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A Novel Doxorubicin Prodrug with GRP78 Recognition and Nucleus-Targeting Ability for Safe and Effective Cancer Therapy Guo-Bin Ding, Junqing Sun, Peng Yang, Binchun Li, Ying Gao, and Zhuoyu Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00830 • Publication Date (Web): 05 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017
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Molecular Pharmaceutics
A Novel Doxorubicin Prodrug with GRP78 Recognition and Nucleus-Targeting Ability for Safe and Effective Cancer Therapy Guo-Bin Ding,*,†,‡ Junqing Sun,†,‡ Peng Yang,†,‡ Binchun Li,† Ying Gao§ and Zhuoyu Li*,†,‡,§
†
Institute of Biotechnology, the Key Laboratory of Chemical Biology and Molecular
Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China ‡
Institutes of Biomedical Sciences, Shanxi University, Taiyuan 030006, China
§
School of Life Science, Shanxi University, Taiyuan 030006, China
E-mail:
[email protected] (G.B. Ding);
[email protected] (Z. Li)
Abstract Glucose-regulated protein of 78 kDa (GRP78) has become an attractive and novel target for tumor therapy. Design and construction of powerful delivery systems that could efficiently transport doxorubicin (DOX) to tumor cell nucleus remains a formidable challenge for improving tumor therapeutic index and mitigating side effects to normal tissues. Herein, a novel doxorubicin prodrug (NDP) with GRP78 recognition and nucleus-targeting ability was synthesized by a facile chemical route. NDP exhibited an enhanced antiproliferative activity against colorectal cancer cells and could efficiently enter the cell nucleus. Furthermore, it is inspiring to note that NDP displayed a much stronger inhibitory efficacy against the growth of colorectal cancer xenografts in nude mice than free DOX, and showed superior in vivo safety. Together, the work provides a novel GRP78 and nucleus-targeting strategy and the NDP holds great promise to be used as a potent and safe chemotherapeutic agent.
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KEYWORDS: GRP78, bifunctional peptide, nucleus targeting, novel doxorubicin prodrug, antitumor efficacy
1. INTRODUCTION As a major endoplasmic reticulum (ER) molecular chaperone, GRP78 (glucose-regulated protein of 78 kDa) involves in various cellular activities such as protein folding and assembly, protein quality control, Ca2+ binding and ER stress signalling regulation.1,2 Apart from its location in ER, recent evidence shows that GRP78 is overexpressed on the surface of cancer cells but not normal cells, which renders GRP78 a highly attractive target for targeted tumor therapy.3 Many GRP78-binding peptides have been identified by phage display technique, among which the WIFPWIQL (amino acid sequence) sequence exhibiting the highest efficacy.4 Nevertheless, there is few reports on the design and development of GRP78-targeted drug delivery systems.5 Doxorubicin (DOX) has become one of the most widely used anticancer chemotherapeutic agents for the treatment of a large variety of solid tumors and acute leukemias.6 However, its short half-life, low specificity and the severe systemic toxicity to normal tissues greatly hinder its clinical effectiveness.7,8 Over the past few decades, numerous nanoscale
drug
delivery
systems
including
polymeric
micelles,9–11 liposomes,12
nanoparticles,13,14 microparticle,15 polymer-drug conjugates16 and protein17 were developed for the targeted delivery of DOX to improve its therapeutic efficacy and minimize undesirable side effects.18,19 A special type of polymer-drug conjugates, named prodrug, has been developed and investigated extensively in recent years. Typically, a chemotherapeutic prodrug consists of 2 ACS Paragon Plus Environment
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Molecular Pharmaceutics
three components: i) an antitumor drug that exerts therapeutic effect, ii) a targeting moiety that enhances the selectivity of the prodrug and iii) a polymer carrier or spacer that binds the drug and targeting moieties together and improves the solubility of the prodrug.20 In the past few years, the prodrug strategy has been widely employed in targeted delivery of DOX.21,22 Most of the DOX prodrugs developed to date can only deliver DOX to the cytoplasm rather than nucleus.23,24 However, similar to therapeutic genes and some other first-line anticancer agents (camptothecin and cisplatin), DOX has to localize in the nucleus to elicit its pharmacological effect.23,25 Thus, it is indispensable to design and develop a more efficient DOX prodrug with nucleus-targeting ability. Herein, we report a novel DOX prodrug (NDP) that could recognize tumor cell-surface GRP78 and achieve nucleus-targeted DOX delivery. As depicted in Figure 1(a), the NDP is composed of three elements: (1) DOX, (2) a bifunctional peptide (WIFPWIQLPKKKRKVC) that is constituted by a GRP78 binding sequence WIFPWIQL and a nuclear localization signal (NLS) motif PKKKRKVC,26 and (3) a polymer carrier—heterofunctional poly(ethylene glycol) (Maleimide-PEG-COOH). We choose PEG as a polymer carrier because it shows some attractive characteristics such as non-toxic, highly water-soluble and flexible, and has been approved by FDA for human use.27 When the NDP was intravenously injected into the tumor-bearing mice via tail vein, it could accumulate in tumors via the GRP78-mediated active targeting and EPR effect, and achieve a targeted delivery of DOX to the tumor cell nucleus under the guidance of the bifunctional peptide, as illustrated schematically in Figure 1(b). The in vitro antitumor effect, nucleus-targeting ability and in vivo inhibitory efficacy against tumor growth in DLD1 tumor-bearing mice of NDP were systematically evaluated. 3 ACS Paragon Plus Environment
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Figure 1. Schematic figure illustrating (a) synthesis of novel doxorubicin prodrug (NDP) and (b) in vivo targeted and effective delivery of DOX to the tumor cell nucleus.
2. 2. EXPERIMENTAL SECTION 2.1. Materials Heterofunctional
poly(ethylene
glycol)
(PEG),
maleimide
PEG
carboxyl
(Mal-PEG-COOH, Mn=3.5 kDa) was obtained from JenKem technology Co. Ltd. (Beijing, China). Doxorubicin hydrochloride (DOX·HCl, >98%) was purchased from Dalian Meilun Biological
Technology
Co.
Ltd.
(Dalian,
China).
The
bifunctional
peptide
(WIFPWIQLPKKKRKVC, ≥98%) was synthesized by Bankpeptide Biological Technology Co.
Ltd.
(Hefei,
China).
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) were provided by Suzhou
Highfine
Biotech
Co.
3-(4.5-dimethyl-thiazol-2-yl)-2.5-diphenyltetrazolium
Ltd. bromide
(Suzhou, (MTT)
China). and
4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich. All other organic solvents used were of analytical grade. 4 ACS Paragon Plus Environment
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Molecular Pharmaceutics
2.2. Characterization Gel permeation chromatography (GPC) measurements were performed on a Waters 410 GPC using tetrahydrofuran (THF) as eluent with a flow rate of 1.0 mL/min. The molecular weights were calibrated with polystyrene (PS) standards. Fourier transform infrared (FTIR) spectral studies were carried out on a Thermo Scientific Nicolet iS50 spectrometer with a resolution of 0.5 cm-1. 1H NMR spectra were recorded on a Bruker Avance III 600 MHz NMR spectrometer at room temperature with tetramethylsilane (TMS) as the internal reference and DMSO-d6 as the solvent. The intracellular distribution behavior of DOX and NDP was observed by an Olympus FV1000-IX81 confocal laser scanning microscope. 2.3. Synthesis of NDP Mal-PEG-COOH (116 mg, 0.033 mM) and HATU (38 mg, 0.1 mM) were dissolved in anhydrous N,N-dimethylformamide (DMF) (1 mL) and stirred for 2 h, then DOX·HCl (29 mg, 0.05 mM) and DIPEA (52 µL, 0.3 mM) was added to the solution. After magnetic stirring for 42 h, the mixture was transferred to a dialysis bag (Mw cutoff: 3500 Da) and dialyzed against deionized water for 48 h in the dark to remove DMF and the unreacted DOX, then the Mal-PEG-DOX solution was lyophilized. Next, Mal-PEG-DOX (105 mg, 0.026 mM) and bifunctional peptide (80 mg, 0.039 mM) were dissolved in 1 mL water in a brown glass vial, and stirred for 16 h followed by dialysis (Mw cutoff: 3500 Da) against deionized water for 24 h to remove the free bifunctional peptide. Finally, the mixture in the dialysis bag was freeze-dried to give red powder. To determine the content of DOX in NDP, the freeze-dried NDP was redissolved in water and the absorbance of DOX at 480 nm was measured to calculate drug content in the solution using a previously established calibration curve. 5 ACS Paragon Plus Environment
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2.4. In Vitro Antitumor Activity of NDP
In vitro cytotoxicity of NDP was evaluated via an MTT assay in two human colon cancer cell lines (DLD1 and SW620) and one normal human colon epithelial cell line (FHC), and was compared with that of free DOX. Cells were seeded into 96-well plates at a density of 5×103 cells per well and cultured in RPMI-1640 or F-12 medium supplemented with 10% FBS at 37 °C in a humidified incubator containing 5% CO2 for 24 h. Then, free DOX or NDP with various DOX concentrations (0.01, 0.1, 1, 10 and 100 µM) were added, and five parallel wells were used for each sample at a specific concentration. After incubation for 24 or 48 h, 20 µL of MTT solution (5 mg/mL in PBS) was added to each well and further incubated for 4 h. Finally, 150 µL DMSO was added to each well to dissolve the formazan precipitate and cell viability was determined on a microplate reader at 570 nm. 2.5. Nucleus-Targeting Ability of NDP DLD1 cells were seeded in 24-well plates (a sterile cover slip was placed in each well) and allowed to adhere for 24 h. Free DOX or NDP dissolved in cell culture medium were added to the wells at an equivalent DOX concentration of 10 µM. After co-incubation for 12, 24 or 48 h, the supernatant was carefully removed, cells were washed three times with cold PBS and fixed with 4% formaldehyde at 4 °C for 30 min. Then cells were stained with DAPI for 5 min and washed three times with PBS. The slides were mounted in gelvatol and subjected to confocal fluorescence analysis. 2.6. Animals and Tumor Model Four-week old male BALB/c mice were purchased from China Institute for Radiation Protection, and maintained under an aseptic environment with free access to enough sterile 6 ACS Paragon Plus Environment
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Molecular Pharmaceutics
diet and water throughout the experiments. All experiments were conducted in accordance with guidelines set by national regulations for the care and use of laboratory animals, and were approved by the Institutional Animal Care and Use Committee of Shanxi University. A colorectal cancer xenograft tumor model was established by subcutaneous injection of DLD1 cells (about 5×106 cells suspended in 100 µL saline) into armpits of right anterior limbs of the nude mice. Tumor size was monitored with a digital caliper and calculated according to the formula: V (mm3)=(L×W2)/2, where L and W (mm) were the largest and smallest diameters of the tumor, respectively. 2.7. In Vivo Antitumor Efficacy The tumor-bearing mice were randomly divided into three groups (n=6): control, free DOX and NDP in a way that each group has an average tumor size of about 110 mm3. And treatment was initiated on the same day, taken as day 1. Free DOX and NDP were dissolved in saline and were administered via tail vein injection at an equivalent doxorubicin dose of 5 mg/kg for four times (at day 1, 7, 14 and 21). The control group received saline (5 mL/kg) only. Tumor volume and body weight were recorded every three days for a period of 28 days. The tumor growth inhibition ratio was calculated with the equation: (Vc-Vt)/Vc×100%, where Vc was the average tumor volume of the control group, while Vt was the average tumor volume of treated group.28 2.8. Tissue Damage Assay Seven days after the last treatment, all mice were euthanized. The blood of each mouse was obtained, centrifuged at 3000 rpm for 5 min and the supernatant serum was collected. The blood biochemistry indicators including cardiac function parameters creatine kinase (CK), 7 ACS Paragon Plus Environment
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creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) level; liver-associated alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and renal function parameters blood urea nitrogen (BUN) and creatinine (Cr) levels were determined by the corresponding commercial ELISA kits (Beijing Leadman Biochemistry Co., Ltd, Beijing, China) according to the standard protocols provided by the suppliers. 2.9. Histopathological Analysis After blood sampling, the tumor, heart, liver and kidney of the mice were isolated. These excised tissues were fixed with freshly prepared 4% (V/V) formaldehyde in PBS (pH 7.4) for 24 h and embedded in paraffin. The paraffin-embedded tumor and organ tissues were sectioned into 5 µm slices and stained with hematoxylin and eosin (H&E). The histological changes were observed by a digital microscope (Nikon Eclipse Ti). 2.10. Immunohistochemistry Study Tumor tissues dissected from 3 groups of mice were subjected to immunohistochemistry to detect the expression of Ki-67, Caspase-3 and BCL-2. Tumor sections were incubated with primary antibodies against Ki-67, Caspase-3 and BCL-2, washed three times, incubated with horseradish peroxidase-conjugated anti-rabbit IgG at 37 °C and washed with PBS for three times. Then the sections were incubated with peroxidase substrate, washed twice with PBS, mounted, and analyzed with a digital microscope (Nikon Eclipse Ti). Five fields in each slide were selected randomly and observed at a magnification of × 200. 2.11. Statistical Analysis All data are expressed as the mean±SD (standard deviations). Statistical significance between two groups was conducted by using a two-tailed Student’s t-test. The differences 8 ACS Paragon Plus Environment
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Molecular Pharmaceutics
were considered statistically significant when P
