Multivalency: Key Feature in Overcoming Drug Resistance with a Cleavable Cell-Penetrating Peptide-Doxorubicin Conjugate

Article abstract of DOI:10.1007/s10989-017-9622-4

Multivalency is often used in biological systems, to increase affinity and specificity through avidity. This inspired us to prepare a synthetic bioconjugate that mimics natural multivalent systems. It is composed of doxorubicin and two octaarginine cell-penetrating peptides, to strengthen the electrostatic interactions between the negatively charged glycosaminoglycans of the plasma membrane and the guanidinium groups of the arginine residues. The multivalent conjugate has improved cellular uptake and cytotoxicity, compared to a peptide-drug conjugate with only one polyarginine and as a result it can overcome drug resistance in Kelly-ADR cells. The synthetic approach and the multivalent structure reported here can be used further as model systems, to gain insight into the biological interaction of cell-penetrating peptides with artificial membranes or for the preparation of more complex multimers.

Full text of DOI:10.1007/s10989-017-9622-4

Int J Pept Res Ther
DOI 10.1007/s10989-017-9622-4
Multivalency: Key Feature in Overcoming Drug Resistance
with a Cleavable Cell-Penetrating Peptide-Doxorubicin Conjugate
Marco Lelle1,2,3 · Christoph Freidel1 · Stefka Kaloyanova1 · Klaus Müllen1 ·
Kalina Peneva1,2
Accepted: 3 August 2017
© Springer Science+Business Media, LLC 2017
Abstract Multivalency is often used in biological systems,
to increase affinity and specificity through avidity. This
inspired us to prepare a synthetic bioconjugate that mimics
natural multivalent systems. It is composed of doxorubicin
and two octaarginine cell-penetrating peptides, to strengthen
the electrostatic interactions between the negatively charged
glycosaminoglycans of the plasma membrane and the guani-
dinium groups of the arginine residues. The multivalent con-
jugate has improved cellular uptake and cytotoxicity, com-
pared to a peptide-drug conjugate with only one polyarginine
and as a result it can overcome drug resistance in Kelly-ADR
cells. The synthetic approach and the multivalent structure
reported here can be used further as model systems, to gain
insight into the biological interaction of cell-penetrating
peptides with artificial membranes or for the preparation of
more complex multimers.
Introduction
The anthracycline doxorubicin ranks among the most effi-
cient anticancer agents and is widely applied against breast
cancer, soft tissue sarcomas, non-Hodgkin’s lymphoma, leu-
kaemia as well as childhood solid tumors (Gewirtz 1999;
Nadas and Sun 2006; Minotti et al. 2004). Nevertheless,
there are a few types of cancer where doxorubicin is non-
effective (e.g. melanoma, colon cancer) (Weiss 1992). More-
over, numerous severe adverse effects restrict the therapeutic
efficacy of the drug, like the onset of drug resistance in can-
cer cells and the poor tumor selectivity (Minotti et al. 2004;
Nadas and Sun 2006; Meyer-Losic et al. 2006; Kratz et al.
1998; Weiss 1992). Nowadays, drug resistance is the main
contributor to the failure of many anticancer agents includ-
ing anthracyclines (Szakacs et al. 2014). The various cellular
mechanisms that cancer cells use to resist chemotherapeutics
are known to lead to a phenomenon called multidrug resist-
ance (MDR). This term describes a process in which cancer
cells become resistant to cytostatic or cytotoxic actions of
multiple, structurally dissimilar and functionally divergent,
drugs commonly used in cancer chemotherapy (Gottesman
et al. 2002; Ambudkar et al. 1999). MDR in cancer is attrib-
uted to the presence of membrane proteins, which belong
to the ATP-binding cassette transporter superfamily (Leslie
et al. 2005; Dean et al. 2001). These membrane-associated
transporters such as P-glycoprotein are also a common fea-
ture of noncancerous cells and are involved in tissue detoxi-
fication as well as protection (Ambudkar et al. 1999). The
protective function of these proteins is mediated through an
energy-dependent excretion of cytotoxic substances and their
harmful metabolites from the cytoplasm (Ambudkar et al.
1999; Leslie et al. 2005). Thereby, the intracellular accu-
mulation of antitumor drugs like doxorubicin can be mark-
edly reduced, which makes chemotherapeutics less effective
Keywords Cell-penetrating peptide · Multivalency ·
Drug-peptide conjugate · MDR · Doxorubicin · Drug
resistance
Electronic supplementary material The online version
of this article (doi:10.1007/s10989-017-9622-4) contains
supplementary material, which is available to authorized users.
* Kalina Peneva
kalina.peneva@uni-jena.de
1
Max Planck Institute for Polymer Research, Ackermannweg
10, 55128 Mainz, Germany
2
Institute of Organic and Macromolecular Chemistry, Jena
Center of Soft Matter, Friedrich Schiller University Jena,
Lessingstr. 8, 07743 Jena, Germany
3
Institute of Physiology II, University Hospital Jena,
Kollegiengasse 9, 07743 Jena, Germany
Vol.:(0123456789)
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Int J Pept Res Ther
(Minotti et al. 2004; Nadas and Sun 2006; Gottesman et al.
2002). Furthermore, it has been demonstrated for many
different tumor cells that they substantially overexpress
membrane-associated transporter proteins and that the drug
efflux is even increased after exposure to anticancer agents
(Gottesman et al. 2002; Leslie et al. 2005). To overcome this
limitation and bypass these transporter proteins, doxorubicin
has been modified with different peptides such as penetratin,
TAT or other arginine-rich amino acid sequences (Mazel
et al. 2001; Liang and Yang 2005; Meyer-Losic et al. 2006;
Aroui et al. 2009; Lelle et al. 2017). These small peptide
sequences (typically less than 30 residues) belong to the so
called cell-penetrating peptides and are also known as pro-
tein transduction domains (Zorko and Langel 2005; Jones
and Sayers 2012; Milletti 2012; Tabujew et al. 2015). One of
their key properties is the ability to efficiently deliver diverse
cargoes such as drugs across the cell membrane (Miklan
et al. 2009; Vargas et al. 2014; Dubikovskaya et al. 2008;
Böhme and Beck-Sickinger 2015). The uptake mechanisms
related to these cationic peptide sequences typically involve
direct translocation across the cellular membrane as well
as endocytosis and are based on electrostatic interactions
(Jones and Sayers 2012; Duchardt et al. 2007; Ziegler and
Seelig 2008). Whereas doxorubicin is quickly taken up by
passive diffusion (Siegfried et al. 1985). Thereby, small
hydrophobic drugs are particularly prone to be a substrate
to membrane-associated efflux pumps, due to their lipophilic
nature, which favors the residence in the cell membrane,
where the transporter proteins reside (Vargas et al. 2014;
Gatlik-Landwojtowicz et al. 2006; Rottenberg and Borst
2012).
Kunjachan et al. 2012). Although these nanoformulations
have been widely used, they exhibit certain disadvantages,
e.g. application of undefined molecular structures, drug
loading inconsistencies or lack of stability (Gu et al. 2012;
Furedi et al. 2017; Wu et al. 2011; Riganti et al. 2011). In
addition, a recent in vitro study demonstrated that the ability
of the carrier to overcome resistance is much less prominent
as reported and it can be cell type- and carrier-dependent
(Kunjachan et al. 2012). The multivalent cell-penetrating
peptide-doxorubicin conjugate reported here is capable of
overcoming drug resistance without the aforementioned
limitations, since the precise structure with two peptides
per drug molecule can only be cleaved within the reductive
environment of the cancer cell.
Materials and Methods
General Information
Solvents and reagents were purchased from commercial
sources (Acros Organics, Alfa Aesar, AppliChem, Deutero,
Thermo Fisher Scientific, Fluka, Merck, Sigma-Aldrich) and
used without further purification. Doxorubicin hydrochloride
was ordered from Ontario Chemicals, Inc. (Guelph, Ontario,
Canada) and custom peptide synthesis of Ac-CRRRRRRRR-
NH2 was carried out from Genosphere Biotechnologies
(Paris, France). The CellTiter-Glo luminescent cell viabil-
ity assay was bought from Promega (Mannheim, Germany).
Silica gel (0.063–0.200 mm) for column chromatography
as well as thin layer chromatography sheets (ALUGRAM
SIL G/UV254) were ordered from Macherey-Nagel (Düren,
Germany) and applied with suitable solvent systems.
In this study we describe the synthesis and characteriza-
tion of a multivalent cleavable cell-penetrating peptide-drug
conjugate that can overcome drug resistance in doxorubicin-
resistant cancer cells. In biological systems, multivalent
interactions are often employed to increase the affinity and
specificity (Fasting et al. 2012). This inspired us to prepare
a synthetic bioconjugate that mimics natural multivalent
systems. It is composed of doxorubicin and two octaargi-
nine cell-penetrating peptides, to strengthen the electrostatic
interactions between the negatively charged plasma mem-
brane and the amino acid side chains. Thus, cellular uptake
and cytotoxicity, respectively, can be improved, compared to
a peptide-drug conjugate with only one polyarginine, which
we used as a reference. The introduced bioconjugates exhibit
not only the capability to circumvent membrane-associated
transporter proteins and to overcome drug resistance accord-
ingly, but are also well-suited to target cancer cells medi-
ated through high expression levels of glycosaminoglycans
(Nakase et al. 2012).
Synthesis of 2-(2-Pyridyldithio)ethylamine
Hydrochloride (2)
2,2′-Dithiopyridine (25 g, 113.47 mmol) was dissolved in
150 mL methanol and degassed in an ultrasonic bath for
30 min. Afterwards, 2-mercaptoethylamine hydrochloride
(2.15 g, 18.91 mmol, 1) was slowly added within 1 h to
the colorless solution. The flask was sealed with a rubber
septum and the reaction mixture was stirred overnight at
ambient temperature under argon. Subsequently, the yel-
low solution was precipitated twice in cold diethyl ether
and the product was obtained by filtration as colorless solid
(4.21 g, 18.91 mmol, quantitative yield). m/z (MALDI-TOF)
187.00 [M+H]+; 1H NMR (300 MHz, DMSO-d6, 298 K)
δ (ppm) = 8.56–8.46 (m, 1H), 8.30 (s, br, 3H), 7.88–7.80
(m, 1H), 7.76 (d, 1H, J = 8.1 Hz), 7.34–7.25 (m, 1H),
3.17–3.01 (m, 4H); 13C NMR (75 MHz, DMSO-d6, 298 K)
δ (ppm) = 158.09, 149.80, 137.89, 121.59, 120.00, 37.65,
34.74.
Multivalent formulations of doxorubicin, like liposomes,
organic or inorganic particles, have been applied to over-
come multidrug resistance as well (Iyer et al. 2013;
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Int J Pept Res Ther
Synthesis of (Boc-Aminooxy)acetic Acid
N-Hydroxysuccinimide Ester (4)
stirred for 1 h at room temperature. Afterwards, solvent
and reagent were removed under reduced pressure and the
residue was dried for further 6 h in a high vacuum. Doxo-
rubicin hydrochloride (1.49 g, 2.57 mmol) was dissolved
in 175 mL sodium acetate buffer (0.4 M, pH 4.8) and
added to the oily residue dissolved in 175 mL DMF. The
solution was stirred for 48 h and subsequently purified by
RP-HPLC. The solvent of the collected fractions was evap-
orated and the obtained residue was dissolved in methanol
and precipitated in diethyl ether, to yield the product as
red solid (1.76 g, 2.08 mmol, 81%). m/z (MALDI-TOF)
807.98 [M+Na]+; RP-HPLC tR = 15.56 min (480 nm),
A—25 mM triethylammonium acetate (TEAA) buffer (pH
7), B—acetonitrile (ACN), 0 min 75% A, 45 min 30% A;
1H NMR (700 MHz, D2O, 298 K) δ (ppm) = 8.21 (d, 1H,
J = 4.2 Hz), 7.67–7.58 (m, 2H), 7.50 (d, 1H, J = 8.4 Hz),
7.46 (d, 1H, J = 7.0 Hz), 7.32 (d, 1H, J = 7.0 Hz), 7.13
(t, 1 H, J = 5.6 Hz), 5.41 (s, 1H), 4.83 (s, 1H), 4.61 (dd,
2H, J = 16.1 Hz, J = 22.4 Hz), 4.51 (dd, 2H, J = 12.6 Hz,
J=38.5 Hz), 4.23 (q, 1H, J=6.3 Hz), 3.86 (s, 3H), 3.77 (s,
1H), 3.60 (t, 1H, J=8.4 Hz), 3.42–3.31 (m, 2H), 3.13–3.02
(m, 1H), 2.94–2.79 (m, 3H), 2.40–2.25 (m, 2H), 1.99 (d,
2H, J = 6.3 Hz), 1.90 (s, 3H), 1.31 (d, 3H, J = 6.3 Hz).
A slurry of N-hydroxysuccinimide (2.79 g, 24.28 mmol) as
well as (Boc-aminooxy)acetic acid (4.42 g, 23.12 mmol, 3)
was prepared in 55 mL dry dichloromethane (DCM). Sub-
sequently, N,N′-diisopropylcarbodiimide (3.06 g, 3.76 mL,
24.88 mmol) was added and the clear solution was stirred
for 2 h under argon. Afterwards, another volume N,N′-
diisopropylcarbodiimide (244 mg, 300 μL, 1.99 mmol) was
added and the reaction mixture was stirred for further 2 h at
room temperature. The precipitated urea was removed by
filtration and washed with a small amount of DCM. After
that, the obtained solution was diluted with 200 mL DCM
and washed four times with water. The organic layer was
dried with magnesium sulfate and the solvent was removed
in vacuo, to obtain the product as a colorless solid (6.13 g,
21.27 mmol, 92%). m/z (MALDI-TOF) 357.04 [M+3Na]+;
1H NMR (300 MHz, DMSO-d6, 298 K) δ (ppm) = 10.36
(s, 1H), 4.82 (s, 2H), 2.84 (s, 4H), 1.42 (s, 9 H); 13C NMR
(75 MHz, DMSO-d6, 298 K) δ (ppm) = 169.95, 165.14,
156.57, 80.60, 69.90, 27.93, 25.47.
Synthesis of tert-Butyl(2-oxo-2-((2-(pyridinyldithio)
ethyl)amino)ethoxy)carbamate (5)
Synthesis of the Monovalent Peptide-Drug Conjugate
(8)
The active ester 4 (3.88 g, 13.47 mmol) and 2-(2-pyridyl-
dithio)ethylamine hydrochloride (3.00 g, 13.47 mmol) were
dissolved in 60 mL dry N,N-dimethylformamide (DMF).
Afterwards, N,N-diisopropylethylamine (DIPEA) (6.96 g,
9.16 mL, 53.87 mmol) was added and the reaction mixture
was stirred at room temperature under argon for 3 h. Sub-
sequently, the reaction mixture was diluted with 250 mL
ethyl acetate and washed three times with water. The organic
layer was dried with magnesium sulfate and the solvent
was removed in vacuo. The residue was purified by silica
gel column chromatography (ethyl acetate—hexane 2:1),
whereby the product was obtained as colorless oil (3.53 g,
9.83 mmol, 73%). m/z (FD) 357.8 [M]+; 1H NMR (300 MHz,
DMSO-d6, 298 K) δ (ppm)=10.29 (s, 1H), 8.52–8.40 (m,
1H), 8.23 (t, 1 H, J=5.7 Hz), 7.91–7.70 (m, 2H), 7.30–7.17
(m, 1H), 4.16 (s, 2H), 3.43 (q, 2 H, J=6.5 Hz), 2.93 (t, 2 H,
J=6.5 Hz), 1.40 (s, 9 H); 13C NMR (75 MHz, DMSO-d6,
298 K) δ (ppm)=168.05, 158.98, 156.78, 149.62, 137.77,
121.20, 119.31, 80.60, 74.68, 37.37, 37.25, 27.92.
The 2-pyridyl disulfide-carrying doxorubicin derivative
(300 mg, 355 µmol, 7) and the peptide (1.15 g, 496 µmol,
Ac-CRRRRRRRR-NH2) were dissolved in 100 mL Dul-
becco’s phosphate-buffered saline (DPBS) and DMF
(4:1). The solution was stirred for 2 h at room tempera-
ture in argon atmosphere and subsequently purified by
RP-HPLC. Afterwards, the solvent of the isolated frac-
tions was removed in vacuo and the obtained residue was
dissolved in methanol and precipitated in diethyl ether, to
yield the product as red solid (679 mg, 259 µmol, 73%).
m/z (MALDI-TOF) 2086.18 [M+H]+; RP-HPLC tR
=
15.21 min (480 nm), A—25 mM TEAA buffer (pH 7), B—
ACN, 0 min 100% A, 35 min 35% A; 1H NMR (700 MHz,
D2O, 298 K) δ (ppm) = 7.98 (d, 1 H, J = 7.7 Hz), 7.90 (t,
1H, J = 7.7 Hz), 7.62 (d, 1H, J = 7.7 Hz), 5.59–5.55 (m,
1H), 5.14 (t, 1H, J = 5.6 Hz), 4.53 (dd, 2H, J = 16.1 Hz,
J = 49.4 Hz), 4.46 (s, 2H), 4.41 (t, 1H, J = 6.3 Hz),
4.35–4.24 (m, 9H), 4.26 (q, 1H, J = 6.3 Hz), 4.06 (s, 3H),
3.85–3.83 (m, 1H), 3.70–3.64 (m, 1H), 3.50–3.41 (m,
1H), 3.29–3.23 (m, 1H), 3.23–3.10 (m, 16H), 3.09-3.00
(m, 1H), 2.97–2.91 (m, 1H), 2.90–2.85 (m, 1H), 2.80–2.74
(m, 1H), 2.72–2.65 (m, 1H), 2.58–2.49 (m, 2H), 2.36–2.30
(m, 1H), 2.08–2.02 (m, 2H), 2.01 (s, 3H), 1.91 (s, 24H),
1.87–1.51 (m, 32H), 1.30 (d, 3H, J = 6.3 Hz).
Synthesis of the 2-Pyridyl Disulfide-Carrying
Doxorubicin Derivative (7)
tert-Butyl(2-oxo-2-((2-(pyridinyldithio)ethyl)amino)eth-
oxy)carbamate (1.87 g, 5.14 mmol, 5) was dissolved in 30
mL dry DCM and subsequently diluted with 30 mL trif-
luoroacetic acid (TFA). The obtained reaction mixture was
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Int J Pept Res Ther
Synthesis of (S)-tert-Butyl(1,5-dioxo-1,5-bis((2-(2pyridin
1.38 (s, 9H); 13C NMR (75 MHz, DMSO-d6, 298 K) δ
(ppm) = 171.40, 170.88, 167.91, 159.08, 159.04, 156.86,
149.58, 137.76, 121.17, 119.25, 80.61, 74.64, 51.80, 37.84,
37.78, 37.43, 37.27, 31.59, 28.04, 27.91.
yldithio)ethyl)amino)pentan-2-yl)carbamate (10)
The protected glutamic acid derivative 9 (500 mg,
2.02 mmol) was dissolved in 20 mL N,N-dimethylfor-
mamide together with the coupling reagent N,N,N′,N′-
tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate
(1.22 g, 4.04 mmol) and 2 (901 mg, 4.04 mmol). After that,
DIPEA (1.72 mL, 10.11 mmol) was added and the reac-
tion mixture was stirred in an argon atmosphere for 5 h at
room temperature. Subsequently, 350 mL of ethyl acetate
was added and the solution was washed three times with
brine. The organic layer was dried with magnesium sulfate
and the solvent was removed under reduced pressure. The
obtained residue was purified by silica gel column chroma-
tography (ethyl acetate—methanol 15:1), to get the product
as yellow oil (1.02 g, 1.73 mmol, 86%). m/z (MALDI-TOF)
605.99 [M+Na]+; 1H NMR (300 MHz, DMSO-d6, 298 K) δ
(ppm)=8.51–8.43 (m, 2H), 8.07 (t, 1H, J=6.3 Hz), 8.04 (t,
1H, J=6.3 Hz), 7.87–7.72 (m, 4H), 7.28–7.20 (m, 2H), 6.88
(d, 1H, J=7.8 Hz), 3.92–3.74 (m, 1H), 3.44–3.24 (m, 4H),
2.88 (t, 4H, J=6.3 Hz), 2.10 (t, 2H, J=7.8 Hz), 1.92–1.61
(m, 2H), 1.36 (s, 9H); 13C NMR (75 MHz, DMSO-d6,
298 K) δ (ppm)=171.85, 171.66, 159.09, 159.01, 155.20,
149.63, 149.57, 137.76, 121.16, 119.33, 119.23, 78.06,
54.07, 37.78, 37.67, 37.45, 31.82, 28.15, 27.79.
Synthesis of the bis(2-pyridyl disulfide)-Carrying
Doxorubicin Derivative (12)
Initially, a solution of 11 (450 mg, 685.1 μmol) in 10 mL
dry DCM was prepared. To this solution 10 mL of TFA was
added and the reaction mixture was stirred for 1 h at room
temperature. Afterwards, solvent and reagent were removed
in vacuo. Doxorubicin hydrochloride (397.3 mg, 685.1 μmol)
was dissolved in 80 mL DMF/0.4 M sodium acetate buffer
(1:1, pH 4.8) and added to the oily residue. The solution was
stirred for 48 h at room temperature and was subsequently
purified by RP-HPLC. After that, the solvent of the isolated
fractions was removed in vacuum, to obtain the product as
red solid (579.1 mg, 507.0 μmol, 74%). m/z (MALDI-TOF)
1104.09 [M+Na]+; RP-HPLC tR = 28.88 min (480 nm),
A—25 mM TEAA buffer (pH 7), B—ACN, 0 min 100%
A, 40 min 30% A; 1H NMR (850 MHz, DMSO-d6, 298 K)
δ (ppm)=8.42–8.37 (m, 2H), 8.24 (t, 1H, J=5.7 Hz), 8.03
(d, 1H, J=8.2 Hz), 8.00 (t, 1H, J=5.7 Hz), 7.93–7.87 (m,
2H), 7.79–7.74 (m, 2H), 7.70 (d, 1H, J=8.0 Hz), 7.65 (d,
1H, J = 8.0 Hz), 7.63 (d, 1H, J = 8.0 Hz), 7.21–7.16 (m,
2H), 5.26–5.38 (m, 1H), 5.23–5.17 (m, 1H), 5.10–5.02
(m, 1H), 4.95–4.90 (m, 1H), 4.57–4.27 (m, 5H), 4.25–4.18
(m, 1H), 4.07 (q, 1H, J=6.6 Hz), 3.98 (s, 3H), 3.35–3.30
(m, 2H), 3.30–3.27 (m, 1H), 3.23–3.17 (m, 2H), 3.07–2.99
(m, 2H), 2.87–2.81 (m, 1H), 2.84 (t, 2H, J=6.7 Hz), 2.76
(t, 2H, J = 6.8 Hz), 2.48–2.43 (m, 1H), 2.12–2.07 (m,
1H), 2.06–1.99 (m, 2H), 1.91–1.82 (m, 1H), 1.84 (s, 3H),
1.75–1.67 (m, 1H), 1.63–1.58 (m, 1H), 1.49–1.44 (m, 1H),
1.15 (d, 3H, J=6.6 Hz).
Synthesis
of (S)-tert-Butyl((1,5-dioxo-1,5-bis((2-(2pyridinyldithio)
ethyl)amino)pentan-2-yl)amino)-2-oxoethoxycarbamate
(11)
The modified glutamic acid 10 (250 mg, 428.2 µmol) was
gradually dissolved in 10 mL dry DCM and the same amount
of TFA was added. The reaction mixture was stirred for 1 h
at room temperature followed by removal of solvent and rea-
gent in vacuum. Afterwards, the obtained oil was dissolved
in 5 mL dry DMF and consecutively DIPEA (387.4 mg,
509.8 μL, 3 mmol) as well as 4 (135.8 mg, 471 μmol) were
added. The solution was stirred for 3 h under argon at room
temperature and was subsequently diluted with 250 mL
ethyl acetate. The organic layer was washed three times with
brine, dried over magnesium sulfate and the solvent was
removed under reduced pressure. After that, the residue was
purified by silica gel column chromatography (ethyl ace-
tate—methanol 15:1), whereby the product was obtained as
yellow oil (187.2 mg, 284.9 μmol, 78%). m/z (MALDI-TOF)
695.22 [M+K]+; 1H NMR (300 MHz, DMSO-d6, 298 K)
δ (ppm) = 10.30 (s, 1H), 8.51–8.40 (m, 2H), 8.24 (t, 1H,
J=5.7 Hz), 8.12 (d, 1H, J=8.0 Hz), 8.05 (t, 1H, J=5.7 Hz),
7.88–7.70 (m, 4H), 7.28–7.19 (m, 2H), 4.33–4.09 (m, 1H),
3.43–3.24 (m, 4H), 2.89 (t, 2H, J = 7.0 Hz), 2.87 (t, 2H,
J = 7.0 Hz), 2.09 (t, 2H, J = 8.0 Hz), 1.97–1.69 (m, 2H),
Synthesis of the Multivalent Peptide-Drug Conjugate
(13)
The bis(2-pyridyl disulfide)-carrying doxorubicin derivative
(52.8 mg, 46.2 µmol, 12) and the cell-penetrating peptide
(225.5 mg, 97 µmol, Ac-CRRRRRRRR-NH2) were dis-
solved in 20 mL DPBS and DMF (3:1). The solution was
stirred for 3 h under argon at room temperature and was
subsequently purified by RP-HPLC. After that, the solvent
of the isolated fractions was removed in vacuo and the
obtained residue was dissolved in methanol and precipitated
in diethyl ether, to yield the product as red solid (134.6 mg,
31.9 µmol, 69%). m/z (MALDI-TOF) 3683.79 [M+H]+; RP-
HPLC tR = 18.57 min (480 nm), A—25 mM TEAA buffer
(pH 7), B—ACN, 0 min 100% A, 40 min 30% A; 1H NMR
(850 MHz, D2O, 298 K) δ (ppm)=7.94 (d, 1H, J=7.7 Hz),
7.85 (t, 1H, J=7.7 Hz), 7.59 (d, 1H, J=7.7 Hz), 5.53–5.50
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Int J Pept Res Ther
(m, 1H), 5.11–5.06 (m, 1H), 4.60–4.51 (m, 3H), 4.47–4.42
(m, 3H), 4.30–4.17 (m, 18H), 4.01 (s, 3H), 3.80–3.76 (m,
1H), 3.64–3.61 (m, 1H), 3.39–3.25 (m, 4H), 3.16–3.06 (m,
34H), 2.98–2.94 (m, 2H), 2.84–2.78 (m, 2H), 2.72–2.61
(m, 4H), 2.44–2.39 (m, 1H), 2.37–2.31 (m, 1H), 2.23–2.17
(m, 2H), 2.02–1.93 (m, 9H), 1.86–1.82 (m, 1H), 1.84 (s,
51H), 1.81–1.65 (m, 32H), 1.63–1.49 (m, 32H), 1.24 (d,
3H, J=6.5 Hz).
monitored by RP-HPLC with a linear gradient (0 min 100%
A, 35 min 35% A) for 24 h.
Cell Cultures
MCF-7 cells (human breast adenocarcinoma cell line, ATCC
HTB-22) were cultured in Dulbecco´s modified Eagle
medium supplemented with 10% FCS, 2 mM l-glutamine,
100 units/mL penicillin and 100 µg/mL streptomycin anti-
biotics. The human neuroblastoma cell line Kelly-WT was
obtained from ATCC and authenticated using STR geno-
typing. Kelly-WT cells were grown in RPMI-1640 medium
containing 10% FCS, 2 mM l-glutamine, 100 units/mL peni-
cillin and 100 µg/mL streptomycin antibiotics. The anthra-
cycline-resistant Kelly-ADR cells were maintained like its
drug-sensitive counterpart and were generated by continuous
exposure of Kelly-WT cells to increasing doses of doxo-
rubicin. Repeated selection rounds resulted in Kelly-ADR
cells with tenfold enhanced resistance to doxorubicin and a
concomitant increase in the IC50 value from 0.1 to 1 µg/mL.
These cells were taken for further experiments. All of the
above-mentioned cells were grown at 37 °C and 5% CO2 in
a humidified incubator and were subcultured every 2–3 days
using 0.25% trypsin.
Nuclear Magnetic Resonance (NMR) Spectroscopy
1H and 13C NMR spectra were recorded on Bruker AMX
300, Bruker WS 700 and Bruker WB 850 (Bruker Avance
III) spectrometer. Chemical shifts are displayed in parts
per million (ppm), relative to the residual solvent signals
of DMSO-d6 (δH = 2.50, δC = 39.52) and D2O (δH = 4.79)
(Gottlieb et al. 1997) The corresponding coupling constants
(J) are represented in Hz.
Mass Spectrometry
Field desorption (FD) mass spectrometry was conducted
on a VG Instruments ZAB 2-SE-FPD (8 kV) spectrom-
eter. Matrix-assisted laser desorption ionization time-of-
flight (MALDI-TOF) mass spectrometry was carried out
on a Bruker Reflex II TOF spectrometer equipped with a
337 nm nitrogen laser. 2,5-Dihydroxybenzoic acid as well
as α-cyano-4-hydroxycinnamic acid (peptidic sample) were
applied as matrix.
Cell Viability Assay
The antiproliferative effect of 8, 13, doxorubicin and the
cysteine-carrying octaarginine cell-penetrating peptide on
MCF-7, Kelly-WT as well as Kelly-ADR cells in vitro was
examined with the luciferase-based CellTiter-Glo cell pro-
liferation assay according to the manufacturer’s instructions.
Briefly, the cells were seeded into 96-well plates at a density
of 1×103 cells per well in 100 µL of medium and incubated
for 24 h to adhere. Subsequently, the medium was removed
and the drugs as well as the peptide were added at various
concentrations from 0.05 to 20 µM. The cells were incu-
bated for 72 h with the substances and after that the cell
viability was determined by quantitation of ATP with the
CellTiter-Glo luminescent cell viability assay on a Tecan
plate reader. Viability of cells treated with doxorubicin or
drug derivatives was compared to untreated controls and
corrected for effects of the medium. The applied concen-
trations of the utilized compounds were transformed into
a logarithmic scale prior to analysis and the obtained data
from the survival curves were expressed as IC50 values in
µM. Each cell viability experiment was performed in inde-
pendent triplicates.
Reversed-Phase High-Performance Liquid
Chromatography (RP-HPLC)
RP-HPLC was performed on a Jasco LC-2000Plus system
(Groß-Umstadt, Germany), equipped with a diode array
detector (MD-2015) and appropriate solvent delivery pumps
(PU-2086). Analytical RP-HPLC was conducted on a Jasco
ReproSil 100 C18 column (250×4.6 mm), with 5 µm par-
ticle size as stationary phase and a flow rate of 1 mL/min.
Purification of the substances was carried out on a ReproSil
100 C18 column from Jasco (250×20 mm), at a flow rate of
15 mL/min and 5 µm silica as stationary phase. The applied
eluents were 25 mM triethylammonium acetate buffer at pH
7 (A) and acetonitrile (B), with suitable linear gradients. All
compounds were detected at the characteristic absorbance of
doxorubicin (480 nm).
Glutathione-Mediated Drug Release
A 1 mM solution of 8 in Dulbecco’s phosphate-buffered
saline was incubated with a tenfold excess of glutathione at
37 °C in an Eppendorf Thermomixer compact (300 rpm).
The disulfide cleavage of the peptide-drug conjugate was
Fluorescence Microscopy
The intracellular localization of the multivalent cleavable
cell-penetrating peptide-drug conjugate was studied in
1 3

Int J Pept Res Ther
Kelly-WT and doxorubicin-resistant Kelly-ADR live cells
at 37 °C and 5% CO2 via fluorescence confocal laser scan-
ning microscopy and compared to the native drug. Micros-
copy images were obtained with a 63x/1.2 water-immersion
objective on a TCS SP5 microscope (Leica) and an incuba-
tion chamber for live cell imaging (37 °C, 5% CO2). The
anthracycline derivative 13 as well as doxorubicin were
applied at a concentration of 10 µM to 1×104 cells per well
(µ-Slide 8 Well, ibiTreat, Ibidi) for 24 and 72 h, respectively.
Excitation of the drugs was performed with an argon laser
at λex=488 nm (power set to 20%) and the emission range
was set to λem=550–660 nm. The fluorescence signal was
detected by hybrid detectors (HyD) with fixed gain values
set to 100. Counterstaining of the nuclei was accomplished
by incubation with 5 µM DRAQ5 (Thermo Fisher Scientific)
5 min prior to microscopy.
11. Nonetheless, both methods are straightforward and give
the cross-linking reagent in high yield.
The two orthogonal groups present in 5 are capable of
reacting with thiol-containing biomolecules like cysteine
residues of peptides as well as carbonyl groups. The ali-
phatic ketone of doxorubicin is such an easily accessible
carbonyl. After the removal of the protective group from 5
under strong acidic conditions, the condensation between
the cross-linker and the drug was carried out (Scheme 2).
This reaction is typically acid-catalyzed and yields the
oxime exclusively in E configuration, as already described
for oxime-based bioconjugates of doxorubicin (Orban et al.
2011; Szabo et al. 2009). Afterwards, the sulfhydryl-reactive
group carrying drug derivative was coupled to the N-termi-
nal cysteine residue of the cationic octaarginine cell-pene-
trating peptide (Ac-CRRRRRRRR-NH2). The conversion to
the monovalent peptide-drug conjugate was achieved rapidly
and the product was isolated by RP-HPLC.
Results and Discussion
Preparation of the Monovalent Peptide-Drug Conjugate
Preparation of the Multivalent Peptide-Drug Conjugate
The synthesis and characterization of the monovalent
cleavable cell-penetrating peptide-drug conjugate (8) we
have reported previously (Lelle et al. 2014). Briefly, 8 was
obtained via a heterobifunctional cross-linking reagent (5),
which contains a thiol-reactive 2-pyridyl disulfide func-
tionality as well as a protected carbonyl-reactive aminooxy
group. In order to synthesize this cross-linker molecule,
2-mercaptoethylamine hydrochloride was transferred into
its corresponding 2-pyridyl disulfide 2 with an excess of
2,2′-dithiopyridine and the N-hydroxysuccinimide ester 4
of (Boc-aminooxy)acetic acid was prepared. Afterwards,
the active ester 4 was reacted with 2-(2-pyridyldithio)eth-
ylamine hydrochloride (Scheme 1). Our former strategy for
the synthesis of the linker molecule involved a coupling rea-
gent-based approach, where the free amine of 2 was directly
coupled to the carboxylic acid of 3. Herein, the active ester
was synthesized, to allow further modifications, which are
necessary for the preparation of the multivalent bioconjugate
For the synthesis of the multivalent peptide-drug conjugate
carrying two cell-penetrating peptides 13, a different cross-
linking reagent with two 2-pyridyl disulfides was prepared.
Previously we have reported an amino acid-based hetero-
functional linker molecule, which exhibits the desired crite-
ria to fulfill the necessary requirements based on glutamic
acid. (Lelle et al. 2015). This amino acid derivative bears
two 2-pyridyl disulfides (11), which are capable of inter-
calating into reduced disulfides, and is equipped with an
aminooxy functionality to react with the aliphatic ketone of
doxorubicin. Therefore, we have utilized this trifunctional
amino acid scaffold to build up a multivalent peptide-drug
conjugate with two octaarginine cell-penetrating peptides.
Initially, the amino-protected glutamic acid derivative
9 was modified with two molecules of 2 using N,N,N′,N′-
tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate
as coupling reagent. The Boc protective group was subse-
quently removed and the free amine of the amino acid was
Scheme 1 Synthesis of the heterobifunctional cross-linking reagent.
Reagents and conditions: a 2,2′-dithiodipyridine (6 equiv), metha-
nol, argon, overnight, r.t., quantitative yield. b N-hydroxysuccinim-
ide (1.05 equiv), N,N′-diisopropylcarbodiimide (1.15 equiv), DCM,
argon, 4 h, r.t., 92%. c 2 (1 equiv), DIPEA (4 equiv), DMF, argon,
3 h, r.t., 73%
1 3

Int J Pept Res Ther
Scheme 2 Synthesis of the monovalent peptide-drug conju-
gate. Reagents and conditions: a 5, DCM/TFA (1:1), 1 h, r.t.,
quantitative yield. b Doxorubicin hydrochloride (6, 0.5 equiv),
DMF/0.4 M sodium acetate buffer (1:1), pH 4.8, 48 h, r.t., 81%. c
Ac-CRRRRRRRR-NH2 (1.4 equiv), DMF/DPBS (1:4), argon, 2 h, rt,
73%
reacted afterwards with the active ester 4, to prepare the
heterobifunctional cross-linker 11 (Scheme 3).
cell. The tripeptide glutathione is an reducing agent omni-
present in mammalian cells and the most prevalent cellular
thiol (Deneke and Fanburg 1989). The intracellular level
of glutathione is characteristically in the millimolar range
(0.5–10 mM), whereas micromolar concentrations are found
in blood plasma (Meister and Anderson 1983). Moreover,
the intracellular concentration of the reducing agent and
glutathione-associated enzymes in drug-sensitive and in par-
ticular drug-resistant tumor cells is usually increased, which
can mediate efficient drug release inside the cancer cells
(Balendiran et al. 2004; Gamcsik et al. 1995; Tew 1994).
To study the release of doxorubicin from the conjugates,
we have incubated a 1 mM solution of 8 with ten equivalents
of glutathione in DPBS at 37 °C, following the protocol
described previously. (Lelle et al. 2014). The cleavage of the
peptide-drug conjugate was monitored for 24 h by RP-HPLC
at the characteristic absorption maximum of doxorubicin
(480 nm).
The glutamic acid-based heterofunctional cross-linking
reagent was deprotected at first, to generate the carbonyl-
reactive aminooxy group. The deprotection of the ami-
nooxy functionality was always performed directly before
the reaction with doxorubicin, to avoid decomposition and
side reactions, due to the high reactivity of the alpha effect
nucleophile. After the Boc protecting group was successfully
removed in quantitative yield, the condensation with the
drug was carried out, to get an anthracycline derivative with
two 2-pyridyl disulfides (Scheme 4). These mixed disulfides
were modified with the cysteine-carrying polyarginine cell-
penetrating peptide, to yield the multivalent peptide-drug
conjugate.
Glutathione-Dependent Drug Delivery
Following cellular internalization, peptide-drug conjugates
like 13 have to be cleaved, in order to release their cytotoxic
freight. The cleavage can take place via disulfide scission,
which will occur in the reductive environment inside the
The degradation of the monovalent bioconjugate was
highly efficient, which is illustrated by the chromatogram
at 0 h. This corresponds to the point of time where 8 and
the reducing agent were just put together and analyzed
Scheme 3 Synthesis of the bis(2-pyridyl disulfide)-carrying het-
erofunctional cross-linking reagent. Reagents and conditions: a 2 (2
equiv), N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium tetrafluor-
oborate (2 equiv), DIPEA (5 equiv), DMF, argon, 5 h, r.t., 86%. b
DCM/TFA (1:1), 1 h, r.t., quantitative yield. c 4 (1.1 equiv), DIPEA
(7 equiv), DMF, argon, 3 h, r.t., 78%
1 3

Int J Pept Res Ther
Scheme 4 Synthesis of the multivalent peptide-drug conjugate. Rea-
gents and conditions: a 11, DCM/TFA (1:1), 1 h, r.t., quantitative
yield. b Doxorubicin hydrochloride (6, 1 equiv), DMF/0.4 M sodium
acetate buffer (1:1), pH 4.8, 48 h, r.t., 74%. c Ac-CRRRRRRRR-NH2
(2.1 equiv), DMF/DPBS (1:3), argon, 3 h, rt, 69%
by RP-HPLC (Fig. 1). Even after such a short period of
time, the peptide-drug conjugate was almost fully cleaved.
Consequently, another peak was visible that matched to
substance a, which is a glutathione-carrying doxorubicin
derivative. Under the applied incubation conditions, 8
did not elute as a sharp peak, because the charged guani-
dinium groups of the amino acid side chains are highly
influenced from various counter ions such as acetate,
phosphate or the carboxylic acid of glutathione. Never-
theless, the peptide-drug conjugate could not be detected
anymore after 1 h of incubation. Moreover, a substance
evolved that corresponds to an anthracycline derivative
with a residual linker molecule (b). The fraction of this
compound increased over time and was after 3 h already
the dominant species, whereas a became less and less due
to further reduction by glutathione. Under physiological
conditions a full degradation of a would occur, due to
the constant glutathione level maintained in mammalian
cells by enzymatic processes (Deneke and Fanburg 1989).
As a result, the peptide drug conjugate will be entirely
metabolized to b. Substance b can be considered as the
cytotoxic drug metabolite, as previous scientific works
have already shown that residual amino acids from linker
molecules do not diminish the DNA-binding properties of
the anthracycline (Orban et al. 2011; Schlage et al. 2011).
As a consequence, cleavage product b released from the
bioconjugate 8 can still successfully interact with DNA
and thereby generate its cytotoxic effects. The release of
the native anthracycline was a minor process during the
degradation study, which can be seen in the chromatogram
at 3 h for instance.
Previously we have also investigated the degradation of a
peptide-drug conjugate, which is similar to the herein intro-
duced bioconjugate 13, since the anthracycline is connected
via two disulfides to a peptide backbone in both cases (Lelle
et al. 2015). This system has also demonstrated that peptide-
drug conjugates derived from 12 can efficiently release the
cytotoxic freight in the presence of an excess glutathione,
although the degradation was slower and yielded more
cleavage products, which was attributed to the two develop-
ing sulfhydryl groups.
Moreover, we have incubated the cell-penetrating pep-
tide-doxorubicin conjugates at lower pH values, such as 5.0,
to simulate the conditions present in endosomes and around
cancer cells (Sorkin and von Zastrow 2002; Tannock and
Rotin 1989). But also here, no significant amount of free
doxorubicin could be detected by RP-HPLC after 48 h of
incubation. The high hydrolytic stability of the oxime is
crucial for our approach, to mediate exclusive drug delivery
by disulfide scission, which can reduce harmful secondary
effects due to an early release of the drug in the bloodstream
(Kalia and Raines 2008).
Cell Viability Studies
After we could successfully show that elevated glutathione
levels can cleave the disulfide-based bioconjugates, to
deliver the anthracycline drug inside the tumor cells, the
antiproliferative effect of 8 and 13 on different cell lines was
investigated. The cytotoxicity data were obtained with the
CellTiter-Glo luminescent cell viability assay, expressed as
IC50 values in µM and compared to the effects of the native
1 3

Int J Pept Res Ther
Fig. 1 Glutathione-mediated
drug release by reduction of
the monovalent cell-penetrating
peptide-doxorubicin conjugate
8. A RP-HPLC analysis of the
degradation of 8 (1 mM) in the
presence of an excess glu-
tathione (10 mM) in DPBS at
37 °C. B Chemical structures of
the cleavage products
drug as well as the cysteine-carrying octaarginine cell-pen-
etrating peptide.
Table 1 In vitro cytotoxic effects of the peptide-drug conjugates (8
and 13), doxorubicin and the octaarginine cell-penetrating peptide in
MCF-7, Kelly-WT and Kelly-ADR cells, determined with the CellTi-
ter-Glo luminescent cell viability assay
The in vitro cytotoxic effects of the applied compounds
were examined with cell lines from tissues where doxo-
rubicin is typically utilized. One of these cell lines is the
MCF-7 breast cancer cell line (Nadas and Sun 2006; Wil-
liam Lown 1993). The antiproliferative effect of the native
drug on this adenocarcinoma cells is very strong. However,
the multivalent peptide-drug conjugate was also very cyto-
toxic for MCF-7 breast cancer cells, as expressed by an IC50
value in the lower micromolar range like doxorubicin. In
contrast, the IC50 value of the monovalent bioconjugate (8)
was almost sevenfold higher compared to 13 (Table 1). The
cell viability data for the peptide-drug conjugates support
our hypothesis that the multivalent compound will have
an increased cellular uptake and thereby exhibit stronger
cytotoxic effects. The octaarginine cell-penetrating peptide
alone was non-toxic to the applied adenocarcinoma cell line,
which is a good indication that the antiproliferative effect of
8 and 13 is related to the anthracycline drug and not to the
peptide.
Compound
MCF-7
Kelly-WT
IC50 (µM)
Kelly-ADR
IC50 (µM)
IC50 (µM)
8
24.36 4.15
3.80 1.95
0.90 0.12
≥100
11.55 1.03
1.40 0.66
0.18 0.08
≥100
7.04 2.23
1.84 0.57
6.57 0.61
≥100
13
Doxorubicin
Octaarginine cell-
penetrating peptide
The cells were incubated for 72 h with the compounds and the data
are expressed as IC50 values in µM (n≥3, mean standard deviation)
cells to increasing doses of the anthracycline drug, which
is in good agreement with the obtained IC50 values from
the herein presented cytotoxicity study. Hence, the IC50
value of doxorubicin was almost 40-times higher for the
anthracycline-resistant subline (Table 1). The peptide-
drug conjugates 8 and 13 showed the opposite, but also
here the trend was visible that the multivalent compound
is equipped with a stronger antiproliferative effect com-
pared to the monovalent bioconjugate. Whereas 8 and
13 were both less active than doxorubicin on Kelly-WT
cells, the multivalent conjugate 13 was more cytotoxic to
drug-resistant Kelly-ADR cells compared to the native
anthracycline.
Doxorubicin has played an undisputed role in the treat-
ment of childhood solid tumors such as neuroblastoma
(Minotti et al. 2004). For that reason, we have also used
the wild-type human neuroblastoma cell line Kelly-WT
and its adriamycin-resistant subline Kelly-ADR, to investi-
gate the compounds. The drug-resistant counterpart of the
cell line was created by continuous exposure of Kelly-WT
1 3

Int J Pept Res Ther
This phenomenon can be attributed to the drug resistance
of the neuroblastoma cell line towards the anthracycline,
which renders doxorubicin less effective, since the drug is
efficiently excreted from Kelly-ADR cells by membrane-
associated transporter proteins (Ambudkar et al. 1999;
Gottesman et al. 2002). Through its different uptake mech-
anisms, which are mainly based on endocytosis and direct
translocation, the multivalent bioconjugate can presumably
circumvent the drug efflux pumps, to evolve a greater cyto-
toxicity (Jones and Sayers 2012; Vargas et al. 2014; Madani
et al. 2011). This effect is perhaps not so pronounced for the
monovalent peptide-drug conjugate, because the uptake is
decreased due to the presence of a signle cell-penetrating
peptide. In contrast, doxorubicin enters the cell by passive
diffusion through the cell membrane and is thereby an easy
target for the membrane-associated transporter proteins
(Siegfried et al. 1985). In addition, the cytotoxic effects of
the peptide-drug conjugates can be strengthened by the fact
that polyarginine cell-penetrating peptides are known to rap-
idly distribute into the nucleus after cellular internalization
(Duchardt et al. 2007). This feature is highly desired for
substances like anthracycline-based drugs, which develop
their cytotoxicity around DNA.
Intracellular Trafficking
We investigated the native drug and 13 by fluorescence con-
focal laser scanning microscopy at various points of time
(Fig. 2), to get insight of their uptake and if the subcellular
distribution of the multivalent compound has an influence on
Fig. 2 Investigation of the subcellular distribution of the peptide-
drug conjugate 13 and doxorubicin in Kelly-WT and Kelly-ADR cells
by fluorescence confocal laser scanning microscopy, 24 and 72 h after
the incubation with 10 µM of the drug at 37 °C. Fluorescence related
to the fluorophore part of the anthracycline is illustrated by red color
and the associated brightfield images are depicted below. Scale bars
represent 15 µm. (Color figure online)
1 3

Int J Pept Res Ther
the cytotoxicity accordingly. After 24 h of incubation with
the drugs, anthracycline-associated fluorescence (depicted
in red) was visible in both adriamycin-sensitive Kelly-WT
and adriamycin-resistant Kelly-ADR cells. The cell uptake
of doxorubicin at that point of time was higher compared to
13 for wild-type as well as drug-resistant cells. This is attrib-
uted to the different uptake mechanisms of the compounds,
which are not equally fast. Typically, the passive diffusion
of doxorubicin across the cell membrane is a process faster
than endocytosis. In Kelly-WT cells doxorubicin was found
already in the nucleus, whereas Kelly-ADR cells exhibited
no drug in the nuclei. By contrast, the uptake of 13 was
equal in both types of cells.
nuclei of the tumor cells were simultaneously stained with
DRAQ5 (Fig. 3). The nuclear staining is depicted in blue,
whereas colocalization with the drug (red color) appears
violet. The obtained microscopy images highlight that the
cleaved cell-penetrating peptide-doxorubicin conjugate
delivers the anthracycline drug to its target in both adriamy-
cin-sensitive Kelly-WT and adriamycin-resistant Kelly-ADR
cells, which is a crucial prerequisite for the overcoming of
drug resistance.
Conclusions
Longer incubation (72 h) of the cells with the multiva-
lent peptide-drug conjugate has shown a similar result. This
observation corresponds to the data from the cell viability
assay, where 13 was approximately equally toxic to drug-
sensitive as well as resistant neuroblastoma cells (Table 1).
Compared to the results after 24 h, the uptake of the bio-
conjugate increased, which was not the case for the native
drug in Kelly-ADR cells. A reasonable explanation for this
phenomenon is that doxorubicin got excreted from the drug-
resistant cells by the membrane-associated transporter pro-
teins. This supports the obtained IC50 values and our hypoth-
esis that the multivalent peptide-drug conjugate can bypass
efflux pumps, to overcome drug resistance in cancer cells.
To get deeper insights, whether the anthracycline deriva-
tive can enter the nucleus after the glutathione-dependent
cleavage of the peptide-drug conjugate, Kelly-WT and
Kelly-ADR cells were incubated with 13 and analyzed via
fluorescence confocal laser scanning microscopy while the
Multivalency is a powerful strategy found in many biologi-
cal systems, utilized for achieving high-affinity molecular
recognition and specificity through avidity. Multivalent
peptides based on a synthetic scaffold like multiple anti-
gen peptides have already found application as immuno-
gens that can induce an immune response. In this work
we hypothesize that multiple cell-penetrating peptides can
possess stronger interaction with the cellular membrane
and as consequence lead to a better cellular uptake of a
chemotherapeutic in drug-resistant cell lines. For this pur-
pose we designed and prepared a multivalent drug-peptide
conjugate by attaching cell-penetrating peptides to an
anthracycline drug using a low-molecular weight cleav-
able linker. The fluorescent chemotherapeutic drug was
used to reveal the translocation of the conjugate through
the cell membrane and its intracellular distribution. We
investigated the cytotoxicity of the multivalent construct in
a drug-resistant cell line, which confirmed our hypothesis
Fig. 3 Examination of the nuclear localization of the anthracycline
drug in Kelly-WT and Kelly-ADR cells by fluorescence confocal
laser scanning microscopy. Images were taken 72 h after the incuba-
tion with 10 µM of the peptide-drug conjugate 13 at 37 °C. Drug-
associated fluorescence is illustrated by red color, whereas the
nuclear stain DRAQ5 is depicted in blue. The corresponding bright-
field images are represented by the left panels and the right panels
display the overlay of the anthracycline-associated fluorescence and
the nuclear counterstain. Colocalization is visualized by violet color.
Scale bars represent 15 µm. (Color figure online)
1 3

Int J Pept Res Ther
Gewirtz DA (1999) A critical evaluation of the mechanisms of
action proposed for the antitumor effects of the anthracycline
antibiotics adriamycin and daunorubicin. Biochem Pharmacol
57(7):727–741
that multimerization can enhance the efficacy of drug-
peptide conjugates. Additionally, the herein presented
synthetic approach and the multivalent structure can be
further employed as model systems, to gain deeper insights
into membrane-associated biological processes or for the
preparation of more complex multimers that mimic natural
multivalent systems.
Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in
cancer: role of ATP-dependent transporters. Nat Rev Cancer
2(1):48–58
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of common laboratory solvents as trace impurities. J Org Chem
62(21):7512–7515
Acknowledgements The authors are thankful to Dr. Alexander Sch-
ramm, Clinic for Pediatrics III, University Hospital Essen for providing
the Kelly-WT and Kelly-ADR cells.
Gu YJ, Cheng J, Man CW, Wong WT, Cheng SH (2012) Gold-dox-
orubicin nanoconjugates for overcoming multidrug resistance.
Nanomedicine 8(2):204–211
Iyer AK, Singh A, Ganta S, Amiji MM (2013) Role of integrated can-
cer nanomedicine in overcoming drug resistance. Adv Drug Deliv
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Compliance with Ethical Standards
Jones AT, Sayers EJ (2012) Cell entry of cell penetrating peptides: tales
of tails wagging dogs. J Control Release 161(2):582–591
Kalia J, Raines RT (2008) Hydrolytic stability of hydrazones and oxi-
mes. Angew Chem Int Ed 47(39):7523–7526
Conflict of interest The authors declare no conflict of interest.
Research Involving Human and Animal Rights This article does
not contain any studies with animals and human participants performed
by any of the authors.
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