Novel cleavable cell-penetrating peptide–drug conjugates: synthesis and characterization


  • Marco Lelle,

    1. Department of Synthetic Chemistry, Max Planck Institute for Polymer Research, Mainz, Germany
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  • Stefanie U. Frick,

    1. Department of Synthetic Chemistry, Max Planck Institute for Polymer Research, Mainz, Germany
    2. Department of Dermatology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany
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  • Kerstin Steinbrink,

    1. Department of Dermatology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany
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  • Kalina Peneva

    Corresponding author
    1. Department of Synthetic Chemistry, Max Planck Institute for Polymer Research, Mainz, Germany
    • Correspondence to: Kalina Peneva, Department of Synthetic Chemistry, Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. E-mail:

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We report the first drug conjugate with a negatively charged amphipathic cell-penetrating peptide. Furthermore, we compare two different doxorubicin cell-penetrating peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH, namely the positively charged octaarginine and the negatively charged proline-rich amphipathic peptide. These conjugates were prepared exploiting a novel heterobifunctional crosslinker to join the N-terminal cysteine residue of the peptides with the aliphatic ketone of doxorubicin. This small linker contains an activated thiol as well as aminooxy functionality, capable of generating a stable oxime bond with the C-13 carbonyl group of doxorubicin. The disulfide bond formed between the peptide and doxorubicin enables the release of the drug in the cytosol, as confirmed by drug-release studies performed in the presence of glutathione. Additionally, the cytotoxicity as well as the cellular uptake and distribution of this tripartite drug delivery system was investigated in MCF-7 and HT-29 cell lines. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.


Cell-penetrating peptides, also known as protein transduction domain (PTD), have attracted interest as carriers for intracellular drug delivery. Originally, these short peptide sequences, typically with 5 to 30 amino acids, were derived from the trans-activating transcriptional activator of the HIV 1 [1, 2]. Cell-penetrating peptides have been used to transport numerous cargos across the cell membrane in vitro and in vivo [3] (e.g. drugs [4], nucleic acids [5], polymers [6], quantum dots [7], and proteins [8]). Although they have been studied for more than 25 years now [9], and have demonstrated considerable potential as carriers, some issues remain still unclear. For instance, cellular uptake efficiency can strongly depend on the different incubation conditions and the applied cell lines [10]. The mechanism of uptake is intensively debated, although it is believed that the majority of PTDs enter the cell by some form of endocytosis, and many parameters can easily influence cellular internalization. The applied peptide concentration can alter the uptake from energy-dependent endocytosis at low concentration to direct translocation at elevated concentration [11, 12]. The work of Hirose et al. demonstrated that a particular combination of PTD and a small hydrophobic dye can lead to direct penetration, which highlighted the importance of the choice of payload when investigating or comparing different PTDs [13]. The influences of the cargo and the combination of the peptide carrier could also explain some of the inconsistencies that exist in the PTD field, and it underlines that studies that aim to unveil the potential of particular peptide should first consider the cargo and the mode of attachment.

The aim of this study was to synthesize and characterize cell-penetrating peptide–drug conjugates, exploiting a novel crosslinking method and doxorubicin as a model drug. In addition, we compare two peptide carriers, which belong to different classes of PTDs, in order to study the influence of the nature of the peptide over the uptake efficiency and drug efficacy.

The two applied cell-penetrating peptides differ in their net charge at physiological pH and their secondary structure. First, a polyarginine, consisting of nine amino acids, eight arginines, and an N-terminal cysteine, was selected. Octaarginine is widely used in the context of arginine-rich PTDs, like HIV-1 Rev, HIV-1 transcriptional activator, HTLV-II Rex, and BMV Gag [2, 14]. Second, a proline-rich amphipathic cell-penetrating peptide [15, 16] containing three VELPPP units with a negative net charge was chosen. Various proline-rich sequences have been described in the literature and have shown the capacity to permeate cells and no cytotoxic effects, even at high concentrations [16-18]. Furthermore, the anionic cell-penetrating peptide bears a CGGW motif at its N-terminus, required for drug modification at the terminal cysteine.

As a model drug, we have chosen doxorubicin (DOX) for the attachment to the two PTDs, due to its intrinsic fluorescence, which enables the determination of cellular uptake, and intracellular distribution applying flow cytometry and fluorescence microscopy. Anthracycline drugs have a wide spectrum of activity against hematopoietic and solid tumors [19]; however, a reversible dose-dependent hematologic toxicity represents the acute dose-limiting toxicity, and their prolonged administration can induce cardiomyopathy and congestive heart failure [20]. Another major drawback of doxorubicin is the onset of drug resistance; P-glycoprotein overexpressed by resistant cells is extruding the drug, causing low intracellular retention [21]. Therefore, doxorubicin and its analogues have been modified in order to improve the drug's biological properties on the one hand and to reduce the severe side effects on the other hand. Doxorubicin modification can be performed either in a noncovalent fashion, encapsulated in liposomes, e.g. [22], or by alteration of the drugs structure with the addition of a crosslinker. Heterobifunctional crosslinkers are extensively applied in drug modifications, and their utilization has proven to be very important for the attachment of diverse carriers, such as monoclonal antibodies [23, 24], proteins [25], polymers [26], and peptides [27], to doxorubicin. Established conjugation technique is applied at the C-13 keto group by hydrazones, due to their fast hydrolysis in acidic environment existing in biological compartments like endosomes and lysosomes [28, 29]. However, the insufficient stability of the doxorubicin hydrazone conjugates has been reported even at physiological pH (7.4) [27], leading to the release of the free drug in the bloodstream. In order to overcome these difficulties, we have chosen a crosslinker capable of creating an oxime bond on doxorubicin's ketone, due to the higher hydrolytic stability of the oxime group [30]. Additionally, thiol-containing carriers, like albumin proteins, have also been conjugated to anthracyclines utilizing functional groups that are highly specific for sulfhydryl groups, e.g. maleimides and pyridyl disulfides [31]. The application of pyridyl disulfides is advantageous, because a disulfide bond between the linker and the cargo is formed, which can be reduced in the cytoplasm by glutathione to deliver the freight [14]. Furthermore, pyridyl disulfides can serve as a protective group during synthesis to avoid undesired dimerization as well as an activating group for the thiol to facilitate disulfide formation [32]. In contrast, maleimides react with a thiol via Michael addition; thus, a covalent bond is created that cannot be cleaved under physiological conditions. Therefore, we have selected a heterobifunctional crosslinker that contains a protected aminooxy group and pyridyl disulfide.

Materials and Methods


All solvents, chemicals, and reagents were bought from commercial sources and used without further purification. Doxorubicin hydrochloride was purchased from Ontario Chemicals, Inc. (Guelph, Ontario, Canada). For custom peptide synthesis of Ac-CRRRRRRRR-NH2 (CPP) and Ac-CGGWVELPPPVELPPPVELPPP-NH2 (CPPamph) Genosphere Biotechnologies (Paris, France) and Severn Biotech Ltd. (Kidderminster, Worcestershire, United Kingdom) were employed. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay was from Trevigen Inc. (Gaithersburg, MD, USA).


NMR spectroscopy

Proton (1H) and carbon (13C) NMR measurements were recorded on Bruker (Billerica, MA, United States of America) DPX 250, Bruker AMX 300, and Bruker WS 700 spectrometers in suitable deuterated solvent.

Mass spectrometry

Field desorption (FD) MS was performed on a VG Instruments (Manchester, UK) ZAB 2-SE-FPD (8 kV) spectrometer. MALDI-TOF MS was conducted on a Bruker Reflex II-TOF spectrometer, utilizing a 337 nm nitrogen laser. The analyzed samples were either embedded in α-cyano-4-hydroxycinnamic acid (peptidic sample) or in 2,5-dihydroxy benzoic acid (DOXoxm) as matrix.

High-performance liquid chromatography

The RP-HPLC was conducted on a JASCO LC-2000Plus System (Groß-Umstadt, Germany), with appropriate diode array detector (MD-2015), solvent delivery pumps (PU-2086), and columns. Analytical HPLC was accomplished with a ReproSil (JASCO, Groß-Umstadt, Germany) 100 C18 column (250 × 4.6 mm) with 5 µm particle size as a stationary phase and a flow rate of 1 ml/min. Purification of the products was performed on a ReproSil 100 C18 column (250 × 20 mm) with a flow rate of 15 ml/min and 5 µm silica as a stationary phase. The applied eluents were 25 mm triethylammonium acetate buffer (pH 7) (A) and acetonitrile (B) with appropriate gradients. All the substances were detected at 480 nm.


2-(2-Pyridyldithio)ethylamine hydrochloride

The synthesis of 2-(2-pyridyldithio)ethylamine hydrochloride was carried out according to a modified procedure invented by Sigler in 1987 [33]. 2,2′-Dithiodipyridine (25 g, 113.47 mmol) was dissolved in 150 ml methanol and degassed in an ultrasonic bath for 30 min. To this solution, 2-mercaptoethylamine hydrochloride (2.15 g, 18.91 mmol) was slowly added within 30 min. Afterwards, the flask was sealed with a septum, and the reaction mixture was stirred overnight at room temperature (RT) under argon. The yellow solution was precipitated twice in diethyl ether, and the product was obtained as a white crystalline solid, which was characterized by 1H and 13C NMR (yield: 93%). MS (MALDI-TOF): m/z (rel. int.) = 187.00 (100%) [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.

2-((tert-Butoxycarbonylaminooxy)-N-(2-(2-pyridyldithio)))ethyl acetamide

Initially, 2-(2-pyridyldithio)ethylamine hydrochloride (2.50 g, 11.22 mmol), 2-(tert-butoxycarbonylaminooxy)acetic acid (2.15 g, 11.22 mmol), and N,N,N′,N′-tetramethyl-O–(N-succinimidyl)uronium tetrafluoroborate (TSTU) (3.38 g, 11.22 mmol) were dissolved in 60 ml of dry DMF; N,N-diisopropylethylamine (6.68 ml, 39.28 mmol) was added; and the solution was stirred in argon atmosphere at RT for 5 h. Subsequently, 400 ml of ethyl acetate was added, and the dilution was washed three times with 0.2 m hydrochloric acid. The organic layer was dried with magnesium sulfate and the solvent removed in vacuo. Afterwards, the residue was purified by column chromatography on silica (ethyl acetate–hexane 2 : 1), in order to obtain colorless oil, which was analyzed by NMR spectroscopy and FD MS (yield: 73%). MS (FD, 8 kV): m/z (rel. int.) = 357.8 (100%) [M]+; 1H NMR (250 MHz, DMSO-d6, 298 K): δ (ppm) = 10.29 (s, 1H), 8.47 (d, 1H, J = 4.5 Hz), 8.23 (t, 1H, J = 5.8 Hz), 7.89–7.72 (m, 2H), 7.30–7.20 (m, 1H), 4.15 (s, 2H), 3.43 (q, 2H, J = 6.5 Hz), 2.93 (t, 2H, J = 6.5 Hz), 1.40 (s, 9H); 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.


In 30 ml DCM, 2-((tert-butoxy carbonylaminooxy)-N-(2-(2-pyridyldithio)))ethyl acetamide (1.87 g, 5.14 mmol) was gradually dissolved, diluted with 30 ml trifluoroacetic acid, and vigorously stirred for 1 h. After the removal of solvent and reagent, the residue was dried in high vacuum for at least 6 h and diluted with 175 ml DMF. Doxorubicin hydrochloride (1.49 g, 2.57 mmol) was dissolved in 175 ml of 0.4 m sodium acetate buffer (pH 4.8) and added to the DMF solution, and the reaction mixture was stirred for 2 days at RT followed by HPLC purification. The solvent of the collected fractions was evaporated, and the obtained solid was dissolved in methanol and precipitated in diethyl ether to yield a red powder, which was characterized by MALDI-TOF MS, HPLC, and NMR spectroscopy (yield: 81%). MS (MALDI-TOF): m/z (rel. int.) = 807.98 (100%) [M + Na]+; HPLC: tR = 15.56 min (480 nm), A: 25 mm TEAA buffer; pH 7 – B: 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 Hz), 7.32 (d, 1H, J = 7 Hz), 7.13 (t, 1H, 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).

DOXoxmCPP and DOXoxmCPPamph

DOXoxm and 1.3 equivalents of the corresponding peptide were stepwise dissolved in Dulbecco's phosphate-buffered saline (DPBS) and DMF. After agitating the solution for 24 h, the product was isolated by HPLC, and the solvent of the desired fractions was removed under vacuum. The work-up of the obtained red residue was carried out according to DOXoxm, and the substance was analyzed by appropriate methods [yield: 61% (DOXoxmCPP); 67% (DOXoxmCPPamph)]. DOXoxmCPP – MS (MALDI-TOF): m/z (rel. int.) = 2086.18 (100%) [M + H]+; 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, 1H, 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); DOXoxmCPPamph – MS (MALDI-TOF): m/z (rel. int.) = 3057.13 (100%) [M + Na]+; HPLC: tR = 23.36 min (480 nm), A: 25 mm TEAA buffer; pH 7 – B: ACN, 0 min, 100% A – 35 min, 35% A.

Fluorescence spectroscopy

Solution fluorescence spectra were recorded on a SPEX-Fluorolog 2 (212) spectrometer (HORIBA, New Jersey, USA). The spectra of doxorubicin and the cell-penetrating peptide–drug conjugates were measured in phosphate (100 mm, pH 7.4) and acetate buffers (100 mm, pH 5) at various concentrations (0.1, 1, 10, and 50 µm). The concentrations of the solutions have been determined with relative standard deviations of 2%. Fluorescence was recorded upon excitation at λ = 488 nm.

CD spectroscopy

The CD spectra were measured with a J-810-S spectropolarimeter (JASCO). The spectra were obtained in a wavelength range of 190 to 250 nm at a spectral bandwidth of 1 nm. Reported spectra were the average of five scans. Spectra were measured at 1 mg/ml of the peptide or peptide–drug conjugate, dissolved in phosphate buffer (100 mm, pH 7.4), and were recorded at 25 °C. The blank was subtracted from each spectrum.

Glutathione-dependent degradation of doxorubicin–peptide conjugates

A 1 mm solution of DOXoxmCPP in DPBS (pH 7.4) was incubated with 10 equivalents of glutathione at 37 °C in an Eppendorf (Hamburg, Germany) Thermomixer compact (300 rpm). The cleavage of the disulfide bond was monitored by HPLC within 24 h. Doxorubicin hydrochloride (1 mm) in DPBS served as a standard reference.

Cell cultures

MCF-7 cells (human breast adenocarcinoma cell line, provided by K. Landfester) were grown in RPMI 1640 medium (Lonza, Verviers, Belgium) and supplemented with 10% heat-inactivated bovine serum (PAA, Laboratories, Pasching Austria), 10 µg/ml human insulin (Insulin detemir – Levemir®, Novo Nordisk, Mainz, Germany), and 2 mm l-glutamine. The human colon carcinoma cell line HT-29 (purchased from DSMZ, Braunschweig Germany) was cultured in McCoy's 5A medium (Biochrom AG, Berlin Germany) containing 10% heat-inactivated bovine serum and 2 mm l-glutamine. Cells were maintained at 37 °C under 5% CO2. Both cell lines were growing as adherent monolayers and were split every 3–6 days with Trypsin EDTA (1 : 250; 1×) (PAA Laboratories).

Flow cytometry analysis

Cellular uptake of DOX and the corresponding peptide conjugates as well as the amount of viable cells was analyzed by flow cytometry. Tumor cells were seeded in 12-well plates at a density of 5 × 104 cells/well, and after 24 h, doxorubicin, DOXoxmCPP, and DOXoxmCPPamph were added with culture medium at a concentration of 0.1, 1, 10, and 50 µm. Cell-associated fluorescence was analyzed employing a BD LSR II flow cytometer (BD Biosciences, Heidelberg, Germany) equipped with a 488 nm laser; emission was detected using a 585/42 nm filter, and the data were analyzed utilizing FlowJo (Treestar, Inc., Ashland, OR, USA). Cell viability was determined by staining the tumor cells with the Fixable Viability Dye eFluor® 780 (eBioscience, Frankfurt, Germany) in order to discriminate between live and dead cells that are positive for the drug.

Cell viability assay

The cytotoxicity of doxorubicin, both peptides, and its conjugates was investigated by an MTT cell proliferation assay with MCF-7 and HT-29 cells according to manufacturer's instructions. Briefly, cells were seeded at 1 × 103 cells/well in 96-well plates 24 h prior to drug exposure. The free drug and the conjugates were added at concentrations of 0.1, 1, 10, and 50 µm in a culture medium containing 10% heat-inactivated serum to the tumor cells and incubated for 72 h at 37 °C under 5% carbon dioxide. All samples were measured in triplicates. The MTT reagent (10 µl/100 µl medium) was added to the cells and incubated for 4 h at 37 °C. Absorbance was detected at 570 nm with a reference wavelength of 650 nm in a microplate reader. Blank wells (culture medium, MTT reagent, and detergent reagent) were subtracted from sample wells, and control cells cultured without drugs were taken as reference to calculate relative survival rate. Analysis of the cytotoxicity of the peptides alone in MTT assay, CPP (MCF-7: 93.53 ± 1.67; HT-29: 87.06 ± 1.68% viable cells) and CPPamph (MCF-7: 96.42 ± 7.23; HT-29: 89.69 ± 6.12% viable cells), respectively, was compared with Dox as positive control (MCF-7: 21.27 ± 7.08; HT-29: 21.08 ± 3.6% viable cells) after incubation with a dosage of 50 µm for 72 h.

Confocal laser scanning microscopy (CLSM): live-cell imaging

Tumor cells were seeded at a density of 1 × 105 cells/well into eight-well Nunc Lab-Tek Chamber Slides (Thermo Scientific, Schwerte Germany) and left to adhere for 24 h. Doxorubicin and the corresponding conjugates were added at 50 µm in a culture medium, and microscopy was performed 5 h after incubation by excitation of the drug at 488 nm. Cell nuclei were stained with Hoechst 33342 (Invitrogen Darmstadt, Germany) and excited using the 405 nm laser line. CLSM was carried out on nonfixed cells using an LSM 710 NLO (Carl Zeiss, Oberkochen Germany), and images were processed with the software ZEN 2009 (Carl Zeiss, Germany).

Results and Discussion

Synthesis of Cell-penetrating Peptide Conjugates

An activated thiol of 2-mercaptoethylamine hydrochloride was prepared initially, with an excess of 2,2′-dithiodipyridine by precipitation from solution (Figure 1). Afterwards, the amine group of this activated thiol-containing molecule was coupled to a protected aminooxyacetic acid derivative via amide bond formation. Deprotection of the aminooxy functionality enables in situ modification of doxorubicins aliphatic ketone, in order to obtain an activated thiol-containing drug molecule (DOXoxm), which was purified by HPLC. The aforementioned chemical modification of the drug (Figure 1, highlighted in red) maintains its solubility in aqueous solution, which enables a reaction with small excess of the cationic or the amphipathic cell-penetrating peptide in short reaction time. Following reaction with the two cysteine-containing peptides (sequence highlighted in green), the peptide–drug conjugates were purified by preparative HPLC with triethylammonium acetate buffer and acetonitrile as eluents in order to avoid degradation of the oxime linkage (depicted in blue) and to maintain acetate counter ions for biological applications. The structure and purity of the two doxorubicin-peptide conjugates DOXoxmCPP and DOXoxmCPPamph were confirmed by NMR, analytical HPLC, and mass spectrometry.

Figure 1.

Synthesis of doxorubicin cell-penetrating peptide bioconjugates (DOXoxmCPP and DOXoxmCPPamph, respectively) with cleavable disulfide linker. (i) 6 equivalents of 2,2′-dithiodipyridine in methanol, room temperature (RT), overnight; (ii) 2-(tert-butoxycarbonylaminooxy)acetic acid, O-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU), DIPEA in DMF, RT, 5 h; (iii) 1. DCM/TFA 1 : 1; RT, 1 h and 2. doxorubicin hydrochloride in DMF/0.4 m sodium acetate buffer (pH 4.8) 1 : 1, RT, 2 days; and (iv) 1.3 equivalents of peptide in Dulbecco's phosphate-buffered saline/DMF 4 : 1, RT, 24 h.

The synthetic approach employed herein represents a short and facile route for the preparation of peptide–drug conjugates in good yield, compared with the procedure used before by Webb et al. [34].

Structural Studies

The secondary structure of the doxorubicin peptide conjugates was studied by CD spectroscopy and compared with the unmodified peptides (Figure 2). Polyarginines like CPP do not possess any secondary structure, as seen In Figure 2, and the modification with the thiol reactive drug derivative does not cause any significant difference in the CD spectra of the conjugate compared with the peptide alone, except in the 215–235 nm region where a S–S bond-related contribution can be expected. Proline-rich sequences, like CPPamph employed here, have a high tendency to develop a well-defined secondary structure in water, the so-called polyproline II helix [18], which enables distinct orientation of hydrophilic and hydrophobic areas on each side along the helical axis. CPPamph exhibits such a structural feature, and the corresponding spectrum displays the characteristic strong negative band at 205 nm [18, 35]. The attachment of doxorubicin via disulfide bond to the N-terminal cysteine of CPPamph shows no changes in the CD spectrum. The amphipathic helix of proline-rich peptides is crucial for efficient cellular uptake; therefore, it is very important that the secondary structure of the peptide is preserved after the conjugation of the drug [36].

Figure 2.

CD spectra of applied peptides and their corresponding doxorubicin conjugate at 1 mg/ml: CPP (0.43 mm), CPPamph (0.42 mm), DOXoxmCPP (0.38 mm), and DOXoxmCPPamph (0.32 mm) in phosphate buffer (100 mm, pH 7.4) at 25 °C.

Spectroscopic Properties

Many uptake and cytotoxicity assays are utilizing the intrinsic fluorescence of the anthracycline drugs. In order to estimate the influence of the peptide over the anthracycline fluorescence, the concentration-dependent emission behavior of doxorubicin and the cell-penetrating peptide conjugates was measured at neutral and acidic pH. Varying the pH from 7.4 to 5 did not influence significantly the emission intensity of doxorubicin and its peptide conjugates (Figure 3). All samples showed increasing fluorescence intensities in the applied concentration range. Doxorubicin and DOXoxmCPP exhibit similar fluorescence at 10 and 50 µm, whereas at lower concentrations, the fluorescence of the oligoarginine–drug conjugate is significantly decreased. At a concentration of 0.1 µm, the fluorescence intensity of the two peptide conjugates is similar to each other and lower than DOX. The emission intensity of DOXoxmCPPamph remained always lower compared with the drug alone over the entire concentration range applied in this study. Proline-rich amphiphatic peptides like CPPamph applied here are prone to aggregation, even at the low micromolar range, as shown by Giralt et al. [18], which can explain the reduced fluorescence of DOXoxmCPPamph.

Figure 3.

Concentration-dependent fluorescence intensities of doxorubicin and the corresponding peptide conjugates in buffer solutions at pH 5 and pH 7.4.

Glutathione is Efficiently Cleaving the Disulfide-linked Doxorubicin

Glutathione is an abundant naturally occurring peptide that serves as an antioxidant and is capable of reducing various disulfide bonds via its cysteine sulfhydryl group [37]. This peptide can be found in almost every mammalian cell, with intracellular concentrations ranging from 1 to 10 mm [38]. Increased intracellular level of glutathione has been reported in many tumor cells, e.g. in bone marrow, colon, breast, and lung cancer.

To investigate the cleavage of the disulfide bond present in all of our drug conjugates, 1 mm of DOXoxmCPP was incubated at 37 °C in DPBS buffer pH 7.4 containing 10 mm of glutathione, and the degradation of the drug–peptide conjugate was followed by analytical RP-HPLC in a time-dependent manner (Figure 4). The absorbance of doxorubicin and the corresponding degradation products of the bioconjugate were monitored at 480 nm.

Figure 4.

(A) Degradation of DOXoxmCPP (1) by reduction with glutathione. The conjugate was incubated in DPBS (pH 7.4) with an excess of glutathione at 37 °C and subsequently analyzed by HPLC over 24 h. (B) Chemical structure of DOXoxmCPP 1 and the corresponding derivatives after cleavage of the peptides – 2 and 3. Retention times of compounds 3 and 4 are 24.17 and 25.03 min, respectively.

The reduction of the conjugate by glutathione is highly efficient, even without prolonged incubation, as it can be seen on the chromatogram at 0 h, which was obtained directly after mixing the two components. Peak 1 corresponds to DOXoxmCPP, and the second developing peak is already doxorubicin coupled via disulfide to glutathione 2. Under the applied incubation conditions, the DOXoxmCPP does not elute as a sharp peak, because the guanidinium groups of the peptide side chains are influenced from various counter ions, such as acetate, phosphate, or the carboxylic acid from glutathione. However, after 1 h incubation, the conjugate is fully degraded, and the doxorubicin glutathione metabolite is the dominating species, with intensity comparable with 1 mm doxorubicin hydrochloride. Moreover, another peak is observed in this chromatogram, which corresponds to a doxorubicin derivative that has the linker moiety with a free thiol 3. Because of the excess of glutathione present and the further incubation, this peak becomes the main product, which is illustrated in the HPLC chromatogram after 4 h. After 3 h, hydrolysis to free doxorubicin (corresponding to peak 4) can be detected and remains the minor process during this study, because hydrolysis of oximes is not favored at neutral pH. The hydrolysis of the oxime linkage in anthracycline bioconjugates is not required in order to obtain DNA binding and cytotoxicity accordingly [27, 39]. In addition, the oxime bond proved to be stable, as no release of doxorubicin was observed from the drug–peptide conjugate in the absence of glutathione after incubation for 24 h in DPBS (pH 7.4) or in 50 mm sodium acetate buffer (pH 4.5).

The thiol-containing doxorubicin species reaches its maximum concentration after 5 h, and prolonged incubation is not changing the degradation dynamics. It is likely that there is no free glutathione present in the solution after a certain time, because further reduction is not visible at 24 h incubation. In cells, a constant glutathione level is maintained by enzymatic processes [40], which would allow full degradation of DOXoxmCPP. As expected, incubation of DOXoxmCPPamph with glutathione showed similar release kinetics of the thiol-containing doxorubicin, as the same coupling approach was utilized for the preparation of the two conjugates (data not shown). Consequently, disulfide cleavage mediated by elevated glutathione has a great potential to serve as a powerful drug-release method, due to the rapid release of toxic metabolites within a few hours, as demonstrated already in antibody–drug conjugates.

Investigation of Cell Uptake and Viability by Flow Cytometry

The cellular uptake of doxorubicin and the conjugates in MCF-7 and HT-29 cells was determined via flow cytometry. Tumor cells were incubated with different concentrations of the drugs in the range of 0.1 and 50 µm for 72 h in a serum-containing medium, followed by flow cytometric analysis to determine the percentage of doxorubicin-positive cells, whereas untreated cells served as control.

Figure 5A illustrates one representative experiment for MCF-7 cancer cells demonstrating the gating strategy for flow cytometry analysis. The uptake of doxorubicin and the corresponding drug conjugates in MCF-7 cells is concentration dependent in the applied range, as reflected by an increasing percentage of doxorubicin-positive cells for all samples. DOXoxmCPP at 10 µm is already taken up by more than 50% of the cells, and at a concentration of 50 µm, almost all the cells are doxorubicin positive. Similar concentration-dependent uptake is given by doxorubicin linked to the amphipathic peptide, which reaches at the highest utilized concentration nearly 80% positive cells. In contrast, the free drug can be detected in almost every tumor cell at a concentration of 1 µm.

Figure 5.

Cellular uptake of doxorubicin and the corresponding peptide conjugates in MCF-7 and HT-29 cells. (A) Flow cytometry histograms after 72 h incubation display uptake by MCF-7 tumor cells from one representative of three independent experiments. Measured doxorubicin intensity (x-axis) is plotted against the cell counts (y-axis). Untreated control cells are shown in filled gray; MCF-7 cells treated with DOX, DOXoxmCPP, and DOXoxmCPPamph are depicted as black lines. Data are represented as percentage of doxorubicin-positive cells. (B) Percentages of DOX (●), DOXoxmCPP (□), and DOXoxmCPPamph (△) uptake by MCF-7 (left) and HT-29 (right) cancer cells. Pooled data of three independent experiments are shown (mean percentages ± standard deviation).

Pooled data of three independent uptake experiments in MCF-7 and HT-29 cells are depicted in Figure 5B. The obtained values for MCF-7 cells are in good agreement with the histograms in Figure 5A. Compared with the breast cancer cells, the uptake of the doxorubicin peptide conjugates in HT-29 cells is even higher, whereas doxorubicin displays no significant difference. The results of the uptake of DOX and the two doxorubicin–peptide conjugates in tumor cells are in line with fluorescence intensity data allowing the analysis per positive cell (provided as supporting information). DOXoxmCPP exhibits in both cancer cell lines an abrupt increase of uptake in the lower micromolar range, which corresponds to the observations in the literature. Above a concentration threshold in the submicromolar range, cationic cell-penetrating peptides as the employed oligoarginine undergo rapid cytosolic uptake independent from endocytosis. Duchardt et al. showed that this highly efficient nonendocytotic pathway originates from spatially confined areas of the plasma membrane and leads to fast release of the PTDs into the cytoplasm [41]. Similarly to MCF-7 cells, the uptake of DOXoxmCPPamph in HT-29 cells is slightly lower, because the utilized amphipathic peptide is internalized by lipid-raft caveolae-mediated endocytosis (LRCvE) [18].

Endocytosis is an active transport across the cell membrane, which explains the reduced cellular internalization compared with the free drug. Contrary to the conjugates, DOX shows rapid cellular association as anticipated, because it is known to diffuse quickly across the membrane [42]. Based on our investigations of the spectroscopic properties of DOXoxmCPPamph, the fluorescence in the applied concentration range (0.1 to 50 µm) is significantly lower compared with doxorubicin and the other peptide derivative, which suggests that the cellular uptake is possibly higher. Therefore, we did not compare the mean fluorescence intensity demonstrating the number of peptide–drug conjugates per cell but the percentages of doxorubicin-positive tumor cells (Figure 5).

In addition to the assessment of the uptake of DOX and the peptide conjugates, the tumor cells were simultaneously stained with the Fixable Viability Dye eFluor® 780 in order to analyze cell viability. The Viability Dye eFluor® 780 penetrates damaged cell membranes and reacts with amine groups in the cytoplasm [43]. The staining is not appropriate to draw definite conclusions on total amount of dead cells, as only percentages of cells rather than the total cell number are addressed, and additionally, cell debris is not included in flow cytometry analysis. MCF-7 and HT-29 cells were incubated with 50 µm doxorubicin, DOXoxmCPP, and DOXoxmCPPamph for 72 h prior to staining. Doxorubicin uptake was plotted against the viability dye in order to investigate percentages of dead doxorubicin-positive cells after drug treatment.

Incubation of MCF-7 cells with DOX reveals that doxorubicin can be detected in all cells, and based on the positive staining with the viability dye, 21% are dead (Figure 6). Incubation with the cell-penetrating peptide conjugates induces a reduced amount of doxorubicin-positive cells in comparison with DOX alone. In the case of DOXoxmCPP, nearly 80% cells are positive for doxorubicin, but half of them are dead, which is a twofold increase compared with the drug alone. DOXoxmCPPamph causes around 10% dead cells, a result that is similar to the control cells cultured without the drug–peptide conjugate. In HT-29 cells, the free drug exhibits similar percentages of uptake, while the percentage of dead cells is not increased compared with the control cells. The overall uptake of DOXoxmCPP and DOXoxmCPPamph is enhanced in HT-29 cells. However, treatment with the octaarginine–drug conjugate does not lead to an increased cell death of HT-29 cells as we find with MCF-7 cells. In addition, DOXoxmCPPamph-treated cells display similar percentages of dead cells as the control cells. Nevertheless, for MCF-7 breast cancer cells, DOXoxmCPP show better results in terms of cell toxicity after incubation for 72 h.

Figure 6.

Cell viability after doxorubicin and drug–peptide conjugate uptake analyzed via flow cytometry. MCF-7 (upper row) and HT-29 (lower row) cancer cells were incubated with 50 µm DOX, DOXoxmCPP, and DOXoxmCPPamph for 72 h and afterwards stained with the Fixable Viability Dye eFluor® 780. Fluorescence intensity of the viability dye (y-axis) is plotted against the measured doxorubicin intensity (x-axis). The percentages of double-positive cells are depicted in the upper right quarter.

In Vitro Cytotoxicity

Doxorubicin binds with high affinity to DNA by intercalation, which leads to DNA damage and subsequent growth inhibition [44, 45]. We performed MTT assays in order to determine the viability of the tumor cells and additionally to assess the proliferative potential of the cells after drug treatment. MCF-7 and HT-29 cells were incubated with doxorubicin and the peptide conjugates at various concentrations in the range of 0.1 and 50 µm for 72 h (in a serum-containing medium), and the results were expressed as IC50-values (Table 1).

Table 1. IC50 values of doxorubicin and the corresponding cell penetrating peptide conjugates in MCF-7 and HT-29 cell lines from one representative of three independent experiments
 MCF-7 IC50 [μM]HT-29 IC50 [μM]

DOXoxmCPP exhibits on HT-29 cells an IC50 value of 19 µm, whereas in the applied breast cancer cell line, the value is significantly lower. This observation is in good agreement with cell viability studies from the previous section, although the uptake in HT-29 cells was higher. The IC50 values of the amphipathic cell-penetrating peptide–doxorubicin conjugate are higher compared with DOXoxmCPP, with slightly increased toxicity against the colon cancer cell line, corresponding to the data obtained from the flow cytometry analysis. Investigation of the cytotoxicity of the peptides alone in MTT assay excluded a toxic effect of CPP and CPPamph in both cancer cell lines (see the Materials and Methods section), even at the highest concentration used in this study. Previous studies have also revealed that the utilized peptides were nontoxic in the applied concentration range, indicating that the cytotoxic effect is due to doxorubicin [18, 46].

Taking into account that the oligoarginine doxorubicin conjugate displays at 50 µm higher toxicity than the free drug as depicted in Figure 6, this demonstrates again that the peptide–drug conjugate possibly require certain threshold concentration in order to undergo rapid cellular internalization, which explains the increased IC50 value, due to the reduced cell uptake at lower micromolar concentrations.

Intracellular Distribution of the Disulfide-linked Doxorubicin Cell-penetrating Peptide Conjugates

DNA intercalation and inhibition of topoisomerases are considered to be the major targets in the anticancer activity of doxorubicin; therefore, it is important that the drug accumulates in the cell nucleus [45]. CLSM was carried out to visualize whether the herein reported doxorubicin conjugates could reach the nucleus after intracellular cleavage by glutathione. Live-cell imaging of HT-29 and MCF-7 cells was performed after treatment with 50 µm DOX, DOXoxmCPP and DOXoxmCPPamph for 5 h (in serum-containing medium), and counterstaining of the nuclei was performed with Hoechst nuclear stain. The fluorescence images in Figure 7 display that both cancer cell lines efficiently take up the free drug within a short period of time. The oligoarginine–drug conjugate exhibits strong cellular uptake in HT-29 and MCF-7 cells as well, which corresponds to the results obtained by flow cytometry. DOXoxmCPP is detected in both cytosol and nucleus. The other peptide conjugate possesses similar properties, even though the cellular uptake appears decreased, as discussed before. For DOXoxmCPPamph, doxorubicin fluorescence occurs in both cell lines almost exclusively in the nucleus, which is illustrated by the counterstaining with the Hoechst dye. The fluorescence of the cytosolic uncleaved peptide–drug conjugate is probably reduced, as seen in our spectroscopic studies. Proline-rich amphipathic cell-penetrating peptides, like CPPamph employed here, are taken up by LRCvE [36]. This internalization pathway is beneficial for peptide conjugates that are susceptible to proteases, because caveolae can be directly transported to the Golgi and/or endoplasmic reticulum, thus avoiding lysosomal degradation [47]. LRCvE of DOXoxmCPPamph can lead to cytosolic delivery of the conjugate and subsequent cleavage by glutathione, thereby releasing doxorubicin species, such as 3, that can enter the cell nucleus.

Figure 7.

Confocal laser scanning microscopy of MCF-7 (upper row) and HT-29 cells (lower row) cells treated with 50 µm doxorubicin, DOXoxmCPP, and DOXoxmCPPamph for 5 h (live-cell imaging). Cell nuclei stained with Hoechst dye appear blue, whereas the drug appears red. The upper row of each cell line depicts additionally the transmitted light channel to visualize cell boarders. Scale bar represents 20 µm.


We synthesized and investigated two doxorubicin cell-penetrating peptide conjugates, where the two peptides differ in their net charge, secondary structure, and origin. To join the carrier with the drug, we employed a novel small crosslinker molecule that does not require modification of the daunosamine sugar of doxorubicin. This linker moiety creates stable oxime bond on doxorubicin's aliphatic ketone and simultaneously carries an activated thiol. Furthermore, the application of the activated thiol group is not only limited to peptide carriers but can also be used for the attachment to cysteine residues of proteins, antibodies, or thiol-containing polymers. Cellular uptake and cytotoxicity studies performed with MCF-7 and HT-29 cancer cell lines demonstrated that both cell-penetrating peptide–drug conjugates, irrespective of their charge, secondary structure, and size, can efficiently transport doxorubicin through the cell membrane. The two conjugates can be efficiently cleaved by glutathione within a very short period of time and deliver the toxic freight into the nucleus. Additionally, the polyarginine–drug conjugate exhibits two crucial advantages compared with the free drug: At elevated concentrations, it displays higher toxicity than doxorubicin in MCF-7 cells, and it can serve as a prototypic tumor-targeting vector in vivo as reported by Futaki et al. [48]. The herein reported molecules are promising drug conjugates, and they will be well suited for further encapsulation in liposomes or nanoparticles which we envision as multistage delivery systems that will help to overcome the rapid clearance of all peptide-based carriers in vivo.


The authors are grateful to Prof. Ruth Duncan for many helpful suggestions and comments and for critically reading the manuscript. We would like to thank also D. Strand and S. Lorenz and the core facility for laser scanning microscopy of the FZI Mainz for their technical assistance in performing the CLSM experiments. This work was funded by the Max Planck Society. K.S. acknowledges funding from the SFB 1066 (B6).