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Jun 2, 2015 - ABSTRACT: The extended use of doxorubicin (DOX) could be limited because of the emergence of drug resistance associated with its ...
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Acid-Sensitive Peptide-Conjugated Doxorubicin Mediates the Lysosomal Pathway of Apoptosis and Reverses Drug Resistance in Breast Cancer Yuan Sheng,†,§ Jinhui Xu,‡,§ Yiwen You,† Feifei Xu,† and Yun Chen*,† †

Nanjing Medical University, Nanjing 211166, China Department of Pharmacy, Suzhou Municipal Hospital, Suzhou 215001, China

‡

S Supporting Information *

ABSTRACT: The extended use of doxorubicin (DOX) could be limited because of the emergence of drug resistance associated with its treatment. To reverse the drug resistance, two thiol-modified peptide sequences HAIYPRHGGC and THRPPMWSPVWPGGC were, respectively, conjugated to DOXO-EMCH, forming a maleimide bridge in this study (i.e., T10-DOX and T15-DOX). The structures and properties of peptide−DOX conjugates were characterized using 1H NMR, 13 C NMR, mass spectrometry, and high-performance liquid chromatography. Their stability was also evaluated. By using MCF-7/ADR cells as an in vitro model system and nude mice bearing MCF-7/ADR xenografts as an in vivo model, the ability of these novel peptide−DOX conjugates to reverse drug resistance was accessed as compared with free DOX. As a result, the IC50 values for T10-DOX and T15-DOX significantly decreased (31.6 ± 1.6 μM and 27.2 ± 0.8 μM), whereas the percentage of apoptotic cell population increased (35.4% and 39.3%). The in vivo extent of inhibition was more evident in the mice groups treated with peptide−DOX conjugates (59.6 ± 8.99% and 46.4 ± 6.63%), which had DOX primarily accumulated in tumor. These conjugates also showed a longer half-life in plasma and cleared much more slowly from the body. Furthermore, T10-DOX may be more effective than T15-DOX with a higher efficacy and a lower side effect. Most importantly, evidence was provided to support the enhanced intracellular drug accumulation and the induction of lysosomal pathway of apoptosis underlying the drug resistance. As an endosomal/lysosomal marker, cathepsin D permealized the destabilized organelle membrane and was detected in the cytoplasm, leading to the activation of the effector caspase-3 in cell apoptosis. This report is among the first to demonstrate that peptide−DOX-like conjugates promote apoptosis through the initiation of the lysosomal pathway. KEYWORDS: transferrin receptor-mediated drug delivery, doxorubicin, peptide conjugates, drug resistance reversal, lysosomal pathway, acid sensitive

1. INTRODUCTION Doxorubicin (DOX) is a widely used anthracycline that has been proven highly effective against a variety of human malignancies including breast cancer.1,2 However, from a clinical perspective, the extended use of DOX could be limited due to the emergence of drug resistance associated with its treatment. A variety of pathways have been suggested for cancer cells to acquire drug resistance.3 Two major pathways include drug efflux and the direct suppression of apoptosis. Drug efflux occurs because of the increased membrane accumulation of various ATP-binding cassette (ABC) transporters including pglycoprotein (P-gp). The suppression of apoptotic pathways is most likely attributed to the accumulation of mutant p53 (mutp53) with “gain of function” and to the increased expression of antiapoptotic proteins such as caspase-3. To date, many efforts have been made to reverse the drug resistance. One attempt is to explore the potential of transferrin receptor (TfR)-mediated endocytosis, which has attractive © XXXX American Chemical Society

features in overcoming the drug resistance and has been employed for DOX delivery in recent years.4 TfR is a transmembrane homodimer that can bind up to two molecules of transferrin (Tf) and internalize them by means of TfR-mediated endocytosis.5 These receptors are generally overexpressed and feature a high turnover rate in tumor cells.6 Most importantly, the emergence of drug resistance is associated with further overproduction of TfR.7 Our lab has recently quantified the levels of TfR as 5.57 fg/cell in normal breast cells MCF-10A, 41.9 fg/cell in parental drug-sensitive cancer cells MCF-7/WT, and 64.0 fg/cell in drug-resistant cancer cells MCF-7/ADR using liquid chromatography− tandem mass spectrometry (LC−MS/MS)-based targeted Received: May 28, 2014 Revised: May 17, 2015 Accepted: May 22, 2015

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Molecular Pharmaceutics proteomics.8 Thus, Tf has been widely applied as a targeting ligand in the active targeting of anticancer agents, proteins, and genes to tumor cells.4 Among these studies, some have linked DOX with Tf via direct glutaraldehyde cross-linking or a spacer such as maleimide spacer.4,9−13 Their results indicated that the conjugates of DOX with Tf (DOX-Tf) can be specifically delivered to cancer cells, improve the selectivity, and overcome drug resistance, thereby leading to a better treatment.14,15 However, the endogenous Tf (e.g., 25 μM in blood) may competitively inhibit the binding of DOX-Tf to TfR. In addition, there is evidence showing that the relatively high molar weight of Tf makes the conjugates primarily localized in the cytoplasm,9 and it is also hard to estimate the conjugation number (average of molecules of DOX per molecule of Tf). Currently, peptides derived from sequence of cell surface proteins have been explored as trafficking moiety in targeted drug delivery due to their ease of synthesis, structural simplicity, and low probability of undesirable immunogenicity.16 In case of Tf, Engler and colleagues first identified two peptide sequences HAIYPRH and THRPPMWSPVWP that specifically bind to the TfR. More importantly, both of these sequences did not compete with Tf for receptor binding.17 On the other hand, the prodrug of DOX, (6-maleimidocaproyl) hydrazone of DOX (DOXO-EMCH) has remarkable antitumor activity in preclinical studies,18 and meanwhile renamed INNO-206, is initiating a sarcoma phase 3 study.19 DOXO-EMCH is thiolreactive, and previous studies have demonstrated that it conjugated rapidly and selectively to a number of proteins in situ (e.g., albumin, Tf) and then was developed into proteinconjugated DOX. Similar conjugation strategies have been employed for several peptide−DOX conjugates.10−13,20−23 On the basis of these points, we conjugated the sequence- and thiol-modified HAIYPRH and THRPPMWSPVWP to DOXOEMCH, forming a maleimide bridge in this study. By using drug-resistant MCF-7/ADR cells as an in vitro model system and nude mice bearing MCF-7/ADR xenografts as an in vivo model, the ability of these novel peptide−DOX conjugates to reverse drug resistance was evaluated as compared with free DOX. In addition to a comprehensive characterization of conjugates, the mechanisms underlying the drug resistance reversal including the intracellular drug accumulation and lysosomal pathway of apoptosis were also explored.

Inc. (Newark, NJ, USA). Formic acid (FA) was provided by Aladdin Chemistry Co., Ltd. (Shanghai, China) and Xilong Chemical Industrial Factory Co., Ltd. (Shantou, China), respectively. Water was purified and deionized using a MilliQ system from Millipore (Bedford, MA, USA). 2.2. Preparation of Peptide-Conjugated DOX (T10DOX and T15-DOX). A 600 μL sample of DOXO-EMCH (2.4 mM) in water/dimethylformamide (v/v, 1:1) was mixed gently with 600 μL of peptide in PBS (1.2 mM) using electromagnetic agitator for 1.5 h at 27 °C in the dark. DOXO−EMCH was added dropwise at 25 μL/min. The crude reaction mixtures were then purified by highperformance liquid chromatography (HPLC). The purification was performed on a SunFire Prep C18 OBD column (10 μm, 18 mm × 150 mm; Waters, USA) at room temperature. The mobile phase consisted of solvent A (0.01% FA in water) and solvent B (ACN). A linear gradient with a flow rate of 10 mL/ min was applied in the following manner: B 5% (0 min) → 20% (5 min) → 50% (15 min) → 80% (20 min) → 20% (25 min) → 5% (30 min) for T10-DOX, and B 10% (0 min) → 20% (5 min) → 20% (15 min) → 50% (20 min) → 90% (25 min) → 10% (30 min) for T15-DOX. The injection volume was 3 mL. The detection wavelength was set to 490 nm. Fractions were collected, and the eluent was evaporated to dryness. To determine the stability of peptide−DOX conjugates and their DOX release profiles at various pH values, the conjugates were prepared in 0.2 M PBS (pH 5.0, 6.0, and 7.4) and incubated at 37 °C. These pH levels simulated the physiological conditions (pH 7.4) as well as the acidic features of endosomes/lysosomes that are more acidic (pH 4.5−6.5). The amount of released DOX was determined using HPLC. 2.3. Cell Culture. MCF-7/WT (ATTC, Manassas, VA) and MCF-7/ADR (Keygen Biotech, Nanjing, China) cells were cultured in DMEM media supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% CO2. The cells were splitted every 5−7 days by lifting cells with 0.25 × trypsin and feeding between splits through the addition of fresh medium. To maintain a highly drug-resistant cell population, MCF-7/ADR cells were periodically reselected by growing them in the presence of 1000 ng/ mL DOX.17 Experiments were carried out using the cells incubated without DOX for 48 h. Cells were counted with a hemacytometer (Qiujing, Shanghai, China). Cell viability was assessed by trypan blue (0.4%) exclusion, which was completed by mixing cell suspension, trypan blue, and 1 × PBS in a ratio of 2:5:3 and counting the percentage of viable cells following a 5 min incubation at 37 °C. 2.4. MTT Assay. The cytotoxicity of peptide-conjugated DOX was determined using MTT test. Free DOX was used as positive control, and the cells treated with solvent only were considered as negative control. MCF-7/ADR cells in exponential growth were seeded in a 96-well plate and incubated for 24 h at 37 °C in a fully humidified atmosphere of 5% CO2. Then, serial dilutions of DOX and peptide−DOX conjugates at equivalent concentrations (final concentration, 5−80 μM) were added and incubated for another 48 h. After an addition of MTT solution (2 mg/mL in PBS) to each well, the cells were incubated for another 4 h and lysed in DMSO. Absorbance was determined at 490 nm using an EL × 800 absorbance microplate reader (Biotek, USA). The IC50 values were calculated from the linear regression of the dose-log response curves using GraphPad Prism 5 software (Graphpad Software, CA, USA).

2. EXPERIMENTAL SECTION 2.1. Chemicals and Reagents. Peptides (HAIYPRHGGC (T10) and THRPPMWSPVWPGGC (T15)) were developed by ChinaPeptides Co., Ltd. (Shanghai, China). DOXO-EMCH was purchased from Medchem Express (Princeton, NJ, USA) with purity of 99.3%. Transferrin, D-(+)-mannitol and N-(2hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypan blue was obtained from Generay Biotech Co., Ltd. (Shanghai, China). Dulbecco’s Modified Eagle Media (DMEM) media and fetal bovine serum were obtained from Invitrogen (Burlington, ON, Canada). Penicillin was supplied by CSPC Zhongnuo Pharmaceutical Co., Ltd. (Shijiazhuang, China). Streptomycin was obtained from Merro Pharmaceutical Co., Ltd. (Dalian, China). Phosphate-buffered saline (PBS) was from Beyotime Institute of Biotechnology (Jiangsu, China). Ethylenediaminetetraacetic acid (EDTA), potassium hydroxide, sodium bicarbonate, and hydrochloric acid were from Sinopharm Chemical Reagent Company (Shanghai, China). Acetonitrile and methanol were HPLC grade and from ROE B

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Figure 1. Structures of DOX, T10-DOX, and T15-DOX.

2.5. Immnofluorescence Microscopy. MCF-7/ADR cells were grown on dish and treated with 1.5 μM free DOX or equivalent conjugates in cell culture media without fetal bovine serum at 37 °C for 12 h. Then, the cells were washed with PBS. Finally, the cells were imaged using a Leica DMI3000B inverted fluorescence microscope (Leica Microsystems, Mannheim, Germany) equipped with an Ar/Kr laser. The combination of BP 560/40 excitation filter, 595 dichroic mirror, and BP 645/75 suppression filter was used for imaging. The fluorescence was detected by a DFC450C CCD digital camera (Leica Microsystems Wetzlar GmbH, Germany), and images were acquired and analyzed using software LAS 4.2 (Leica Microsystems Wetzlar GmbH, Germany). 2.6. Terminal Deoxynucleotidyl Transferase-Mediated Nick-End Labeling (TUNEL) Assay. TUNEL assay was used for microscopic detection of apoptosis. Procedures were followed according to the DeadEnd Fluorometric TUNEL system (Promega, WI, USA). Apoptotic cells exhibit a strong nuclear green fluorescence at ∼520 nm, whereas the cells stained with DAPI exhibit a strong blue fluorescence at ∼620 nm. Images were acquired at room temperature with Leica DMI3000B inverted fluorescence microscope. 2.7. Evaluation of Lysosomal Pathway of Apoptosis Using Western Blotting. The procedure of subcellular fractionation and protein extraction has been described previously.24 In detail, the cells after treated with 20 μM free DOX or equivalent conjugates for 24 h and were pelleted at 1480 × g for 5 min and washed twice with ice-cold PBS. Then, they were resuspended in 1 mL of fractionation buffer (210 mM D-(+)-mannitol, 70 mM sucrose, 5 mM HEPES, and 5 mM EDTA, pH 7.4). The samples were disrupted using 40 strokes in a Dounce homogenizer with 0.5−2.5 × 10−3 inch clearance (Kontes, Vineland, NJ, USA) on ice. The results of viability assay indicated that >95% of the cells were disrupted with this procedure. Following the disruption, the homogenate was centrifuged at 1400 × g for 10 min to pellet nuclei and unbroken cells. Endosomes/lysosomes as well as other organelles were further pelleted by centrifugation at 14000 × g for 20 min. The post supernatant was obtained for Western blotting.

The integrity of endosomes/lysosomes was assessed by estimation of the amount of cathepsin D translocated to the cytoplasmic fraction. After the separation of cytoplasmic fraction by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the membrane was exposed overnight at 4 °C to a monoclonal mouse antihuman cathepsin D antibody (1:1000; Abcam, Cambridge, UK). The protein was then detected using enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. As the most widely studied downstream effector caspase in cell apoptosis, caspase-3 was also measured using antihuman caspase-3 antibody (1:1000; Cell Signaling Technology, Boston, USA) and Western blotting in this study. 2.8. In Vivo Study. Twenty-four athymic nude mice (Balb/ c nu/nu, females) were used. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Nanjing Medical University. MCF-7/ADR cells were first collected in a concentration of 1× 107/mL and inoculated subcutaneously into the right flank of each mouse with 0.1 mL/mouse. When the tumor size reached a volume of 50−100 mm3, these mice were randomly divided into four groups (each group consisted of six mice) for the treatment with 5 mg DOX equivalent/kg of either free dox, T10-DOX, or T15-DOX once every 7 days for three times. Saline was used as negative control. The animals’ weight and tumor dimensions were measured every 3 days. Tumor volume was calculated using an equation: TV = 1/2 × a × b2, where a is the largest diameter, and b is the smallest diameter. Each animal was tagged in the ear and followed individually throughout the experiments, with the animals receiving the desired drug and dosage. After 21 days of initial treatment, the mice were sacrificed. Tumors and organ samples of heart, liver, spleen, kidneys, and lungs were carefully removed and stored in liquid nitrogen. To assess the pharmacokinetic profiles of drugs, free DOX and the peptide−DOX conjugates were injected i.v. through the caudal vein into mice, respectively. Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post injection. C

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Figure 2. continued

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Figure 2. Comprehensive characterization of T10-DOX and T15-DOX. (A) HPLC chromatograms. The HPLC system consisted of a Shimadzu LC20AB solvent delivery pump, a Rheodyne manual valve injector, and a Shimadzu SPD-20A UV−vis detector (Shimadzu Corporation, Tokyo, Japan). The samples were analyzed using a Biobasic HPLC C8 column (5 μm, 4.6 mm × 150 mm; Thermo, USA) at room temperature. The mobile phase consisted of solvent A (0.01% FA in water) and solvent B (ACN). A linear gradient with a flow rate of 1 mL/min was applied in the following manner: B 0% (0 min) → 20% (5 min) → 50% (10 min) → 80% (20 min) → 80% (25 min) → 20% (30 min)→ 20% (35 min)→ 0% (37 min). The detection wavelength was set to 495 nm. The data were acquired and processed with LabSolutions/LC solution version 1.2. (B) The 1H NMR spectra were performed in D2O. Observed signals for T10-DOX are δ 1.16 (d, 3H, H3C−C-5′), 1.28 (m, 14H, Ile and spacer H2C), 1.47 (d, 3H, Ala), 1.56 (m, 4H, Arg), 1.95 (m, 8H, Pro), 2.13 (m, 2H, H2C-9), 2.61(s, 2H, H2C-7), 2.96 (d, 2H, maleimide), 3.07 (m, 10H, His and Tyr), 3.03− 3.50 (m, 15H, Pro), 3.82 (m, 3H, maleimide), 3.90 (s, 3H, H3C−O−C-1), 4.08 (m, 6H, Gly), 4.23 (m, 1H, HC-5′), 4.61 (d, 2H, H2C−CN), 6.70 (m, 6H, Tyr), 7.35 (t, 1H, HC-3), 7.42 (m, 3H, His), 7.55 (d, 2H, HC-2 and HC-4). Observed signals for T15-DOX are δ 1.19 (d, 3H, H3C−C-5′), 1.23 (m, 10H, Thr), 1.28 (m, 7H, spacer H2C), 1.52 (m, 2H, Arg), 1.93 (m, 3H, Pro), 2.01 (m, 8H, Ser), 2.02−2.11 (m, 5H, Met), 2.15 (m, 2H, H2C-9), 2.44 (m, 3H, Met), 2.63 (s, 2H, H2C-7), 2.91(m, 3H, Trp), 3.03 (d, 2H, maleimide), 3.81(m, 2H, Thr), 3.84 (m, 3H, maleimide), 3.89− E

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4.02 (m, 6H, Ser), 4.23 (m, 1H, HC-5′), 4.61 (d, 2H, H2C−CN), 7.19 (m, 4H, Trp), 7.36 (t, 1H, HC-3), 7.46 (m, 3H, His), 7.61 (d, 2H, HC-2 and HC-4). The parent carbons in DOX were numbered consistent with (8S-cis)-10-(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione. (C) The 13C NMR spectra were performed in D2O. Observed signals for T10-DOX are δ 9.99 (Ile), 14.64 (Ile), 15.73 (CH3), 16.01 (Arg), 24.53 (Pro), 27.52 (C-7), 29.37 (Cys), 33.20 (maleimide), 35.17 (C-2′), 42.36 (Pro), 43.30 (maleimide), 48.01(Gly), 49.56 (Gly), 56.30 (CH3O), 64.36 (C-8), 67.02(C-5′), 118.24(C-2), 119.29(C-4), 124.4 (His), 128.36 (Tyr), 130.53 (Tyr), 133.51 (His), 134.36 (His), 155.43 (C-6), 156.70 (Tyr), 160.12 (C-1), 163.05 (Arg), 185.20(C-5). Observed signals for T15-DOX are δ 15.73 (CH3), 16.3 (Val), 16.8 (Met), 24.41 (Pro), 27.48 (C-7), 28.7 (Val), 29.41 (Cys), 33.20 (maleimide), 34.74 (C-2′), 42.31 (Pro), 43.30 (maleimide), 48.03 (Gly), 49.66 (Gly), 55.97 (CH3O), 64.03 (C-8), 66.71 (Ser), 68.14 (C-5′), 109.33 (Trp), 112.58 (Trp), 118.51 (C-2), 119.02 (Trp), 119.84 (C-4), 125.64 (His), 130.80 (His), 155.16 (C-6), 161.88 (C-1), 164.39 (Arg), 186.87 (C-5). (D) The parent ion spectra. The mass spectrometer was interfaced with an electrospray ion source and operated in the positive MRM mode. Q1 and Q3 were both set at unit resolution. The flow of the drying gas was 10 L/min, while the drying gas temperature was held at 350 °C. The electrospray capillary voltage was optimized to 4000 V. The nebulizer pressure was set to 45 psi. The data were collected and processed using the Agilent MassHunter Workstation Software (version B.01.04). (E) The product ion spectra. The collision energy was set at 30 eV. ∗ = unique product ions of DOX, ∗∗ = −DOXOEMCH. (F) In vitro release profiles of DOX at pH 5.0, 6.0, and 7.4. Data are mean ± SD (n = 3).

2.9. Preparation of in Vivo Samples and Biodistribution Study Using LC−MS/MS. All tissue samples were processed immediately after thawing and maintained in an ice bath throughout the procedure. Approximately 50 mg of tissue was weighted and resuspended in 0.2 M disodium hydrogen phosphate. Then, samples were homogenized using a Bio-Gen PRO200 homogenizer (PRO Scientific Inc., Oxford, CT, USA) for 1 min and vortexed for 5 min. Four milliliters of extraction solvent (9:1 chloroform/heptanol) was added. Afterward, the sample was centrifuged at 4000 rpm for 10 min. After a removal of organic layer, the remaining aqueous phase was re-extracted with 1 mL of extraction solvent for two more times. The solutions were combined and evaporated to dryness and then resuspended in 100 μL of acetonitrile/water 50:50. For whole blood samples, they were centrifuged at 2000 × g for 20 min, and the supernatant was collected followed by an addition of ACN three times the volume thereof. After centrifugation, the collected samples were extracted using the procedure described above. An Agilent Series 1200 HPLC system (Agilent Technologies, Waldbronn, Germany) and a 6410 Triple Quad LC−MS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) were used. Calibration standards and QC standards of drugs were prepared in blank tissue homogenate/plasma. Daunorubicin (DAU) was used as internal standard. The chromatographic and mass spectrometric parameters have been described elsewhere.24

Figure 2, panel B shows the 1H NMR spectra of peptide− DOX conjugates. There were characteristic signals of double bond of maleimide group at 6.70 ppm in DOXO-EMCH (Figure 2S, Supporting Information).25 This signal disappeared in the spectra of peptide-conjugated DOX, and new signals representing SCH and CH2 appeared (3.82 and 2.96 ppm in T10-DOX, and 3.84 and 3.03 ppm in T15-DOX), suggesting that the double bond had reacted with the thiol group of peptides. 13C NMR spectra of conjugates were also carried out. Similarly, the peak at 136.6 ppm (Figure 2S) in DOXO-EMCH was replaced by new peaks at 33.2 and 43.3 ppm (Figure 2C and Figure 3S). Thus, the NMR spectra provided some evidence to the formation of peptide−DOX conjugates. Furthermore, precursor ion scan spectra showed the molecular ion peak of T10-DOX and T15-DOX (Figure 2D). Generally, these molecular ion peaks can not offer the structural characterization of molecules. To date, rare mass spectrometric methods have been reported for the identification of peptide conjugates. Theoretically, the key for effective identification is to predict the product ions based on the similarity of the fragmentation to that of the peptides and conjugated drugs.26 Thus, tandem mass spectrometry and collision-induced dissociation (CID) were attempted in this study. As shown in Figure 2, panel E, similar to CID of peptides, the fragmentation of peptide−DOX conjugates mainly resulted from the cleavage of peptide backbone in the positive ion mode. Some differences existing between two conjugates were probably due to the proline effect (i.e., selective cleavage of the amide bond Nterminal to the proline residue)27 and histidine effect (i.e., the number of sequence-type product ions highly dependent on the position of the histidine residue)28 on their mass behavior. However, the characteristic sequence-specific b ions and y ions with and without DOXO-EMCH part were indicative of both conjugates. In addition, unique product ions of DOX (e.g., m/z 543, m/z 394, m/z 376) are also present in their product ion spectra. These results provided further evidence that peptides were conjugated with DOXO-EMCH. Owing to the specific binding of peptides to TfR, the conjugates are internalized by active TfR-mediated endocytosis. The wide pH range in endocytosis suggests the presence of several organelle types (i.e., early endosomes, late endosomes, and lysosomes) since these are known to have different pH values. Their pH are also known to decrease progressively during their maturation process from 6−6.5 for early endosomes, to 5.5−6 for late endosomes, and to 4.5−5.5 for lysosomes.29 Since hydrazone bond is known to be acidsensitive, it is suggested to be cleaved in these acidic organelles.

3. RESULTS 3.1. Comprehensive Characterization of PeptideConjugated DOX. To be thiol-reactive, DOX has been modified at its C-13 keto position with a maleimidocaproyl hydrazide linker yielding DOXO-EMCH. A three amino acid peptide (GGC) containing a cysteine at the carboxyl terminus was linked with HAIYPRH and THRPPMWSPVWP to form T10 and T15. As mentioned earlier, cysteine is thiol-reactive. Another two glycine residues were designed to avoid potential steric hindrance in subsequent reaction. Then, the elongated peptide was conjugated to DOXO-EMCH in a Michael addition. After purification, red target products were obtained. The yields of the conjugates (T10-DOX and T15-DOX, Figure 1) were 75.2% and 72.3%, respectively. After separation, their purity was determined using analytical reversed-phase HPLC and resulted in >98% at 495 nm (peaks for T10-DOX at 10.5 min and T15-DOX at 12.3 min, Figure 2A; peaks for free DOX at 12.0 min and DOXO-EMCH at 13.5 min, Figure 1S, Supporting Information). F

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treatment at the concentration of 20 μM, the number of TUNEL positive MCF-7/ADR cells significantly increased (Figure 4). The percentage of apoptotic cell population was higher by 35.4% for T10-DOX and 39.3% for T15-DOX as compared to free DOX by 22.4%. 3.4. Intracellular Drug Accumulation in MCF-7/ADR Cells. The intracellular DOX levels were imaged after 12 h of treatment (Figure 5A). Following the procedure described previously,24 the amounts of DOX in whole cells and nucleus were also determined (Figure 5B,C). As shown, the peptide− DOX conjugates were internalized and concentrated most heavily in the nuclear envelope. These observations were consistent with previous reports that peptide conjugates inside the acidic environment of endosomes/lysosomes were hydrolyzed at the acid−labile hydrazone linker and released DOX inside the cell.26,30 Then, DOX diffused to the nucleus where it acted in analogy to free DOX. The greater intracellular accumulation of T10-DOX and T15-DOX than free DOX implied that these conjugates bypassed drug efflux pumps. 3.5. Effect of Peptide-Conjugated DOX on Lysosomal Pathway of Apoptosis. Cathepsin D is a lysosomal protease. Under normal conditions, cathepsin D is found only in endosomes/lysosome.31 The permeabilization of endosomes/ lysosomes’ membrane could cause the release of this protein.32,33 In this study, the results of Western blotting indicated that this type of release was significantly higher in the cytoplasmic fractions of MCF-7/ADR cells after treatment of conjugates compared to free DOX (Figure 6A), providing the evidence that the integrity of endosomes/lysosomes was destabilized to a greater extent in the presence of T10-DOX and T15-DOX. Since the released cathepsin D in cytosol can further initiate apoptosis signaling pathways,34 apoptosis-related caspase-3 protein expression was measured. As a result, higher levels of active caspase-3 were found in T10-DOX and T15DOX treated cells (Figure 6B and Table 1S), which was consistent with the observation of cathepsin D release. 3.6. In Vivo Antitumor Effect and Biodistribution Study. To assess the effect of peptide-conjugated DOX on tumor growth, MCF-7/ADR tumor-bearing mice were treated with T10-DOX, T15-DOX, free DOX (5 mg/kg), and saline, respectively. Figure 7, panel A shows tumor growth curves for all groups. On day 21 after treatment, T10-DOX, T15-DOX, and free DOX inhibited tumor growth by 59.6 ± 8.99%, 46.4 ± 6.63%, and 31.9% ± 2.86%, respectively (Figure 7B,C). Overall, tumor growth was inhibited in all drug-treated groups compared to that in the control group, whereas the extent of inhibition was more evident in the groups treated with peptide−DOX conjugates. The tumor weight inhibited by T10-DOX and T15-DOX was 87% and 45% greater than that by free DOX. Evident toxicity was observed in free DOX treated mice, whereas mice treated with conjugates had less weight loss (Figure 7D). In vivo fluorescent images were taken at 6 h after injection. As shown in Figure 8, panel A, DOX was primarily accumulated in the tumor treated with peptide−DOX conjugates compared to the distribution of free DOX that accumulated across the body. Biodistribution study indicated that the DOX amounts in the tumor of mice treated with conjugates significantly increased compared with that of free DOX (P < 0.01) (Figure 8B). There was five-fold and three-fold enhancement for T10-DOX and T15-DOX, respectively. Furthermore, their concentrations in liver, kidney, heart, spleen, and lung were lower than that of free DOX (p < 0.05, Figure 8C).

In this study, pH was simulated as 7.4, 6.0, and 5.0, and the release profiles of DOX are shown in Figure 2, panel F. The HPLC data revealed that peptide-conjugated DOX exhibited a pH-dependent feature. The conjugates were moderately stable at pH 7.4 with a 10% release after 24 h, whereas DOX release was much faster at pH 6.0 and pH 5.0, with approximately 70% and 100% after 6 h, respectively. In addition, T15-DOX had a comparative greater release rate than T10-DOX (Figure 4S). 3.2. Effect of Peptide-Conjugated DOX on the Growth of MCF-7/ADR Cells. To determine the sensitivity of MCF-7/ ADR cells to the cytotoxic effects of peptide−DOX conjugates, the cells were treated with increasing concentrations of conjugates using free DOX as control. As shown in Figure 3,

Figure 3. Cytotoxicity profiles of free DOX, T10-DOX, and T15-DOX for (A) MCF-7/WT and (B) MCF-7/ADR cells.

panel A, both T10-DOX and T15-DOX inhibited the growth of MCF-7/ADR cells in a dose-dependent manner. The IC50 values of T10-DOX and T15-DOX calculated from the doselog response curves were 31.6 ± 1.6 μM and 27.2 ± 0.8 μM, respectively. These values were significantly lower than that of free DOX (41.5 ± 2.4 μM), which provide the evidence that MCF-7/ADR cells were much more sensitive to our designed peptide−DOX conjugates. To further evaluate the cytotoxicity of conjugates on drugsensitive cancer cells, MCF-7/WT cells were employed and exposed to free DOX and peptide−DOX conjugates (Figure 3B). Interestingly, free DOX inhibited the cell proliferation more efficiently than T10-DOX and T15-DOX, with IC50 values as 419.4 ± 20.9 nM for free DOX, 885.0 ± 26.3 nM for T15-DOX, and 1251.4 ± 23.1 nM for T10-DOX. 3.3. Effect of Peptide-Conjugated DOX on Apoptosis and DNA Damage of MCF-7/ADR Cells. Nuclei containing DNA damage were detected by TUNEL assay, and the MCF7/ADR cells were counterstained with DAPI to show the nuclei. Fluorescence microscopy showed that, after 24 h drug G

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Figure 4. Cellular apoptosis of MCF-7/ADR cells detected using TUNEL assay (green). Nuclei were stained with DAPI (blue), and merged images were considered as apoptotic cells.

Figure 5. (A) Fluorescence images of MCF-7/ADR cells after the treatment of free DOX, T10-DOX, and T15-DOX and the measured DOX amounts in (B) the whole cell and (C) nucleus isolated from MCF-7/ADR cells. The amounts were averaged on the number of cells.

4. DISCUSSION In this study, innovative peptide−DOX conjugates T10-DOX and T15-DOX have been successfully synthesized and well characterized. As shown, T10-DOX was a little more stable than T15-DOX. The hydrazone bond in the conjugates was designed for stable covalent attachment under normal circulation and reversible uncoupling in acidic environment. Generally, the pH-dependent release mechanism for hydrazone hydrolysis involves: (1) protonation of the imine nitrogen, (2) nucleophilic attack of a water molecule at the imine carbon, (3) formation of a tetrahedral carbinolamine intermediate, and (4) decomposition of the carbinolamine and cleavage of the C−N

Finally, drug concentration profiles in plasma after the treatment of T10-DOX, T15-DOX, and free DOX were evaluated, and pharmacokinetic parameters of each drug were calculated (Figure 8D). Compared with free DOX, the peptide−DOX conjugates showed a longer half-life t1/2 in plasma (5.21 ± 0.51 h and 5.45 ± 0.49 h vs 3.26 ± 0.45 h) and cleared much more slowly from the body (591 ± 26 mL/h/kg and 550 ± 31 mL/h/kg vs 989 ± 19 mL/h/kg). In addition, the conjugated DOX can be released in plasma, and the drug level can be maintained for more than 20 h. H

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result in different steric hindrance of the imine carbon electrophile toward nucleophilic attack by water, leading to the diversity in stability. Both peptide−DOX conjugates indicated their potential to overcome drug resistance. As described previously, the mechanism of drug resistance is multifactorial and complex. Overexpression of drug efflux pumps and induction of apoptosis pathways are currently the most popular ones. In this study, the increased accumulation of peptide-conjugated DOX in vitro compared to free DOX suggested that the conjugates may bypass the drug efflux. The enhanced distribution in the tumor and reduced accumulation in other organs confirmed the effectiveness of conjugates. This success is associated with their TfR-targeting properties. Overexpression of TfR has been observed on a variety of tumor cells. With TfR-medicated endocytosis, drug-targeted delivery can be achieved. In the previous reports, some Tf modified drugs were proposed. However, the extent of their use was limited probably due to the competition of endogenous Tf. There has been evidence that the peptides we conjugated with DOX have different binding sites on TfR from Tf. The results here also indicated that addition of Tf did not interfere with the endocytosis of conjugates into cells (Figure 5S). On the other hand, endosomes/lysosomes contain an arsenal of hydrolases, including proteases, which are normally enclosed in membrane-bound organelles. However, these proteases can

Figure 6. Western blotting of (A) cathapsin D in cytoplasm and (B) active caspase-3 in cell lysate.

bond.35 Thus, the desired stability for controlled pH-dependent hydrolysis is dependent on a delicate balance of groups adjacent to the carbonyl and hydrazide reaction partners used to form the hydrazone.36 Even though the adjacent chemistries are almost the same for T10-DOX and T15-DOX (i.e., DOX and EMCH) in this study, the inclusive peptide structures may

Figure 7. (A) Tumor volume change, (B) tumor weight on day 21, (C) excised tumor, and (D) body weight change of mice bearing MCF-7/ADR xenografts after the treatment of free DOX, T10-DOX, T15-DOX, and saline. I

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Figure 8. (A) In vivo images of mice at 6 h after administrated with free DOX, T10-DOX, and T15-DOX. The DOX amounts in (B) tumors and (C) other organs. (D) Drug concentration profiles in plasma.

Both T10-DOX and T15-DOX showed markedly improved efficacy (IC50 value of the conjugate was lower than that of free DOX against drug-resistant cancer cells). However, this selectivity seems not retained in drug-sensitive cancer cells as shown earlier. The results can be easily explained by the fact that the cellular uptake of free DOX is through simple diffusion, whereas the uptake of conjugates was mainly through receptormediated endocytosis. The rate of simple diffusion is higher than that of receptor-mediated endocytosis. Therefore, the conjugates may exhibit higher IC50 values than that of free DOX. Thus, peptide−DOX conjugates may have a better efficacy for the treatment of drug-resistant breast cancer. The result of in vitro and in vivo studies suggested that T10DOX may be more effective than T15-DOX, even though T15DOX has a higher intracellular accumulation in MCF-7/ADR cells. T10-DOX has a comparable IC50 value and cytotoxicity with T15-DOX in drug resistance cells, whereas it showed a higher IC50 value in drug-sensitive cells, a greater concentration in tumor tissue samples, and a low accumulation in the organs other than tumor. Higher efficacy and lower side effect were achieved after the treatment of T10-DOX. Previous studies indicated that Tf, HAIYPRH, and THRPPMWSPVWP all bind unique sites on the TfR since they each failed to significantly compete with each other for TfR binding. However, affinity study only demonstrated that THRPPMWSPVWP had high affinity for cells expressing the TfR. To date, no data have been

initiate the intrinsic apoptotic pathway through a sequence of events known as the lysosomal apoptotic pathway if released to the cytosol. Among the possible occurrences, exogenous compounds internalized into these organelles via endocytosis can mediate destabilization of lysosomal membranes.37 For example, a high amount of chloroquine that accumulated in the acidic vesicles can destabilize the endosomes.4 The membrane destabilization facilitated the release of lysosomal proteases, and these proteases can not only trigger lysosomal apoptotic pathway, but also amplify the apoptotic pathways initiated in other cellular compartments.38 By using cathepsin D as an endosomal/lysosomal marker, a greater membrane permealization was demonstrated after the peptide-conjugated DOX treatment. This is a phenomenon first described for peptide− DOX-like conjugates. As reported previously, cathepsins can perform rather specific cleavages of proteins in their native conformation during the brief period of time before they succumb to the more neutral pH conditions of the cytosol. Some of the target molecules in the cytosol (e.g., caspase-3) have been identified, and they are involved in apoptotic signaling.39 In this study, the activation of caspase-3 provided the evidence for this indication. Therefore, both mechanisms (i.e., enhanced drug accumulation and lysosomal pathway of apoptosis) facilitated the drug resistance reversal by peptide− DOX conjugates. J

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(20093234120010), the project sponsored by SRF for ROCS, SEM (39), and the Jiangsu Six-Type Top Talents Program (D) awarded to Y.C. The authors would also like to thank American Journal Experts for proofreading the article.

reported for the affinity of HAIYPRH. The result in this study may provide some evidence that the affinity of HAIYPRH for TfR may be lower than that of THRPPMWSPVWP, whereas its DOX conjugate can lead to a better drug resistance reversal in vivo. The inconsistent accumulation results between the in vitro findings and the in vivo outcomes can be attributed to the fact that in vivo situations are much more complex and thus may lead to varied results. Notably, cardiotoxicity of T10-DOX and T15-DOX was not examined in this study. As widely reported, cumulative doses of free DOX can induce cardiotoxicity, such as widespread myocardial lesions characterized by vacuolization, edema, and myofibrillar and membrane disruption.40 To date, several approaches have been used to reduce the incidence of this toxicity.41 Among them, liposomal encapsulation of DOX such as polyethylene glycol-modified liposomal DOX (PEG liposomal DOX42,43) is significantly less cardiotoxic than free DOX. The slow release of DOX from liposomes is believed to contribute to this observed reduction.42 Recently, the synthesis of PEG-DOXO-EMCH has been described.41 Whether the conjugation of PEG-DOXO-EMCH with peptides can be more effective and safer deserves investigation.

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5. CONCLUSIONS In this study, two peptide−DOX conjugates (i.e., T10-TOX and T15-DOX) were successfully synthesized and comprehensively characterized in a variety of ways. There was evidence indicating that drug resistance reversal was achieved by the enhanced intracellular drug accumulation and the induction of lysosomal pathway of apoptosis. These features were highly probably attributed to the targeting properties of peptide sequences and the acid-sensitive hydrazone linker for ensuring effective release of DOX in cells. However, the safety profile and the pharmacodynamic effect of these conjugates deserve further investigation.

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ASSOCIATED CONTENT

S Supporting Information *

Normalized IOD of cathepsin D, active caspase-3, and internal standard actin in Western blotting. HPLC chromatograms of DOX and DOXO-EMCH. 1H NMR and 13C NMR spectra of DOXO-EMCH. Expanded 13C NMR spectra of T10-DOX and T15-DOX. DOX release profiles of T10-DOX and T15-DOX at pH 5.0 and 6.0. Cellular accumulation of DOX in MCF-7/ ADR cells after the treatment of T10-DOX and T15-DOX with and without Tf for 12 h. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/mp500386y.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-25-86868326. E-mail: [email protected]. Fax: 86-25-86868467. Author Contributions §

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to gratefully acknowledge the National Natural Science Fund (21175071), the Research Fund for the Doctoral Program of Higher Education of China K

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