DUP1 peptide modified micelle efficiently targeted delivery paclitaxel and enhance mitochondrial apoptosis on PSMA-negative prostate cancer cells
- Haining Chen†1,
- Fengbo Wu†1,
- Jing Li†1,
- Xuehua Jiang3,
- Lulu Cai1, 2Email author and
- Xiang Li1Email author
© Chen et al. 2016
Received: 7 December 2015
Accepted: 10 March 2016
Published: 22 March 2016
Prostate tumor cell targeted peptide fragment conjugated to the nano drug delivery system is a promising strategy for prostate cancer therapy. In this work, an amphiphilic copolymer Chol–PEG–DUP1 (PEG–cholesterol conjugated with DUP1 peptide) has been synthesized and characterized by proton nuclear magnetic resonance spectrum (1H NMR). The paclitaxel (PTX) was encapsulated into the Chol–PEG–DUP1 micelles to obtain aqueous formulation with small particle size (within 200 nm) and high drug encapsulating efficiency. The DUP1 modified PTX micelle significantly enhanced the cytotoxicity of paclitaxel to PSMA negative prostate tumor cells (PC-3 cell) as demonstrated by MTT (IC50 = 15.8 μg/mL compared to 68.7 μg/mL of free PTX). Flow cytometry analysis and fluorescence images revealed the DUP1 peptide fragments on the surface of micelles increased drug uptake (2.08-fold) by PC-3 cells. Flow cytometry and immunoblotting analysis showed the DUP1 modified PTX micelle enhanced the mitochondrial apoptosis-inducing capacity of PTX to PC-3 cells. In conclusion, Chol–PEG–DUP1 modified micelle was a reasonable, facile, and economic drug delivery system to target the PSMA-negative prostate cancer.
KeywordsDrug delivery Amphiphilic polymer Paclitaxel PSMA-negative Micelle
Nowadays, prostate cancer is one of the main lethal cause from cancer patients worldwide, and more than one-third of newly diagnosed male cancer in Europe and USA was prostate cancer (Crawford 2003; Parkin et al. 2001). Despite the hormone therapy was effective in early stage, many patients with metastatic potential eventually progress to an androgen-resistant state (Sandblom and Varenhorst 2001; Zitzmann et al. 2005). Although there were several treatment methods applied in the clinic, but none showed a survival benefit in hormone independent prostate cancer patients (Sternberg 2003).
Prostate-specific membrane antigen (PSMA) as a member of trans-membrane folate hydrolase family, which could enhanced the expression level in prostate cancer tissue other than benign or neoplastic epithelial prostate cells (Bostwick et al. 1998; Ross et al. 2003). A weak extra prostatic expression of PSMA has been reported in some other tissues (Renneberg et al. 1999; Silver et al. 1997) Therefore, the Hu591 monoclonal antibody(mAb) targeting the PSMA extracellular domain, has been applied to the prostate cancer therapy (Liu et al. 1997; Nanus et al. 2003). An 111In-labeled monoclonal antibody (Capromab pendetide, ProstaScint) could targeted to PSMA and used for imaging lymph node metastases. Recently, Zitzmann et al., have reported a novel peptide DUP1 with specificity for PSMA-negative prostate tumor cell lines, such as DU-145 and PC-3, which was identified by phage display techniques.
Peptide modified polymeric micelles have been investigated extensively, and these works are quite extraordinary, impressive, and laid a solid foundation for our study. In recent years, Torchilin et al., have reported that the monoclonal antibody (mAb)-modified PEG-PE micelles could recognize and bind to numerous tumor cells but not normal cells in vitro (Torchilin et al. 2003). Several researchers have reported that the synergetically therapeutic efficacy of chemotherapy by polymer–peptide or drug–peptide conjugates, which leads to amplifying apoptosis induction activity in the tumor or enhancing tumor targeting (Liu et al. 2008; Dharap et al. 2005). In our previously studies, we have reported the fibroblast growth factor (FGF) fragment peptide modified micelles could significantly enhanced the cytotoxicity of paclitaxel for murine lewis lung cancer (LLC) cells, which were further confirmed by some subsequent experiments in vitro (Cai et al. 2011). These antibodies or peptides conjugated micelles could enhanced the specific uptake of drugs and/or genes by targeted cells actively. To our knowledge, nanotechnology is widely applied to prepare a novel nano-formulation of hydrophobic drugs for enhancing cancer therapy efficiency (Gong et al. 2013). By encapsulating into nanoscale vectors, hydrophobic drugs could form stably disperse morphology in water (Wagner et al. 2006; Jain and Stylianopoulos 2010; Li et al. 2012). Moreover, amphiphilic polymeric micelles have widely applied in drug delivery system (DDS) for anti-tumor agents, in which the hydrophilic shell helps the escape from the clearance by RES (reticuloendothelial system) and the hydrophobic core wraps drug via hydrophobic interactions or hydrogen bonds (Gong et al. 2010, 2011; He et al. 2012; Li et al. 2012, 2013; Wu et al. 2012; Ma et al. 2013; Zhang et al. 2014; Zeng et al. 2014). Furthermore, their nano-size will improve the anti-tumor effects through the enhanced permeability and retention (EPR) effect (Iyer et al. 2006; Muggia 1999; He et al. 2013).
Materials and cell lines
Cholesterol (Chol) monomethoxy poly(ethylene glycol) (MW 2000, mPEG2000) and poly(ethylene glycol) (MW 2000, PEG2000), was obtained from BoAo Biological Technology (Shanghai, China). The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenltetrazolium bromide (MTT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 4-dimethylaminopyridine (DMAP) 1, 8-Diazabicyclo(5.4.0)undec-7-ene (DBU), Coumarin-6, fmoc–l-phenylalanine (fmoc–phe), and succinic anhydride (suc) were Obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-Maleimidopropionic acid N-succinimidyl ester (BMPS) was purchased from Jiaxing Biomatix Co. Ltd. (Jiaxing, China). Soya phosphatidylcholine (SPC) was from Lucas Meyer (Hamburg, Germany). Paclitaxel (PTX) was purchased from Energy Chem. Co. Ltd. (Shanghai, China).
The DUP-1 peptide (CFRPNRAQDYNTN) was synthesised by standard solid-phase peptide synthesis method using Fmoc chemistry. The DUP1 peptide was purified by preperative high-pressure liquid chromatography on a Novasep LC50, C18-ODS-5 μm, 250 × 50 mm column (Novasep, Pompey, France) using water, acetonitrile and methanol as eluent solvent. All other solvents and reagents were of chemical grade and used without other purification. The ultrapure water was prepared from Milli-Q water system without specification.
PC-3 cell was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). PC-3 cells grew in Roswell Park Memorial Institute 1640 medium (RPMI 1640, Gibco, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco, USA). All cells were maintained at 37 °C in humidified incubator containing 5 % CO2.
Synthesis of cholesterol–poly(ethylene glycol)–DUP1 peptides copolymers
In general, the DMSO solution of Chol–PEG–Phe-NH2 (2.0 g, 0.76 mmol), and BMPS (0.24 g, 0.92 mmol) was stirred 24 h at room temperature to give a 3-maleimidopropionic acid modification on the PEG chain. Then the DUP-1 peptide (CFRPNRAQDYNTN, 1.51 g, 0.92 mmol) was added and continuous stirred 12–24 h. After the reaction was completed, the solution was dialyzed to remove un-reacted peptides (membrane tubing, molecular weight cut off = 1.0 KD). The dialyzed product was lyophilized and the final products Chol–PEG–DUP1 (1.42 g, 47.1 %) was obtained.
Preparation and characterization of Chol–PEG–DUP1–M-PTX
The paclitaxel loaded Chol–PEG–DUP1 micelles (M-PTX) were prepared as following: PTX and copolymer (1:19, w/w) were dissolved in ethanol, and the solution was evaporated in rotary evaporator for 20 min to obtain the co-evaporation. Then, normal saline (NS) was added, and co-evaporation was dissolved to self-assemble into Chol–PEG–DUP1–M-PTX. The obtained Chol–PEG–DUP1–M-PTX was first filtered using a 0.22 μm Millex-LG filter (Millipore Co., Waltham, MA, USA), and then lyophilized into powder form before use.
The Malvern Nano-ZS 90 laser particle size analyzer (Malvern, Worcestershire, UK.) was utilized to the determination of particle size distribution and zeta potential of Chol–PEG–DUP1–M-PTX. All the results were tested in three different samples, and data were expressed as the mean ± standard deviation (SD). The morphological characteristic of Chol–PEG–DUP1–M-PTX was further detected by transmission electron microscope (TEM, H-6009IV, Hitachi, Tokyo, Japan). The Chol–PEG–DUP1–M-PTX sample was negatively staining by phosphotungstic acid before TEM test.
In vitro cytotoxicity
To investigate cytotoxicity of Chol–PEG–DUP1–M-PTX and free PTX, MTT assays were preformed on PC-3 cells. PC-3 cells cultured in 96-well plates were treated with a series of Chol–PEG–DUP1–M-PTX or free PTX for 48 h, respectively. The mean percentage of cell survival relative to that of control cells was determined from data of three individual experiments, and all the data were expressed as mean ± SD.
Apoptosis induction assay of Chol–PEG–DUP1–M-PTX and free PTX were studied on PC-3 cells. PC-3 cells were plated in 6-well plates and grown for 24 h. The cells were exposed to media containing 20 ng/mL of Chol–PEG–DUP1–M-PTX and free PTX for 48 h, respectively. Then, the cells were fixed with pre-chilled 70 % ethanol for 30 min and stained with 0.5 mL of PI (5 μg/mL in PBS) for 10 min. Apoptotic cells were observed under fluorescence microscopy (TE2000-U, Nikon, Tokyo, Japan), which demonstrated cytoplasmic and nuclear shrinkage and chromatin condensation.
Furthermore, flow cytometric (FCM) assay was used to confirm the apoptotic induction effect of Chol–PEG–DUP1–M-PTX. Apoptosis of PC-3 cells treated with PTX -M, free PTX, or blank micelles was determined using FITC-conjugated AnnexinV/PI (BD PharMingen, San Diego, CA, USA) staining by FCM (BD FACS Calibur, BD, San Jose, CA, USA). Both early apoptotic (Annexin V+/PI−) and late apoptotic (Annexin V+/PI+) cells were included in cell apoptosis determinations.
In vitro drug release
The release profiles of PTX from DUP1-modified micelles or free PTX were investigated by the dialysis method. Briefly, 1 mL of free PTX solution or drug loaded micelles were placed into dialysis bags (molecular weight cut off = 3500), then incubated at 37 °C with gentle shaking (100 rpm) in 50 mL of phosphate buffered solution (PBS) (PH 7.4 or PH 5.5, 0.01 M) containing Tween80 (0.5 wt%). After given time intervals, dialysis medium was withdrawn and replaced with the same volume of fresh buffer. The cumulative amount of released PTX were analyzed and quantified by HPLC. All the results were the mean value of three test runs and all data were shown as the mean ± SD.
Cellular uptake of micelles by flow cytometry analysis
In general, the PC-3 cells suspension (6 × 104 cells/well in 1.5 mL) were incubated at 37 °C for 24 h in six-well plates (Corning, NY, USA). Then conventional micelles or DUP1-modified micelles loading the same amount of coumarine-6 (the final concentrations were about 40 ng/mL) were added into each well, respectively. Then the six-well plates were further incubated at 37 °C for 1 h, the culture medium was discarded, the plates were digested with trypsin and the re-suspended cells were washed with cold PBS twice. Finally, each sample was examined by a flow cytometer (EPICS Elite ESP, Beckman Coulter, Brea, CA, USA). The fluoresces of intracellular coumarin-6 was excited at 488 nm with an argon laser, and the emission fluorescence was detected at 525 nm. Files were collected of 10,000 gated events.
The cellular total proteins were extracted using RIPA buffer (SolarBio, Beijing, China) containing 1 % (v/v) PMSF (SolarBio), 0.3 % (v/v) protease inhibitor (Sigma, St. Louis, MO, USA) and 0.1 % (v/v) phosphorylated proteinase inhibitor (Sigma). Celluar lysates were centrifuged at 13,000 rpm at 4 °C for 10 min, the supernatant was collected. The protein concentration was determined using BCA protein assay kit (Pierce, Waltham, MA, USA). The total protein was separated on SDS-PAGE gel and transferred onto a PVDF membrane. Non-specific interactions were blocked using skimmed milk for 2 h at room temperature. The PVDF membranes were incubated with the primary antibodies overnight at 4 °C. After washed several times, the PVDF membranes were incubated in HRP-conjugated goat anti-rabbit and anti-mouse IgG or HRP-conjugated mouse anti-goat IgG (Abmart, Shanghai, China, all at a 1:5000 dilution) for 4–8 h at room temperature. The target proteins were visualized using enhanced chemiluminescence (Millipore, Billerica, MA, USA) according to the manufacturer’s recommendations.
Results and discussion
Synthesis and characterization of paclitaxel loaded micelles with or without DUP1 modification
In vitro drug release profile
Cell uptake and binding study
In vitro cytotoxicity evaluation
In vitro apoptosis induction effect
In conclusion, targeting peptide modified PEG–Chol polymeric micelles, the surfaces of which are conjugated with DUP1 peptide, have been prepared for PSMA negative PCa targeted drug delivery. The reported Chol–PEG–DUP1–M-PTX bearing both small particle size and high encapsulating efficiency. In vitro experiments suggested that Chol–PEG–DUP1–M-PTX had prior cytotoxicity than free drug in the cell proliferation MTT assays, and could inducing more apoptosis. Cellular flow cytometry results and fluorescence spectroscopy images suggest the surface DUP1 modification of the micelles promote the selective uptake by PSMA negative PC-3 cells. Although further in vivo antitumor investigation of Chol–PEG–DUP1–M-PTX is required, the results of our current study represent a meaningful explore in advancing the use of DUP1 peptide modified micelles as a potent strategy to treat PSMA negative prostate cancer. Thus, Chol–PEG–DUP1–M-PTX may serve as a promising candidate for prostate cancer therapy.
HC contributed to the whole process of the synthesis, characterization, and in vitro evaluation of the drug loaded micelle, and the preparation of the first edition of the manuscript. LC contributed to in vitro evaluation of the drug loaded micelle, related cell culture work, interpretation of the results, and manuscript editing. FW and XL supervised the whole procedure and also contributed to the data interpretation and revision of the manuscript. XJ contributed to data collection and discussion. All authors read and approved the final manuscript.
This work was financially supported by the National Natural Science Foundation of China (81402500), China Postdoctoral Science Foundation Funded Project (2014M560720), and Support Foundation of Science and Technology Department of Sichuan Province (2014FZ0039).
The authors declare that they have no competing interests.
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