Drug Delivery, 2010; 17(4): 255–262
ORIGINAL ARTICLE
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Polymeric micelles as a drug delivery system enhance cytotoxicity of vinorelbine through more intercellular accumulation Xiaoyan Lu1,2, Fayun Zhang1, Lei Qin1, Fengying Xiao1, and Wei Liang1 Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, PR China, and Graduate School of the Chinese Academy of Science, 19A Yuquan Road, Beijing 100080, PR China
1 2
Abstract Polymeric micelles had been used as an efficacious carrier system for anti-cancer drug delivery. However, it is not clear whether the molecular mechanism of drug encapsulated in micelles is same as free drug. In this study, the mechanism of vinorelbine loaded in glycol-phosphatidylethanolamine (PEG-PE) micelles (M-Vino) on tumor cells was investigated. Compared with free vinorelbine (Free Vino), M-Vino was more effective in inhibiting the growth of tumor cells in vitro, inducing G2/M phase arrest and apoptosis of tumor cells. M-Vino showed a faster entry and higher accumulation in 4T1 cells than free vinorelbine. Therefore, M-Vino destabilized microtubules, induced cell death, and enhanced its cytotoxicity through more intercellular accumulation of vinorelbine. Keywords: Polymeric micelles; vinorelbine; cell cytotoxicity; apoptosis; drug accumulation
Introduction Vinca alkaloid-based drugs are established in the treatment of cancer, including ovarian, breast, and lung cancer (Ngan et al., 2001; Morris & Fornier, 2008). Vinorelbine, a most commonly used semi-synthetic Vinca alkaloid, destabilizes microtubules by binding to the domain in the β-tubulin sub-unit (Rai & Wolff, 1996; Ngan et al., 2001). Low concentrations of Vinca alkaloid suppress the dynamicity of microtubules, whereas high concentrations lead to a complete disassembly of the microtubule network (Jordan et al., 1985; 1991; Binet et al., 1990; Dhamodharan et al., 1995). Consequently, they inhibit chromosome congression and cell mitosis (Jordan & Wilson, 1998). Common problems associated with the clinical use of vinorelbine are the treatmentinduced systemic toxicity and resistance of tumor cells (Krikorian & Breillout, 1991; Pastan & Gottesman, 1991; Abraham et al., 2009). One of the strategies is the encapsulation of the drug in nanoparticles (Vijayaraghavalu et al., 2007). Recent studies have shown that polymeric micelles are used as an effective chemotherapeutic drug
delivery system (Gou et al., 2009a; b; Wei et al., 2009a; b) Previously, we developed a method to encapsulate doxorubicin and vincristine in PEG-PE micelles with superior efficiency. These drugs encapsulated in PEG-PE micelles have an enhanced cytotoxicity toward tumor cells, with the IC50 being 3–5-fold lower than those of the free drugs (Ling et al., 2006; Tang et al., 2007). However, it is not clear whether the molecular mechanism of drug encapsulated in micelles is same as free drug. In this study, we showed that M-Vino enhanced vinorelbine-induced apoptosis, which involved microtubule network disrupting, G2/M phase arresting, and caspase-3 activating, suggesting an amplified signaling of cell death pathway.
Materials and methods Tumor cell lines and chemicals Cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA). The mouse mammary
Address for Correspondence: Wei Liang, 15 Datun Road, Chaoyang District, Beijing, 100101, PR China. Tel: 86-10-64889861. Fax: 86-10-64845388. E-mail:
[email protected] (Received 09 December 2009; revised 30 January 2010; accepted 01 February 2010) ISSN 1071-7544 print/ISSN 1521-0464 online © 2010 Informa UK Ltd DOI: 10.3109/10717541003702769
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256 Xiaoyan Lu et al. carcinoma cell line 4T1 was cultured in RPMI 1640 medium. Media were supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). All cells were maintained at 37°C in a 5% CO2, 95% humidity incubator. Vinorelbine (vinorelbine tartrate) was provided by Minsheng Corp (Hangzhou, China). PEG-PE was purchased from Avanti Polar Lipids (Alabaster, AL).
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Preparation of micelles encapsulating vinorelbine M-Vino was prepared by vinorelbine incorporated into PEG-PE micelles. In brief, vinorelbine was dissolved and PEG-PE was suspended in double-distilled water at concentrations of 4 mg/mL and 40 mg/mL, respectively. Equal volumes of both solutions were mixed at room temperature. Mannitol was added to this mixture and dissolved at a concentration of 5% (w/v), and then incubated at 50°C for 30 min. The mixture was extruded through a membrane with a pore size of 200 nm, lyophilized, and stored at 4°C. Before use, M-Vino was reconstituted with water, and the final concentration of vinorelbine in micelles was 2 mg/mL, as confirmed using high-performance liquid chromatography (HPLC). The HPLC system used was an LC-10ATvp SHIMADZU UOUID chromatograph equipped with an ASPD-10Avp SHIMADZU UV-VIS detector. The intra-day coefficients of variation (CVs) and inter-day CVs were 1.39% and 0.61%, respectively. The limits of detection and quantification are 1.0 ng/ μL and 3.0 ng/ μL. The injection volume was 20 μL and the column temperature was maintained at 40°C. The concentration of vinorelbine in micelles was quantified by calculating the peak area against the standard sample (r2 = 0.9999). Degraded products of vinorelbine incorporated in micelles were detected using HPLC after defined periods of storage. Morphology and size of M-Vino The morphology and size of micelles encapsulating vinorelbine were examined using transmission electron microscopy (TEM) and dynamic light scattering. M-Vino was reconstituted and diluted to a concentration of 2 mg/mL PEG-PE with deionized water. The morphology of micelles was examined with a JEOL 100 CX electron microscope by stained with 1% uranyl acetate (JEOL USA, Inc, Peabody, MA). The size of the micelles was determind with a Zetasizer 5000 (Malvern Instruments, Malvern, Worcestershire, UK). In vitro cytotoxicity assay Cells (2 × 103 4T1 per well) were plated in 96-well plates with 100 µL RPMI 1640 medium and grown for 24 h. The cells were then exposed to a series of concentrations of free vinorelbine or M-Vino for 72 h, and the
viability of cells was measured using the methylthiazoletetrazolium (MTT) method, as previously described (Mosmann, 1983). Cellular tubulin staining 4T1 cells treated with 10 nM and 100 nM of M-Vino or free vinorelbine for 24 h were washed with PBS and fixed in 4% paraformaldehyde. The cells were permeabilized with 0.2% Triton-X 100 and blocked with 1% bovine serum albumin in HBSS for 1 h at room temperature, and then incubated with a mouse monoclonal anti-α-tubulin antibody (Invitrogen) at a concentration of 1 μg/mL. After washing with HBSS, goat anti-mouse immunoglobulin G (H + L) conjugated with Alexa Fluor 488 (Invitrogen) was added to the blocking solution at a final concentration of 2 μg/mL. Hoechst 333342 (Sigma) (10 μM) was then added for 30 min to visualize nuclei after washing with HBSS (Wu-Wong et al., 2001; Kuo et al., 2004). Cell cycle and apoptosis assay 4T1 cells treated with 10 nM free vinorelbine or M-Vino were washed with PBS and fixed in 70% ethanol. Their DNA was stained with a solution containing 50 μg/mL of propidium iodide (Sigma) and 2 μg/mL of DNasefree RNase (Roche). The cells were analyzed for cell cycle using a FACS Calibur (Becton Dickinson). The MultiCycle software (Phoenix Flow Systems, San Diego, CA) or CellQuest program was used to quantitate the distribution of cells in each cell cycle phase. Induction of apoptosis was determined by treating 4T1 cells with various concentrations of free vinorelbine or M-Vino for 48 h. The treated cells were incubated with Annexin V-FITC and propidium iodide (PI) for 15 min in the dark, immediately followed by flow cytometry (Pourroy et al., 2004). Caspase-3 activity assay 4T1 cells were treated with 10 nM or 1000 nM free vinorelbine or M-Vino for 24 h. The caspase-3 activity was measured using the CaspACE Assay System Fluorometric Kit (Promega Corporation, Madison, WI), according to the manufacturer’s instructions. Western blot analysis The drug treated cells were washed with PBS, and then lysed with RIPA lysis and extraction buffer (Pierce Biotech, USA). The denatured lysates were separated on a 12% polyacrylamide gel. Protein was transferred onto a polyvinylidene difluoride membrane. The membrane was blocked using 5% non-fat milk and probed for 2 h at room temperature with primary antibodies for Cdc2,
Polymeric micelles as a drug delivery system enhance cytotoxicity of vinorelbine 257
Results and discussion
Bid, Bax, β-actin (Santa Cruz Biotechnology), cyclin B1, p-Cdc2 (Tyr15), and p-Cdc2 (Thr161) (Cell Signal). After that, the membranes were washed and probed with a 1:2000 dilution of peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), and then detected by enhanced chemiluminescence (Amersham Life Sciences, Amersham, UK).
Preparation and characterization of M-Vino The approach for preparing PEG-PE micelles containing vinorelbine was much simpler than that we reported previously (Tang et al., 2007). However, the micelles used in this study remained high encapsulation efficiency (99.8%). We characterized the morphology and size of M-Vino by TEM and Zetasizer 5000, respectively. TEM showed that both the empty micelles and M-Vino had acquired a spherical structure (Figures 1A and B). The average diameter of M-Vino was 17.2 ± 0.27 nm (Figure 1D). PEG-PE micelles encapsulating vionrelbine also demonstrated high integrity as observed by TEM after storage for 6 months at 4°C (Figure 1C).
Vinblastine-BODIPY (Free Vinb, Invitrogen)encapsulated PEG-PE micelles (M-Vinb) were prepared according to a previously described method (Tang et al., 2007). 4T1 cells treated with 0.2 µM M-Vinb or Free Vinb for a specific time were collected for quantization of vinblastine-BODIPY-derived fluorescence by a flow cytometer with excitation/emission: 488/530 nm. Also, 4T1 cells treated with various concentrations M-Vinb or Free Vinb for 30 min were collected for quantitatively measuring vinblastine-BODIPY-derived fluorescence. Cells were treated with 0.3 µM M-Vinb or Free Vinb for 30 min, and then incubated with 60 nM LysoTracker Red DND-26 (Invitrogen) and 10 µM Hoechst 333342 for 30 min to visualize lysosomes and nuclei using confocal microscopy, respectively. The excitation/emission wavelengths were 543/590 nm for LysoTracker and 488/525 nm for vinblastine-BODIPY.
Enhancement of the cytotoxicity of vinorelbine by encapsulation in micelles We compared cytotoxic activity of encapsulated vinorelbine with free vinorelbine. 4T1 cells were exposed to a series of equivalent concentrations of free vinorelbine or M-Vino for 72 h, and the percentage of viable cells was quantified. The concentration of M-Vino that caused 50% killing was much lower than that of free vinorelbine (mean = 14.20 ± 0.26 vs 25.65 ± 0.96 nM, p
