1021/mp100243j

ARTICLE
pubs.acs.org/molecularpharmaceutics
Biological Evaluation of a Novel Doxorubicin-Peptide Conjugate for
Targeted Delivery to EGF Receptor-Overexpressing Tumor Cells
Shibin Ai,*,† Jianli Duan,† Xin Liu,† Stephanie Bock,† Yuan Tian,‡ and Zebo Huang*,†,§
†College of Pharmacy, Wuhan University, Wuhan 430071, China
‡Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430021, China
§Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071, China
ABSTRACT: Epidermal growth factor receptor (EGFR) is
overexpressed in a variety of epithelial malignancies and thus
can be used for EGFR-targeted therapy to improve antitumor
efficacy. Therefore we synthesized a novel conjugate of
doxorubicin (DOX) with an EGFR-binding peptide (NH2-
CMYIEALDKYAC-COOH; EBP) via an ester bond at
position 14 of DOX through a glutarate spacer. To confirm
that the DOX-EBP conjugate is capable of targeting tumor
cells overexpressing EGFR, we compared the cellular accu-
mulation, intracellular distribution and in vitro cytotoxicity of
DOX-EBP and free DOX. After treating with equimolar
concentration of DOX-EBP or free DOX, the conjugate
accumulated at significantly higher levels in EGFR-overex-
pressing cells than in non-EGFR-overexpressing cells, while the intracellular accumulation of free DOX was almost the same in all
the cells. However, the intracellular accumulation of DOX-EBP was significantly reduced in EGFR-overexpressing cells
preincubated with inhibitory anti-EGFR monoclonal antibody, demonstrating the involvement of EGFR pathway in the transport
of the conjugate. Confocal fluorescence microscopy reveals that the conjugate was distributed in cytoplasmic and perinuclear areas
during the first 30 min, whereas the free DOX was accumulated in both cytoplasm and nuclei. After 24 h, however, the DOX signal in
the cells treated with DOX-EBP was also distributed in the nuclei, suggesting the release of DOX from the conjugate and entry into
the nuclei. Biodistribution and in vivo antitumor experiments, together with in vitro cytotoxicity, indicate that the therapeutic
competence of DOX-EBP was due to its increased accumulation in EGFR-expressing tumor cells. Furthermore, the survival of
tumor-bearing mice treated with DOX-EBP was significantly higher than that with free DOX. These data demonstrate the
enhanced anticancer efficacy and reduced systemic toxicity of DOX-EBP conjugate with targeting ability to EGFR-overexpressing
tumor cells.
KEYWORDS: epidermal growth factor receptor, peptide, doxorubicin, drug delivery
’ INTRODUCTION
which disposes of a high cellular binding affinity to the targeted
site.11,12 For example, DOX was conjugated to a cyclic pentapep-
tide (CNGRC) via a hydrolyzable spacer to target CD13 on the
surface of the SK-UT-1 cells, and the DOX-CNGRC conjugate
was reported to alter the cellular uptake process of DOX.13 AN-
152, a conjugate of DOX covalently linked with the luteinizing
hormone-releasing hormone (LHRH), was reported to bind
specifically to LHRH receptors and act as a chemotherapeutic
agent after internalization of the ligand-receptor complex into
cancer cells expressing LHRH,9 and a phase II clinical study of
the conjugate was started in 2008.14
Doxorubicin (DOX), an anthracycline antibiotic, has been
widely used in chemotherapy for treatment of leukemia, colon
cancer, breast cancer and many other types of cancer, and has
become one of the most commonly used anticancer drugs.1,2 The
mechanism of DOX to kill tumor cells has mainly been related to
its intercalation into DNA and disruption of topoisomerase II
activity.3 However, due to its lack of specific targeting to tumor
cells, DOX has shown a large-spread field of systemic toxicities in
healthy tissues.4,5 In particular, the extensive use of DOX in the
clinical setting may cause serious irreversible cardiac toxicity.6,7
In order to enhance the anticancer efficacy and to reduce the
systemic toxicity of DOX, a number of studies have used over-
expressed receptors on the surface of tumor cells as DOX target
sites and accordingly designed drug molecules or delivery
systems to distribute DOX to tumor cells.8-10 The strategy
involves the conjugation of DOX with a target-specific vector,
Epidermal growth factor receptor (EGFR) is an important
transmembrane receptor comprising an extracellular ligand-binding
Received: July 27, 2010
Accepted: January 17, 2011
Revised:
January 10, 2011
Published: January 17, 2011
r
2011 American Chemical Society
375
dx.doi.org/10.1021/mp100243j Mol. Pharmaceutics 2011, 8, 375–386
|

Molecular Pharmaceutics
ARTICLE
domain, a transmembrane domain and an intracellular domain
with tyrosine kinase activity.15 Since EGFR is overexpressed or
abnormally activated in a number of malignancies and highly
related to proliferation, angiogenesis, invasion and metastasis of
tumor cells,16 it has been increasingly used as a potential target
for cancer therapeutics. Several EGFR targeting agents have been
developed as anticancer drugs, including (1) monoclonal anti-
bodies such as cetuximab and panitumumab, which target the
extracellular domain of the receptor and inhibit the ligand-
dependent EGFR signal transduction, and (2) small-molecule
inhibitors such as gefitinib and erlotinib, which target the
intracellular tyrosine kinase domain of EGFR.17,18
Epidermal growth factor (EGF) is one of the most important
EGFR ligands with high binding ability and selectivity.19 The
structure of EGF molecule contains 3 loops which are held in
place by 3 disulfide bonds:20,21 loop A, Cys6 and Cys20; loop B,
Cys14 and Cys31; and loop C, Cys33 and Cys42. The B-loop is
implicated in EGFR binding,22-24 and the active region is
CMYIEALDKYAC,25 which may be used as a vector to conjugate
DOX to specifically target tumor cells expressing EGFR.
In this study, we synthesized the EGFR-binding peptide (EBP:
NH2-CMYIEALDKYAC-COOH) and conjugated it with DOX
via an ester bond at position 14 through a glutarate spacer. To
confirm the EGFR-targeted chemotherapy of DOX-EBP con-
jugate, we tested the intracellular accumulation levels of the
conjugate in tumor cells with different EGFR expression levels
and investigated the cellular entry pathway of DOX-EBP
conjugate by blocking EGFR function using an anti-EGFR mon-
oclonal antibody. We also compared the cytotoxicity of the
conjugate and free DOX in tumor cells in vitro as well as their
distribution and antitumor effect in vivo.
stirred at room temperature for 3 h. The resulting N-Fmoc-DOX
crystals were collected by filtration and washed with cold diethyl
ether, and then placed with glutaric anhydride to a round-
bottomed flask containing DMF and DIPEA. The mixture was
stirred at room temperature for 10 h. After removal of the
solvent, the residue was taken up by dichloromethane and
washed with water and brine. The obtained N-Fmoc-DOX-14-
O-hemiglutarate was dissolved in Fmoc-NH-CMYIEALD-
KYAC-COOH solution in DMF. BOP reagent, 1-hydroxyben-
zotriazole and DIPEA were then added and stirred for 1 h. The
solvent was removed, and the residual oil was treated with ethyl
acetate. The resulting solid was dissolved in DMF, and piperidine
was added to remove the protecting groups. The DOX-peptide
product was purified by RP-HPLC on a C18 column and
lyophilized.28 Purity of the product was determined by HPLC
analysis as described below. The product was confirmed by ele-
ctrospray mass spectra.
HPLC Analysis. DOX content in samples was analyzed using
an Agilent 1100 series HPLC systems (Agilent Technologies,
Santa Clara, CA) and a Shimadzu RF-10A XL fluorescence
detector (Shimadzu Inc., Kyoto, Japan). The analytical column
was ODS-C18 (5 μm particle size, 250 ꢀ 4.6 mm, Thermo
Scientific, USA). The mobile phase consisting of a mixture of
acetonitrile, methanol and water (32:50:18, v/v/v) was delivered
at a flow rate of 1.0 mL/min. The column temperature was main-
tained at 40 °C. Fluorescence detection was performed using
excitation and emission wavelengths of 480 and 580 nm,
respectively.
Western Blot Analysis. The expression of EGFR in the
tumor cell lines was analyzed by Western blotting.29 The cells
were grown to near confluence in 25 cm2 culture flasks at a
density of 1 ꢀ 107 cells/flask. Confluent cells were washed with
ice-cold phosphate-buffered saline (PBS), and then treated with
lysis buffer containing 50 mM Tris-HCl (pH 7.4), 120 mM NaCl,
1 mM EDTA, 1% TritonX-100 and 1% (v/v) protease inhibitor
cocktail (Sigma-Aldrich). The samples were homogenized on ice
for 2 min with a Dounce homogenizer, and cellular debris was
removed by centrifugation (3000 rpm; 10 min) at 4 °C. The
lysates were further centrifuged at 12,000 rpm for 1 h at 4 °C, and
the supernatants were stored at -70 °C until use. The protein
concentration was quantitated using the Bio-Rad Detergent
Compatible Protein Assay kit (Bio-Rad, Hercules, CA). An equal
amount of proteins (20 μg per lane) for each sample was
separated by SDS-PAGE (12%) and transferred to polyvinyli-
dene difluoride (PVDF) membranes (Millipore, Bedford, MA).
The membranes were blocked overnight (4 °C) in Tris-buffered
saline containing 0.05% (v/v) Tween-20 (TBS-T) and 5% (m/v)
nonfat dried milk, and were incubated with anti-EGFR antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-β-actin
antibody (Sigma-Aldrich). After removal of the primary anti-
body, the membranes were washed with TBS-T and incubated
for 1 h at room temperature with a secondary horseradish
peroxidase-coupled IgG antibody (Pierce Chemical, Rockford, IL).
The membranes were washed for 3 times with TBS-T. Specific
protein bands were visualized using enhanced chemolumines-
cence (ECL) according to the protocol of the manufacturer
(Amersham Biosciences Inc., Piscataway, NJ).
’ MATERIALS AND METHODS
Chemicals and Reagents. Doxorubicin hydrochloride (DOX
HCl) salt, daunorubicin hydrochloride (internal standard), glu-
taric anhydride, piperidine and 1-hydroxybenzotriazole were
from Sigma-Aldrich (St. Louis, MO, USA). Trifluoroacetic acid
(TFA) was obtained from Rhodia (France). N-(9-Fluorenyl-
methoxycarbonyloxy) succinimide (Fmoc-OSu), benzotriazol-
1-yloxytris(dimethylamino)phosphonium hexafluoropho-
sphate (BOP), N,N-dimethylformamide (DMF) and N,N-diiso-
propylethylamine (DIPEA) were purchased from GL Biochem
(Shanghai, China).
Tumor Cell Lines. Human colon cancer cell line SW480,
human gastric cancer cell line SGC-7901, human breast cancer
cell line MCF-7, and murine melanoma cell line B16 were
obtained from China Center for Type Culture Collection
(CCTCC, Wuhan, China). The cells were cultured in RPMI-
1640 medium (Gibco BRL, GrandIsland, NY) containing 10%
fetal bovine serum (Hyclone, Logan, UT), 105 U/L penicillin
and 100 mg/L streptomycin at 37 °C.
Synthesis of DOX-Peptide Conjugate. The Fmoc pro-
tected EBP (Fmoc-NH-CMYIEALDKYAC-COOH) was syn-
thesized via solid phase methodology using Rink Amide methyl-
benzhydrylamine (MBHA) resin and Fmoc strategy. The synthe-
sized peptide was purified using reversed phase high performance
liquid chromatography (RP-HPLC) on a preparative C18 column
after TFA cleavage.26 The DOX-peptide conjugate was synthe-
sized essentially as described by Nagy et al. (1996) using EBP as
the peptide.27 Briefly, to a solution of DOX HCl in DMF, Fmoc-
OSu and DIPEA were added, and the reaction mixture was
Determination of Cellular DOX Content. Intracellular ac-
cumulation levels of DOX and DOX conjugate in different EGFR
expressing cells were determined by HPLC analysis.30 SW480, SGC-
7901, B16 and MCF-7 cells were grown in 25 cm2 culture flasks
with normal medium to ∼80% confluence (1 ꢀ 107 cells/flask).
376
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
After careful removal of medium, the cells were incubated with
5 mL of fresh serum-free medium containing 0.5 μM free DOX
or DOX-EBP conjugate at 37 °C for indicated times. The cells
were then washed 3 times with ice-cold PBS to remove unbound
DOX or DOX-peptide conjugate, collected by centrifugation,
and counted using a Coulter counter, and daunorubicin was
added as an internal standard for DOX quantification. The cell
pellet was then thoroughly extracted with an equal volume of
chloroform and isopropanol mixture (3:1, v/v) and centrifuged
at 13,000 rpm for 2 min. The drug levels in the cell lysates were
quantified by HPLC analysis as described above. The DOX
concentration was given by the standard curve, and the cellular
drug content was shown as pg of DOX/cell.
EGFR Inhibition Assay. To test the cellular entry pathway of
DOX-EBP conjugate, SW480, SGC-7901, B16 and MCF-7 cells
were preincubated with 5 mL of fresh serum-free medium
containing 5 μg/mL anti-EGFR monoclonal antibody C225
(ImClone Systems, Inc., New York, NY) for 12 h at 37 °C.31
After removal of the pretreatment solution, the cells were washed
once with PBS solution and further incubated with 5 mL of fresh
serum-free medium containing 0.5 μM free DOX or DOX-EBP
conjugate at 37 °C for indicated times. Cellular DOX content was
measured by HPLC as described above, and the C225 prompted
reduction of cellular DOX accumulation was used as an inhibi-
tion index of cellular DOX uptake.
Laser Scanning Confocal Fluorescence Microscopy. Since
DOX and DOX conjugate are autofluorescent at an excitation
wavelength of 488 nm, the detection of DOX and the conjugate
was performed using a laser scanning confocal fluorescence
microscope (Fluoview FV300, Olympus, Japan) at λex = 480
nm and λem = 580 nm.13,14 In order to investigate the intracel-
lular distribution of fluorescent DOX and DOX conjugate in
different EGFR expressing cells, SW480 and MCF-7 cells were
seeded on glass coverslips in 24-well plates (5 ꢀ 104 cells per
well). After 24 h, the cells were washed with serum-free medium,
treatedwith0.5 μM freeDOXorDOX-EBPconjugate (prepared
in RPMI-1640 serum-free medium prior to use) and incubated at
37 °C for indicated times. At the end of the treatment, the cells
were examined under laser confocal fluorescence microscope. To
detect nuclei, the cells were stained with 1 μg/mL of 40,6-
diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) at room
temperature for 10 min and visualized by fluorescence micro-
scopy at λex = 350 nm and λem = 460 nm.32,33 To further
characterize the selective cellular uptake of DOX-EBP conju-
gate via EGFR-mediated pathway, the cells were preincubated
with C225 for 12 h at 37 °C. Following the removal of the
pretreatment solution, the cells were washed with serum-free
medium, treated with free DOX or DOX-EBP conjugate and
incubated at 37 °C for indicated times. The cells were visualized
using the laser scanning confocal fluorescence microscope, and
the fluorescent intensity of cells was analyzed using Image-Pro
Plus 6.0 for Windows (Media Cybernetics, Silver Spring, MD).
In Vitro Cytotoxicity Assay. The cytotoxicity of free DOX
and DOX-EBP conjugate was evaluated using a modified MTT
cell viability assay.34,35 SW480, SGC-7901, B16 and MCF-7 cells
were seeded in 96-well plates at a density of 5 ꢀ 103 cells/well
and grown in normal medium for 24 h. The medium was then
replaced with serum-free medium containing a series of concen-
tration of DOX or DOX-EBP conjugate, and the cells were
further incubated at 37 °C for 5 or 48 h as indicated; the cells
incubated in serum-free medium without any drug were used as
blank controls. After adding 20 μL of 5 mg/mL MTT solution in
PBS to each well, the plates were further incubated for 4 h. The
cells were then solubilized in 100 μL of DMSO solution. The
absorbance was read at 570 nm (test wavelength) and 630 nm
(reference wavelength) on a microplate reader (Bio-Rad Model
450, Hercules, CA, USA).
Tumor Xenograft Model. Inbred C57BL/6 (H-2b) mice (18
to 22 g; 6-8 weeks old) of both sexes were used for B16
xenograft model.36 Mice were obtained from the Experimental
Animal Center of Tongji Medical College, Huazhong University
of Science and Technology (Wuhan, China), and all experi-
mental procedures involving animals were approved by the
Animal Care and Use Committee of Huazhong University of
Science and Technology. Male and female mice were housed
separately in polycarbonate cages, and provided with food and
water ad libitum. Tumors were grown at the lateral flank by sc
injection of 1 ꢀ 107 murine melanoma B16 cells and randomized
to constitute groups for distribution assay and antitumor activity
assay in vivo.37,38
Evaluation of Drug Distribution in Tumor-Bearing Mice.
To evaluate the distribution of DOX-EBP conjugate in vivo, the
C57BL/6 mice were used for the B16 xenograft model. The
model mice were randomly divided into groups, which contained
48 animals each. Control mice were given injections of 100 μL of
PBS. The experimental mice received equimolar amounts of free
DOX or DOX-EBP conjugate (6 mg/kg DOX or equivalent),
which were injected once into the tail vein at 2 weeks after tumor
implantation with an average tumor size of 450 to 550 mm3. At
selected time points (0.25, 0.5, 1, 2, 4, 8, 24, and 48 h) after
injection, blood was collected from the mice and centrifuged to
separate the plasma. The plasma was divided into 2 ꢀ 50 μL
aliquots and immediately frozen at -80 °C. The mice were then
sacrificed by cervical dislocation, and the liver, lungs, kidneys,
heart and tumor tissues were excised, washed with sterilized
saline solution, and cut into small pieces in 2 mL of PBS. The
tissues were homogenized in Ultra-Turrax T18 Homogenizer
(IKA, USA). All tissues were labeled and placed into polypro-
pylene cryovials and immediately frozen at -80 °C until analysis.
Plasma and tissues from nondosed mice (n = 24) were also
collected for the preparation of standard curves. The DOX
content in plasma and tissues was determined by HPLC analysis
as described above.38,39
Evaluation of Antitumor Activity of DOX-EBP Conjugate.
To test the in vivo efficacy of DOX-EBP conjugate, one day after
transplantation of tumors, animals bearing B16 xenografts were
randomly divided into groups. Each group contained nine
animals. The experimental mice were injected once with free
DOX (5 mg/kg) or DOX-EBP conjugate (5 mg/kg DOX
equivalent) into the tail vein at days 1, 8, and 15 after trans-
plantation of tumors; the control mice received injections of
saline. Tumors were measured twice weekly by vernier calipers,
and the tumor volume was calculated using the following for-
mula: volume (mm3) = (length) ꢀ (width)2/2.40 Animal survival
was monitored daily, and survival data were presented in a
Kaplan-Meier plot.41
Statistical Analysis. Data were presented as mean ( SD and
evaluated by Student’s t-test using SPSS 13.0 for Windows (SPSS
Inc., Chicago).
’ RESULTS
Synthesis of DOX-EBP Conjugate. The EGFR-binding
peptide was selected as a vector in the present study and
377
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
Scheme 1. Synthesis of DOX-EBP Conjugate from Doxorubicin Hydrocholoridea
a Reagents: (i) N,N-dimethylformamide (DMF), N-(9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu), N,N-diisopropylethylamine
(DIPEA), trifluoroacetic acid (TFA), and then diethyl ether; (ii) glutaric anhydride, DMF, DIPEA, and then TFA; (iii) Fmoc-NH-CMYIEALD-
KYAC-COOH, DMF, DIPEA, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), and then ethyl acetate; (iv) DMF
and piperidine; TFA, pyridine and DMF; and then ethyl acetate. The ester bond between the 14-OH group of DOX and glutaric acid is indicated by a
dashed line frame.
synthesized using a solid phase methodology.26 DOX was
conjugated via an ester bond at its C14 with EBP through a
glutarate spacer.27 As shown in Scheme 1, N-(9-fluorenylmeth-
oxycarbonyl)-DOX (N-Fmoc-DOX) was prepared by linking
N-(9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu)
with DOX at the 30 amino group. N-Fmoc-DOX-14-O-hemi-
glutarate was obtained using glutaric anhydride,28 identified by
RP-HPLC and further confirmed by MS and NMR. The N-
Fmoc-DOX-14-O-hemiglutarate was then conjugated with
Fmoc-NH-CMYIEALDKYAC-COOH through D-Lys,9 and the
Fmoc group was removed with piperidine. The DOX-peptide
product was purified by RP-HPLC on a C18 column and
lyophilized.29 The purity of the product was determined by
analytical HPLC, and the percentage of surface area was used to
378
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
Figure 1. EGFR expression in SW480, SGC-7901, B16 and MCF-7
cells. Cell lysates were prepared from SW480 (lane 1), SGC-7901 (lane
2), B16 (lane 3) and MCF-7 (lane 4) cells, separated by SDS-PAGE,
and analyzed by Western blotting.
Table 1. The Intracellular Levels of DOX in Selected Cell
Lines after 1 and 12 h of Incubation with Free DOX or
DOX-EBP Conjugatea
intracellular DOX levels (pg/cell)
1 h
DOX-EBP
12 h
DOX-EBP
cells
free DOX
free DOX
SW480
SGC-7901
B16
0.51 ( 0.08
0.57 ( 0.05
0.50 ( 0.08
0.65 ( 0.24
0.19 ( 0.04
0.21 ( 0.06
0.16 ( 0.05
0.05 ( 0.03
0.62 ( 0.07
0.63 ( 0.05
0.65 ( 0.07
0.50 ( 0.06
0.80 ( 0.05
0.93 ( 0.06
0.78 ( 0.05
0.17 ( 0.04
MCF-7
a Cells were grown in 25 cm2 culture flasks with normal medium to
∼80% confluence (1ꢀ107 cells/flask). After careful removal of medium,
5 mL of serum-free medium containing 0.5 μM of free DOX or
DOX-EBP conjugate was added, and the cells were further incubated
at 37 °C for indicated times. The DOX content in the cell lysates was
quantified by HPLC. Values are presented as the mean ( SD (n = 6).
Figure 2. Intracellular accumulation of free DOX and DOX-EBP
conjugate in tumor cells. SW480, SGC-7901, B16 and MCF-7 cells
were treated with free DOX or DOX-EBP for 1 h (A) and 12 h (B). The
intracellular DOX levels were determined by HPLC and expressed as pg
of DOX/cell. Data represent mean ( SD of 6 samples for each cell line.
*p < 0.05 (with versus without C225 pretreatment).
assess the purity (95%). The DOX-EBP product was confirmed
by electrospray mass spectra (direct injection): m/z (ESI), 1944
[M þ H]þ. The yield from DOX HCl to the final product was
40%.
Expression of EGFR in Tumor Cells. The baseline EGFR
expression levels of the four tumor cell lines SW480, SGC-7901,
B16 and MCF-7 were confirmed by Western blot analysis. While
MCF-7 was non-EGFR-overexpressing cells, the other three
were EGFR-overexpressing cell lines (Figure 1). These results
are in agreement with previous reports.42-45
Accumulation of DOX-EBP Conjugate in Tumor Cells
with EGFR Overexpression. To test whether DOX-EBP
conjugate can be selectively delivered into the tumors that
overexpress EGFR, the cellular DOX levels in different EGFR
expressing cells treated with the conjugate were compared with
that treated with free DOX. After incubation with free DOX or
DOX-EBP conjugate, the cellular DOX contents were deter-
mined by HPLC. No difference in intracellular DOX levels was
observed in all the tested cell lines after incubation with free
DOX for 1 h, whereas the DOX levels in EGFR-overexpressing
cells (SW480, SGC-7901 and B16) were significantly higher than
those in non-EGFR-overexpressing cells (MCF-7) when treated
with DOX-EBP conjugate for 1 h (Table 1). Similar results were
also observed after 12 h of incubation with free DOX or the
conjugate (Table 1). These data demonstrate a distinct cellular
entry of DOX-EBP conjugate from that of the free DOX.
Entry of DOX-EBP Conjugate into Tumor Cells through
EGFR-Mediated Pathway. Anti-EGFR monoclonal antibody
C225 can block the binding sites of EGFR to its ligands15,31,46
and thus can be used to inhibit cellular entry of drugs through
EGFR-mediated pathway. Therefore, to investigate whether the
cellular uptake of DOX-EBP conjugate was mediated by EGFR,
an EGFR inhibition assay was conducted through competitive
blockade of EGFR with C225. The cells were preincubated with
C225 for 12 h and then treated with free DOX or DOX-EBP.
After 1 h of incubation with free DOX, no significant difference of
cellular DOX level was observed in all tested cell lines with or
without C225 preincubation (Figure 2A). However, after 1 h of
incubation with DOX-EBP conjugate, the intracellular DOX
accumulation was reduced in all the cells preincubated with C225
as compared to those of corresponding cells without C225
pretreatment (Figure 2A). Similar trend was also found for the
12 h treatment with free DOX or DOX-EBP after C225
preincubation (Figure 2B), demonstrating the effectiveness of
C225 to inhibit cellular uptake of DOX-EBP and thus the
involvement of EGFR in the cellular delivery of the conjugate.
Cellular Uptake and Intracellular Localization of DOX-
EBP Conjugate. To further compare the cellular uptake path-
ways of free DOX and DOX-EBP conjugate, EGFR-over-
expressing SW480 cells and non-EGFR-overexpressing MCF-7
cells were incubated with free DOX or the conjugate and
evaluated by confocal fluorescence microscopy. As shown in
Figure 3A, DOX was present in nuclei as well as in cytoplasmic
region of both SW480 and MCF-7 cells after 30 min of incuba-
tion with free DOX, as revealed by superimposition of red
fluorescence from DOX and blue fluorescence from DAPI.
The DOX fluorescence intensity was also the same in both
SW480 and MCF-7 cells as assessed by Image-Pro Plus software
(Figure 3A). After the cells were incubated with DOX-EBP
379
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
Figure 3. Intracellular distribution of free DOX and DOX-EBP conjugate in SW480 and MCF-7 cells after 30 min of drug exposure. The cells were
treated with equimolar free DOX (A) or DOX-EBP conjugate (B) for 30 min and visualized under confocal microscope. Merged images display the
overlay of nuclear DAPI staining (blue) and DOX autofluorescence (red). Purple represents DOX in nuclear region. To detect the effect of anti-EGFR
antibody C225, the cells were preincubated with or without C225 for 12 h and then treated with equimolar free DOX or DOX-EBP for 30 min prior to
visualization (C).
conjugate for 30 min, however, DOX was clearly present in
cytoplasm and perinuclear region of both cell lines, but the DOX
fluorescence intensity in EGFR-overexpressing SW480 cells was
much stronger than that in non-EGFR-overexpressing MCF-7
cells (Figure 3B). On the other hand, although preincubation of
the cells with anti-EGFR monoclonal antibody C225 had no
effect on the intracellular distribution of free DOX, the fluores-
cence originated from DOX-EBP conjugate was inhibited by
the preincubation (Figure 3C). Together, these results further
suggest that the small, hydrophobic DOX entered the cytoplasm
in a passive manner and rapidly diffused into the nucleus (within
30 min), whereas the DOX conjugate was only visible in
cytoplasmic and perinuclear areas, but not in the nucleus, during
the first 30 min, indicating a distinct entry pathway of the
conjugate from the free DOX.
After 24 h of incubation with free DOX, the intracellular
distribution of DOX signal was almost the same as that of 30 min
in both SW480 and MCF-7 cells (Figure 4A). However, after the
same period of incubation with DOX-EBP conjugate, a stronger
DOX fluorescence in both cytoplasm and nuclei was detected in
380
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
Figure 4. Intracellular distribution of free DOX and DOX-EBP conjugate in SW480 and MCF-7 cells after 24 h of drug exposure. The cells were
treated with equimolar free DOX (A) or DOX-EBP conjugate (B) for 24 h and visualized under confocal microscope. Merged images display the
overlay of nuclear DAPI staining (blue) and DOX autofluorescence (red). Purple represents DOX in nuclear region. To detect the effect of anti-EGFR
antibody C225, the cells were preincubated with or without C225 for 12 h and then treated with equimolar free DOX or DOX-EBP for 24 h prior to
visualization (C).
SW480 cells, but only a weak DOX signal in the cytoplasm and an
even weaker signal in the nuclei were visible in MCF-7 cells
(Figure 4B), which was different from that of the first 30 min
treatment with the conjugate (Figure 3B). Preincubation with C225
had no effect on the fluorescence distribution of free DOX in both
cell lines, but showed an inhibitory effect on the fluorescence
intensity when treated with DOX-EBP conjugate (Figure 4C).
These data clearly demonstrate the buildup of DOX fluorescence in
the nuclei after prolonged incubation with the conjugate (24 h), and
also suggest EGFR-mediated uptake of DOX-EBP conjugate.
In Vitro Cytotoxicity of DOX-EBP Conjugate. In order to
compare the cytotoxic effects of DOX-EBP conjugate with free
DOX, we selected the four cell lines based on EGFR expression
levels as shown in Figure 1. The cells were incubated with a series
of concentrations of free DOX or DOX conjugate, and the cell
viability was evaluated using the MTT method. As shown in
Table 2, the IC50 values of DOX-EBP conjugate were much
higher than those of free DOX for all tested cell lines after
incubation for 5 h, i.e. the cytotoxicity of the conjugate was lower
than that of free DOX. However, after 48 h of incubation, the
cytotoxicity of the conjugate was much higher than that of free
DOX in all the EGFR-overexpressing cell lines (SW480, SGC-
7901 and B16) although free DOX was still more cytotoxic than
DOX-EBP conjugate in the non-EGFR-overexpressing MCF-7
381
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
Table 2. Cytotoxicity of Free DOX and DOX-EBP Conju-
enhance the therapeutic efficacy of DOX against EGFR-expres-
sing tumors and to improve the survival rate of the animals
bearing tumor xenograft. As shown in Figure 6A, the reduction of
tumor volume in C57BL/6 mice bearing B16 cells was apparent
in both free DOX and DOX conjugate groups: at the end of the
experiment on day 28, the mean volume of the tumors in the
mice treated with free DOX (1585 ( 102 mm3) and DOX-EBP
conjugate (668 ( 180 mm3) was significantly smaller than that in
the control group (1990 ( 222 mm3), i.e. about 20% and 66%
reduction respectively. These data demonstrate that the
DOX-EBP conjugate was more efficacious than free DOX in
the inhibition of tumor growth.
gate on Tumor Cellsa
IC50 (μM) of DOX equivalent
5 h
DOX-EBP
48 h
free DOX DOX-EBP
cells
free DOX
SW480
12.45 ( 1.62
64.37 ( 2.68 0.56 ( 0.16 0.08 ( 0.03
82.16 ( 3.24 0.38 ( 0.10 0.05 ( 0.08
76.50 ( 4.74 0.72 ( 0.19 0.16 ( 0.09
SGC-7901 18.50 ( 2.18
B16
15.58 ( 1.42
MCF-7
17.75 ( 2.38 165.68 ( 3.22 0.45 ( 0.20 6.25 ( 0.65
a Cells were seeded in 96-well plates at a density of 5 ꢀ 103 cells/well and
incubated for 24 h, and then exposed to free DOX or DOX-EBP for 5
and 48 h. Cell viability was assessed by the MTT assay. Data are
expressed as mean IC50 ( SD (n = 3).
Due to the lack of selectivity, the systemic cytotoxic chemother-
apy of free DOX would result in adverse toxicity. Indeed, although
free DOX did inhibit tumor growth compared to the saline control
(Figure 6A), the survival of mice bearing tumor xenograft did not
significantly improve (Figure 6B): the average survival time was
34 ( 3 days for free DOX treatment and 32.5 ( 2.5 days for the
control. On the other hand, however, DOX-EBP conjugate
significantly increased the survival time of animals (39.5 ( 3.5
days) under the same conditions (Figure 6B), demonstrating the
targeting effect of the DOX-EBP conjugate in chemotherapy.
cells (Table 2). These results clearly show that the DOX-EBP
conjugate was much more potent than the free DOX after 48 h
treatment in EGFR-overexpressing cells.
In Vivo Distribution of DOX-EBP Conjugate. Following a
single iv injection of free DOX or DOX-EBP, samples from
blood, liver, lung, kidney, heart and tumor tissues of C57BL/6
mice bearing B16 cells were taken and DOX level was measured
by HPLC. As shown in Figure 5A, the free DOX rapidly di-
sappeared from the circulation, whereas the DOX-EBP con-
jugate maintained at high level for a much longer time (up to 48
h). During the drug circulation, the highest level of DOX content
in plasma was 22.94 ( 1.68 μg/mL at 0.25 h after injection of
DOX-EBP conjugate, whereas that of free DOX was 6.52 (
0.13 μg/mL (n = 6, mean ( standard deviation).
As the primary site of DOX metabolism, the liver contained
the highest level of DOX among all the organs tested. The DOX
content in liver reached the highest level (26.03 ( 1.84 μg/g
tissue) after the mice were treated with free DOX for 30 min,
while the peak appeared at 8 h after administration of
DOX-EBP conjugate (Figure 5B). The highest value of DOX
content in liver of mice treated with free DOX was 4 times higher
than that treated with DOX-EBP conjugate (Figure 5B). Simi-
larly, the DOX levels in lung, kidney and heart of mice treated
with free DOX were much higher than those treated with
DOX-EBP conjugate (Figure 5C-E).
When DOX-EBP conjugate was injected, the DOX content
in tumor tissues was increased with time during the first 8 h and
peaked at 25.46 ( 2.19 μg/g tissue, and decreased to 6.68 ( 2.52
μg/g tissue at 48 h (Figure 5F). For free DOX treatment,
however, the DOX content rapidly peaked at 8.69 ( 2.17 μg/g
tissue within 30 min, and decreased to 1.23 ( 0.75 μg/g tissue at
48 h (Figure 5F). The area under the DOX concentration-time
curve (AUC), which is a key therapeutic index of a drug, was
calculated (Kinetic software version4.4.1, ThermoElectron Corp.,
Waltram, MA) for tumor tissues. As shown in Figure 5F, the
AUC0f48 in tumor tissues after DOX-EBP treatment (861.8 μg
h/g) was much higher than that of free DOX (149.6 μg h/g),
indicating that DOX concentration in the tumor tissues was
significantly higher after administering animals with DOX-EBP
than with free DOX.
’ DISCUSSION
In order to increase the therapeutic efficacy of doxorubicin
(DOX) while decreasing its systemic toxicity, a strategy was
chosen to enhance tumor specificity of DOX by conjugation to
EGFR-binding peptide (EBP), which may help deliver DOX
directly to EGFR-overexpressing neoplastic cells. This study
investigated the ability of the DOX-EBP conjugate to bind to
EGFR-overexpressing cells as well as its antitumor efficacy in vitro
and in vivo.
For targeted delivery, a drug can be linked to a peptide carrier
via covalent, ionic or hydrophobic bond, which may result in
alteration of structure and activity of the drug. A range of peptides
conjugated to DOX through different sites and linkers have been
studied. For example, DOX has been conjugated to Vectocell
peptides through ester, thioether and amide chemical linkers at
position 14 and 30 amino group of DOX, and the conjugation via
chemically stable bonds either at position 14 (thioether) or at
position 30 (amide) of DOX resulted in significant or complete
loss of activity while the conjugate via ester bond at C14 position
increased antitumor efficacy compared to free DOX.48 Another
example is the conjugation of DOX to LHRH via DOX-14-O-
hemiglutarate, which has fully preserved the binding affinity of
the peptide carrier and the cytotoxic activity of DOX.49 We have
shown in this study that the conjugation of DOX via the 14-OH
group to EBP has increased both in vitro and in vivo antitumor
activity (Table 2 and Figure 6).
To efficiently target EGFR-overexpressing tumor cells, the
DOX-EBP conjugate must preserve the binding ability of EGF
to EGFR. As shown in Table 1, the accumulation of free DOX in
both EGFR-overexpressing and non-EGFR-overexpressing cells,
as determined by HPLC, was almost the same; in contrast, the
accumulation of the DOX-EBP conjugate in EGFR-overexpres-
sing cells (SW480, SGC-7901 and B16) was much higher than
that in non-EGFR-overexpressing cells. Fluorescent microscopy
further demonstrates the high affinity of DOX-EBP conjugate
in EGFR-overexpressing cells: the fluorescent intensity in
SW480 cells was much higher than that in MCF-7 cells
after incubation with DOX-EBP conjugate (Figure 3B and
In Vivo Antitumor Therapeutic Efficacy. Since EGFR is
commonly expressed at high levels in a variety of solid
tumors,46,47 it can be used as a target to improve the antitumor
effect of drugs and decrease their systemic cytotoxicity. There-
fore we tested whether the DOX-EBP conjugate was able to
382
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
Figure 5. Distribution of free DOX and DOX-EBP conjugate in B16 bearing mice. Murine melanoma B16 cells were injected subcutaneously into
C57BL/6 mice, and the tumor was formed in 2 weeks. The animals were then treated with a single dose of free DOX (4) (6 mg/kg) or DOX-EBP (X)
(6 mg/kg DOX equivalent), and samples from blood and tissues were taken at indicated times. DOX concentration was determined in plasma (A), liver
(B), lung (C), kidney (D), heart (E) and tumor tissues (F) by HPLC after drug administration.
Figure 4B). These data suggest that the binding ability of the EBP
to EGFR was preserved in the conjugate.
after preincubation with C225 (Figure 3C and Figure 4C),
demonstrating the inhibition of cellular uptake of the conjugate.
These results suggest that the effect of DOX-EBP conjugate was
mediated through EGFR.
For a DOX-peptide conjugate to become effective after
entering tumor cells, the DOX must be released from the
conjugate and enter the nucleus. The availability of active
DOX to the nucleus, the drug action site, may become more
prominent because the primary mechanism of cytotoxicity of
DOX is through intercalation into DNA and disruption of
topoisomerase II action.3 Although DOX can be detected in
After targeting EGFR-overexpressing cells, the DOX-EBP
conjugate may enter tumor cells through EGFR-mediated route.
As shown in Figure 2, the competitive anti-EGFR monoclonal
antibody C225 can significantly reduce the binding of DOX-
EBP conjugate to EGFR, inhibit its entry and result in reduced
cellular accumulation of the conjugate in tumor cells. Fluorescent
microscopy further shows that the fluorescent signal of DOX-
EBP conjugate in both EGFR-overexpressing (SW480) and
non-EGFR-overexpressing (MCF-7) cells became very weak
383
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
DOX would be released from the conjugate after extension of time,
and penetrate the nuclear envelope and enter the nucleus.
As expected, the fluorescent signal of DOX in the nuclei of
SW480 cells was stronger after prolonged incubation (24 h) with
DOX-EBP (Figure 4B). In contrast, there was no obvious
difference between the fluorescent distribution in SW480 cells
treated with free DOX for 30 min and 24 h (Figure 3A and 4A).
Consequently, the cytotoxic activity of the DOX-EBP conjugate
was much higher than that of the original free DOX in SW480 cells
after extended incubation (48 h; Table 2).
The binding of DOX-EBP conjugate to EGFR-overexpressing
cells suggests that it may have a targeting effect in mice bearing EGFR-
overexpressing tumors, therefore we examined the distribution and
the antitumor activity of the conjugate in vivo. As shown in Figure 5,
the administration of DOX-EBP resulted in different biodistribution
compared with free DOX. The high content and long circulation time
of the conjugate in the bloodstream (Figure 5A) imply the potential
of the novel chemotherapeutic agent to achieve a better tumor
targeting effect in vivo. As shown in Figure 5F, the DOX content
was indeed higher in EGFR-overexpressing tumors of B16 cells
bearing mice treated with the conjugate than that with free DOX.
The substantial delay of the peak concentration of DOX-EBP (at 8 h
for DOX-EBP vs 0.5 h for DOX) also suggests that the conjugation
of DOX with EBP can enhance its therapeutic efficacy. As expected,
after 4 weeks of treatment with DOX-EBP, the tumor volume was
reduced 66% compared to the saline control, while the reduction was
only 20% when treated with free DOX (Figure 6A). Taken together,
these data demonstrate that the DOX-EBP conjugate had exhibited
longer circulation time and greater accumulation in EGFR over-
expressing tumors, and thus lead to increased therapeutic efficacy.
Similar results have also been reported for DOX conjugated with
other peptides.52,53
The adverse effect is a major concern for free DOX as a
therapeutic agent, and the nonselective toxicity of DOX is closely
related to its distribution in normal tissues at high levels. The
DOX-EBP conjugate, however, effectively lowered the distribu-
tion of DOX in normal tissues, in particular with no significant
accumulation in the heart tissue, which is extremely sensitive to
DOX toxicity (Figure 5B-E). Furthermore, treatment with
DOX-EBP conjugate significantly extended the survival time
of mice bearing tumor xenograft (Figure 6B), demonstrating
lower systemic toxicity and higher therapeutic efficacy.
Figure 6. Therapeutic efficacy of free DOX and DOX-EBP in B16
tumor xenograft model. Murine melanoma B16 cells (1 ꢀ 107 cells)
were injected subcutaneously in C57BL/6 mice. On days 1, 8, and 15
after transplantation of tumors (n = 9), free DOX (4) or DOX-EBP
(X) was injected via tail vein at 5 mg/kg body weight (DOX or DOX
equivalent). Control mice received injections of saline (9). (A)
Comparison of tumor volume. (B) Survival analysis of B16 tumor-
bearing mice (Kaplan-Meier survival curve).
both cytoplasm and nucleus after incubation with free DOX
(Figure 3A), the conjugate was clearly distributed in cytoplasm
and perinuclear zone, but not in nucleus, after incubation for 30
min (Figure 3B). Similar phenomena were also observed with
other peptide carriers conjugated with DOX. For example, the
conjugates of a cyclic pentapeptide (CNGRC)13 and a cell-
penetrating peptide (CGGGYGRKKRRQRRR)50 with DOX
exhibited a cytoplasmic distribution, similar to DOX-EBP
(Figure 3B). This may be because the large hydrophilic peptides
have high hydrogen bonding potential and thus are not readily
partitioned into the cell membranes as they are energetically
unfavorable to expel hydrogen-bonded water molecules.51
Therefore, the DOX conjugates inside the cells cannot directly
diffuse into the nucleus. On the other hand, during the early
incubation period, the active DOX released from the conjugate
inside the cytoplasm was not sufficient to be partitioned to the
nucleus, therefore its in vitro cytotoxicity was not as high as that of
free DOX (Table 2). These data demonstrate that the entry and
partition routes of the conjugate are distinct from that of free DOX.
Since DOX-EBP conjugate in the current study is an ester
conjugate linked via a glutaric acid spacer, it is sensitive to
hydrolysis by esterase. Therefore, although the conjugate cannot
directly diffuse toward the nucleus, it may be hydrolyzed by cellular
esterase to release active DOX in the cytoplasm.13 As a result, more
In conclusion, we report the conjugation of DOX with an
EGFR-binding peptide (EBP: NH2-CMYIEALDKYAC-COOH)
via an ester bond at C14 through a glutarate spacer, and
demonstrate that the DOX-EBP conjugate efficiently retained
the EGFR binding ability. The conjugate displayed targeting
competence toward EGFR-overexpressing tumor cells, and thus
exhibited potent antitumor effect in vivo.
’ AUTHOR INFORMATION
Corresponding Author
*S.A. and Z.H.: College of Pharmacy, Wuhan University, 185
East Lake Road, Wuhan 430071, China. Phone: 86-27-
68759006. Fax: 86-27-68759850. E-mail: aishibin@tom.com
(S.A.); zbhuang@whu.edu.cn (Z.H.).
’ ACKNOWLEDGMENT
This study was supported by the National Mega Project on
Major Drug Development (Grant No. 2009ZX09301-014-1),
384
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
the National Natural Science Foundation of China (Grant No.
30971456) and in part by the National High-Tech R & D
Program of China (863 Program; Grant No. 2007AA10Z344).
(21) Montelione, G. T.; W€uthrich, K.; Nice, E. C.; Burgess, A. W.;
Scheraga, H. A. Solution structure of murine epidermal growth factor:
determination of the polypeptide backbone chain-fold by nuclear
magnetic resonance and distance geometry. Proc. Natl. Acad. Sci. U.S.A.
1987, 84, 5226–5230.
(22) Koide, H.; Muto, Y.; Kasai, H.; Kohri, K.; Hoshi, K.; Takahashi,
S.; Tsukumo, K.; Sasaki, T.; Oka, T.; Miyake, T. A site-directed
mutagenesis study on the role of isoleucine-23 of human epidermal
growth factor in the receptor binding. Biochim. Biophys. Acta 1992, 1120,
257–261.
(23) Wingens, M.; Walma, T.; van Ingen, H.; Stortelers, C.; van
Leeuwen, J. E.; van Zoelen, E. J.; Vuister, G. W. Structural analysis of an
epidermal growth factor/transforming growth factor-alpha chimera with
unique ErbB binding specificity. J. Biol. Chem. 2003, 278, 39114–39123.
(24) Ogiso, H.; Ishitani, R.; Nureki, O.; Fukai, S.; Yamanaka, M.;
Kim, J. H.; Saito, K.; Sakamoto, A.; Inoue, M.; Shirouzu, M.; Yokoyama,
S. Crystal structure of the complex of human epidermal growth factor
and receptor extracellular domains. Cell 2002, 110, 775–787.
(25) Komoriya, A.; Hortsch, M.; Meyers, C.; Smith, M.; Kanety, H.;
Schlessinger, J. Biologically active synthetic fragments of epidermal
growth factor: localization of a major receptor-binding region. Proc.
Natl. Acad. Sci. U.S.A. 1984, 81, 1351–1355.
(26) Wilson, C. L.; Monteith, W. B.; Danell, A. S.; Burns, C. S.
Purification and characterization of the central segment of prothymosin-
alpha: methodology for handling highly acidic peptides. J. Pept. Sci. 2006,
12, 721–725.
(27) Nagy, A.; Schally, A. V.; Armatis, P.; Szepeshazi, K.; Halmos, G.;
Kovacs, M.; Zarandi, M.; Groot, K.; Miyazaki, M.; Jungwirth, A.; Horvath, J.
Cytotoxic analogs of luteinizing hormone-releasing hormone containing
doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more
potent. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 7269–7273.
’ REFERENCES
(1) Gewirtz, D. A. A critical evaluation of the mechanisms of action
proposed for the antitumor effects of the anthracycline antibiotics
adriamycin and daunorubicin. Biochem. Pharmacol. 1999, 57, 727–741.
(2) Lal, S.; Mahajan, A.; Chen, W. N.; Chowbay, B. Pharmacoge-
netics of target genes across doxorubicin disposition pathway: a review.
Curr. Drug Metab. 2010, 11, 115–128.
(3) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L.
Anthracyclines: molecular advances and pharmacologic developments
in antitumor activity and cardiotoxicity. Pharmacol. Rev. 2004, 56, 185–
229.
(4) Thornton, K. Chemotherapeutic management of soft tissue
sarcoma. Surg. Clin. North Am. 2008, 88, 647–660.
(5) Raschi, E.; Vasina, V.; Ursino, M. G.; Boriani, G.; Martoni, A.; De
Ponti, F. Anticancer drugs and cardiotoxiciy: Insights and perspectives in
the era of targeted therapy. Pharmacol. Ther. 2010, 125, 196–218.
(6) Mordente, A.; Meucci, E.; Silvestrini, A.; Martorana, G. E.;
Giardina, B. New developments in anthracycline-induced cardiotoxicity.
Curr. Med. Chem. 2009, 16, 1656–1672.
(7) Ferreira, A. L.; Matsubara, L. S.; Matsubara, B. B. Anthracycline-
induced cardiotoxicity. Cardiovasc. Hematol. Agents Med. Chem. 2008, 6,
278–281.
(8) Langer, M.; Kratz, F.; Rothen-Rutishauser, B.; Wunderli-Allen-
spach, H.; Beck-Sickinger, A. G. Novel peptide conjugates for tumor-
specific chemotherapy. J. Med. Chem. 2001, 44, 1341–1348.
(9) Nagy, A.; Schally, A. V. Targeting of cytotoxic luteinizing
hormone-releasing hormone analogs to breast, ovarian, endometrial,
and prostate cancer. Biol. Reprod. 2005, 73, 851–859.
(28) Chen, Q.; Sowa, D. A.; Gabathuler, R. Synthesis of doxorubicin
conjugates through 14-hydroxy group to melanotransferrin P97. Synth.
Commun. 2003, 33, 2389–2398.
(10) de Groot, F. M.; Broxterman, H. J.; Adams, H. P.; van Vliet, A.;
Tesser, G. I.; Elderkamp, Y. W.; Schraa, A. J.; Kok, R. J.; Molema, G.;
Pinedo, H. M.; Scheeren, H. W. Design, synthesis, and biological
evaluation of a dual tumor-specific motive containing integrin-targeted
plasmin-cleavable doxorubicin prodrug. Mol. Cancer Ther. 2002, 1, 901–
911.
(11) Qian, Z. M.; Li, H.; Sun, H.; Ho, K. Targeted drug delivery via
the transferrin receptor-mediated endocytosis pathway. Pharmacol. Rev.
2002, 54, 561–587.
(12) Schally, A. V.; Nagy, A. New approaches to treatment of various
cancers based on cytotoxic analogs of LHRH, somatostatin and bombe-
sin. Life Sci. 2003, 72, 2305–2320.
(13) van Hensbergen, Y.; Broxterman, H. J.; Elderkamp, Y. W.;
Lankelma, J.; Beers, J. C.; Heijn, M.; Boven, E.; Hoekman, K.; Pinedo,
H. M. A doxorubicin-CNGRC conjugate with prodrug properties.
Biochem. Pharmacol. 2002, 63, 897–908.
(14) Emons, G.; Sindermann, H.; Engel, J.; Schally, A. V.; Gr€undker,
C. Luteinizing hormone-releasing hormone receptor-targeted che-
motherapy using AN-152. Neuroendocrinology 2009, 90, 15–18.
(15) Dutta, P. R.; Maity, A. Cellular responses to EGFR inhibitors
and their relevance to cancer therapy. Cancer Lett. 2007, 254, 165–177.
(16) Herbst, R. S. Review of epidermal growth factor receptor
biology. Int. J. Radiat. Oncol. Biol. Phys. 2004, 59, 21–26.
(17) Harari, P. M.; Allen, G. W.; Bonner, J. A. Biology of interac-
tions: antiepidermal growth factor receptor agents. J. Clin. Oncol. 2007,
25, 4057–4065.
(18) Okamoto, I. Epidermal growth factor receptor in relation to
tumor development: EGFR-targeted anticancer therapy. FEBS. J. 2010,
277, 309–315.
(19) Schneider, M. R.; Wolf, E. The epidermal growth factor
receptor ligands at a glance. J. Cell Physiol. 2009, 218, 460–466.
(20) Cooke, R. M.; Wilkinson, A. J.; Baron, M.; Pastore, A.; Tappin,
M. J.; Campbell, I. D.; Gregory, H.; Sheard, B. The solution structure of
human epidermal growth factor. Nature 1987, 327, 339–341.
(29) Bai, L.; Zhu, R.; Chen, Z.; Gao, L.; Zhang, X.; Wang, X.; Bai, C.
Potential role of short hairpin RNA targeting epidermal growth factor
receptor in growth and sensitivity to drugs of human lung adenocarci-
noma cells. Biochem. Pharmacol. 2006, 71, 1265–1271.
(30) Bidwell, G. L., 3rd.; Davis, A. N.; Fokt, I.; Priebe, W.; Raucher,
D. A thermally targeted elastin-like polypeptide-doxorubicin conjugate
overcomes drug resistance. Invest. New Drugs 2007, 25, 313–326.
(31) Overholser, J. P.; Prewett, M. C.; Hooper, A. T.; Waksal, H. W.;
Hicklin, D. J. Epidermal growth factor receptor blockade by antibody
IMC-C225 inhibits growth of a human pancreatic carcinoma xenograft
in nude mice. Cancer 2000, 89, 74–82.
(32) Abbosh, P. H.; Montgomery, J. S.; Starkey, J. A.; Novotny, M.;
Zuhowski, E. G.; Egorin, M. J.; Moseman, A. P.; Golas, A.; Brannon,
K. M.; Balch, C.; Huang, T. H.; Nephew, K. P. Dominant-negative
histone H3 lysine 27 mutant derepresses silenced tumor suppressor
genes and reverses the drug-resistant phenotype in cancer cells. Cancer
Res. 2006, 66, 5582–5591.
(33) Pang, Z.; Feng, L.; Hua, R.; Chen, J.; Gao, H.; Pan, S.; Jiang, X.;
Zhang, P. Lactoferrin-conjugated biodegradable polymersome holding
doxorubicin and tetrandrine for chemotherapy of glioma rats. Mol.
Pharmaceutics 2010, 7, 1995–2005.
(34) Cao, N.; Feng, S. S. Doxorubicin conjugated to D-alpha-
tocopheryl polyethylene glycol 1000 succinate (TPGS): conjugation
chemistry, characterization, in vitro and in vivo evaluation. Biomaterials
2008, 29, 3856–3865.
(35) Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J. Immunol.
Methods 1983, 65, 55–63.
(36) Han, L.; Wang, W.; Fang, Y.; Feng, Z.; Liao, S.; Li, W.; Li, Y.; Li,
C.; Maitituoheti, M.; Dong, H.; Lai, W.; Gao, Q.; Xi, L.; Wu, M.; Wang,
D.; Zhou, J.; Meng, L.; Wang, S.; Ma, D. Soluble B and T lymphocyte
attenuator possesses antitumor effects and facilitates heat shock protein
70 vaccine-triggered antitumor immunity against a murine TC-1 cervical
cancer model in vivo. J. Immunol. 2009, 183, 7842–7850.
385
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386

Molecular Pharmaceutics
ARTICLE
(37) Mamot, C.; Drummond, D. C.; Noble, C. O.; Kallab, V.; Guo,
Z.; Hong, K.; Kirpotin, D. B.; Park, J. W. Epidermal growth factor
receptor-targeted immunoliposomes significantly enhance the efficacy
of multiple anticancer drugs in vivo. Cancer Res. 2005, 65, 11631–11638.
(38) Etrych, T.; Chytil, P.; Mrkvan, T.; Sírovꢀa, M.; Ríhovꢀa, B.;
Ulbrich, K. Conjugates of doxorubicin with graft HPMA copolymers
for passive tumor targeting. J. Controlled Release 2008, 132, 184–192.
(39) Al-Abd, A. M.; Kim, N. H.; Song, S. C.; Lee, S. J.; Kuh, H. J. A
simple HPLC method for doxorubicin in plasma and tissues of nude
mice. Arch. Pharm. Res. 2009, 32, 605–611.
(40) Wu, G.; Barth, R. F.; Yang, W.; Kawabata, S.; Zhang, L.; Green-
Church, K. Targeted delivery of methotrexate to epidermal growth
factor receptor-positive brain tumors by means of cetuximab (IMC-
C225) dendrimer bioconjugates. Mol. Cancer Ther. 2006, 5, 52–59.
(41) Goldstein, N. I.; Prewett, M.; Zuklys, K.; Rockwell, P.;
Mendelsohn, J. Biological efficacy of a chimeric antibody to the
epidermal growth factor receptor in a human tumor xenograft model.
Clin. Cancer Res. 1995, 1, 1311–1318.
(42) Pohl, M.; Stricker, I.; Schoeneck, A.; Schulmann, K.; Klein-
Scory, S.; Schwarte-Waldhoff, V.; Hasmann, M.; Tannapfel, A.;
Schmiegel, W.; Reinacher-Schick, A. Antitumor activity of the HER2
dimerization inhibitor pertuzumab on human colon cancer cells in vitro
and in vivo. J. Cancer Res. Clin. Oncol. 2009, 135, 1377–1386.
(43) Liao, X.; Che, X.; Zhao, W.; Zhang, D.; Long, H.; Prakash, C.;
Li, H. Effects of propranolol in combination with radiation on apoptosis
and survival of gastric cancer cells in vitro. Radiat. Oncol. 2010, 5, 58.
(44) Ueno, Y.; Sakurai, H.; Tsunoda, S.; Choo, M. K.; Matsuo, M.;
Koizumi, K.; Saiki, I. Heregulin-induced activation of ErbB3 by EGFR
tyrosine kinase activity promotes tumor growth and metastasis in
melanoma cells. Int. J. Cancer 2008, 123, 340–347.
(45) Hirsch, D. S.; Shen, Y.; Wu, W. J. Growth and motility
inhibition of breast cancer cells by epidermal growth factor receptor
degradation is correlated with inactivation of Cdc42. Cancer Res. 2006,
66, 3523–3523.
(46) Lurje, G.; Lenz, H. J. EGFR signaling and drug discovery.
Oncology 2009, 77, 400–410.
(47) Mitsudomi, T.; Yatabe, Y. Epidermal growth factor receptor in
relation to tumor development: EGFR gene and cancer. FEBS. J. 2010,
277, 301–308.
(48) Meyer-Losic, F.; Quinonero, J.; Dubois, V.; Alluis, B.;
Dechambre, M.; Michel, M.; Cailler, F.; Fernandez, A. M.; Trouet, A.;
Kearsey, J. Improved therapeutic efficacy of doxorubicin through con-
jugation with a novel peptide drug delivery technology (Vectocell).
J. Med. Chem. 2006, 49, 6908–6916.
(49) Halmos, G.; Nagy, A.; Lamharzi, N.; Schally, A. V. Cytotoxic
analogs of luteinizing hormone-releasing hormone bind with high
affinity to human breast cancers. Cancer Lett. 1999, 136, 129–136.
(50) Liang, J. F.; Yang, V. C. Synthesis of doxorubicin-peptide
conjugate with multidrug resistant tumor cell killing activity. Bioorg.
Med. Chem. Lett. 2005, 15, 5071–5075.
(51) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug
Delivery Rev. 2001, 46, 3–26.
(52) Szepeshazi, K.; Schally, A. V.; Halmos, G. LH-RH receptors in
human colorectal cancers: unexpected molecular targets for experimen-
tal therapy. Int. J. Oncol. 2007, 30, 1485–1492.
(53) Wu, W.; Luo, Y.; Sun, C.; Liu, Y.; Kuo, P.; Varga, J.; Xiang, R.;
Reisfeld, R.; Janda, K. D.; Edgington, T. S.; Liu, C. Targeting cell-
impermeable prodrug activation to tumor microenvironment eradicates
multiple drug-resistant neoplasms. Cancer Res. 2006, 66, 970–980.
386
dx.doi.org/10.1021/mp100243j |Mol. Pharmaceutics 2011, 8, 375–386