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Poly(L-lactide)-Vitamin E TPGS Nanoparticles Enhanced the Cytotoxicity of Doxorubicin in Drug-Resistant MCF-7 Breast Cancer Cells Po-Yu Li, Ping-Shan Lai,* Wen-Chou Hung, and Wei-Jhe Syu Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan Received May 12, 2010; Revised Manuscript Received July 12, 2010
Multiple drug resistance (MDR) seriously reduces the efficacy of many chemotherapeutic agents for cancer. P-Glycoprotein, an efflux pump overexpressed on the cell surface, plays an important role in drug resistance, but several surfactants, such as vitamin E TPGS, can inhibit P-glycoprotein. In this study, a polylactide-surfactant block copolymer poly(L-lactide)-vitamin E TPGS (PLA-TPGS) was synthesized using bidentate sulfonamide zinc ethyl complex as an efficient catalyst, and its self-assembled nanoparticles were used as carriers of doxorubicin. We first found that the activity of P-glycoprotein in drug-resistant breast cancer MCF-7/ADR cells was decreased after incubation with PLA-TPGS nanoparticles. In addition, the nuclear accumulation and cytotoxicity of doxorubicin were significantly increased by encapsulation into the nanoparticles. The enhanced efficacy of the doxorubicinloaded PLA-TPGS nanoparticles may result from the combination of inhibition of efflux and increased entry of doxorubicin into the nucleus in drug-resistant MCF-7/ADR cells. Therefore, this innovative delivery system has potential to act as a nanomedicine for therapy of both drug-sensitive and drug-resistant cancer.
Introduction Multiple drug resistance (MDR) is a major clinical problem that seriously reduces the efficacy of many chemotherapeutic agents. The most extensively studied mechanism of MDR is the overexpression of ABC-transporter P-glycoprotein (P-gp) as cell surface efflux pumps that can successfully purge a wide spectrum of chemotherapeutic agents from cells and thereby decreasing their intracellular accumulation in drug-resistant cells.1-3 Inhibition of ABC-transporters to reverse MDR in cancer cells has been studied extensively.4 Besides small molecule inhibitors, surfactants such as Tween 80, Cremophor EL, several Pluronics, and D-R-tocopheryl polyethylene glycol 1000 succinate (TPGS, a water-soluble succinate ester of vitamin E) are known to modulate efflux pump activity via possible mechanisms that include competitive inhibition of substrate binding, alteration of membrane fluidity, and inhibition of efflux pump ATPase activity.5,6 Recently, the paclitaxel nanocrystals using TPGS as a sole excipient have been demonstrated that can overcome the drug-resistance in human ovarian NCI/ADRRES cancer cells.7 Moreover, the use of surfactant-capped nanoparticle-based drug delivery may provide a new strategy to overcome the MDR phenotype.8 Polymer-based nanotechnology drug delivery systems can deliver their payloads selectively at the target sites with improved longevity in the circulation, thereby enhancing the therapeutic effect of the drugs.9,10 In addition to their activity as inert carriers, these polymeric nanomaterials can act as biological response modifiers.11 Pluronic block copolymers cause various functional alterations in cells as they are incorporated into cellular membranes and subsequently translocated into the cells.11 These block copolymers were thought to sensitize multidrug resistant tumors that were refractory to anthracyclines and other chemotherapeutic agents. A form of doxorubicin encapsulated into mixed micelles of pluronic block * To whom correspondence should be addressed. Tel.: +886-4-22840411, ext. 428. Fax: +886-4-22862547. E-mail:
[email protected].
copolymers, SP1049C, has shown promise in a phase II study in patients with advanced esophageal carcinoma.12 TPGS is an amphiphilic compound consisting of a lipophilic alkyl tail and hydrophilic polar head portion that can be an excellent emulsifier and can also greatly improve drug encapsulation efficiency because of its bulky structure and large surface area.13 In addition, TPGS can inhibit P-gp-mediated drug efflux5 and increase the oral bioavailability of anticancer drugs.14 It has been reported that the mechanism of inhibition of the cellular efflux pumps by TPGS involves inhibition of ATPase rather than a competitive inhibition or a nonspecific effect on the fluidity of the cell membrane.15 Recently, a poly(L-lactide)TPGS (PLA-TPGS) copolymer has been reported to provide a platform for biomedical applications, including drug or protein delivery,16-20 fluorescent cancer imaging,21 and magnetic resonance imaging.22 We hypothesized that PLA-TPGS nanoparticles might be a promising platform for enhancing the therapeutic efficacy of the chemotherapeutic agents such as doxorubicin in drugresistant cells. To test this, PLA-TPGS copolymers was synthesized using high reactive zinc complex as catalyst and their conditions for encapsulation of doxorubicin was determined. Here we report the intracellular localization of doxorubicin-loaded PLA-TPGS nanoparticles and their effects on P-gp activity and cell viability in drug-resistant breast cancer (MCF7/ADR) cells.
Experimental Section Synthesis and Characterization of PLA-TPGS Copolymers. The PLA-TPGS diblock copolymer was synthesized by ring-opening polymerization (ROP) of the L-lactide monomer (Sigma-Aldrich, U.S.A.) with TPGS (D-R-tocopheryl polyethylene glycol 1000 succinate, C33O5H54 [CH2CH2O]23, Eastman Chemical Company, U.S.A.) under a bidentate sulfonamide zinc ethyl complex, [(MPTHQ)ZnEt]2 (where MPTHQ ) 2-(2-methoxyphenyl)-3-tosyl-1,2,3,4-tetrahydroquinazoline; Supporting Information, Figure S1). In brief, a mixture of [(MPTHQ)Z-
10.1021/bm1005195 2010 American Chemical Society Published on Web 08/19/2010
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Figure 1. Schematic representation of PLA-TPGS synthesis under [(MPTHQ)ZnEt]2 catalyst.
nEt]2 (0.048 g, 0.050 mmol), TPGS (0.600 g, 0.400 mmol), and L-lactide (2.304 g, 16.0 mmol) in toluene (20.0 mL) was stirred at room temperature for 3.5 h. After the reaction was quenched by the addition of methanol (5.0 mL), n-hexane (100.0 mL) was added to the above mixture to give a white solid. The white solid was dissolved in CH2Cl2 (10.0 mL), recrystallized by n-hexane (100.0 mL), and then dried under vacuum. Finally, the PLA40-TPGS ([LA]0/[TPGS] ) 40) copolymer was obtained. All manipulations were carried out under a dry nitrogen atmosphere. The copolymers were characterized by 1H NMR (Varian Mercury-400, U.S.A.). Chemical shifts are given in ppm from the internal tetramethylsilane (TMS) or center line of CHCl3 and gel permeation chromatography (GPC) performed on a Hitachi L-7100 system equipped with a differential Bischoff 8120 RI detector using THF as an eluent. The molecular weight and polydispersity of the PLATPGS copolymer were calculated with polystyrene as a reference standard. The synthetic procedure of PLA-TPGS copolymer under [(MPTHQ)ZnEt]2 catalyst was shown in Figure 1. Preparation and Characterization of PLA-TPGS Nanoparticles with or without Doxorubicin Loading. PLA-TPGS or doxorubicinloaded PLA-TPGS nanoparticles were prepared by the solvent evaporation method.23 In brief, the amphiphilic PLA-TPGS copolymers (10 mg), with or without 1 mg doxorubicin (Calbiochem, EMD Biosciences Inc., U.S.A.), were dissolved in THF (5 mL; Tedia, U.S.A.), and then distilled water (20 mL) was poured into the solution. The mixed solutions were incubated under vacuum at 40 °C for 1 h to evaporate the solvent, cooled to room temperature, and then filtered through a 0.45 µm Millipore filter. The solution was freeze-dried for 2 days to obtain the nanoparticle powder. The size distribution and stability of the PLA-TPGS or doxorubicin-loaded PLA-TPGS nanoparticles were measured by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, Ltd., U.K.) with a 633 nm laser source. The morphologies of the resulting nanoparticles were evaluated by transmission electron microscopy (TEM, JEOL JEM-1400, JEOL Ltd., Japan). The loading efficiency of doxorubicin in the PLA-TPGS nanoparticles was quantified by determining the absorbance at 485 nm with a UV-visible spectrophotometer (U-3000, Hitachi, Japan). Critical Aggregation Concentration (CAC) of PLA-TPGS Copolymers. The CAC of the PLA-TPGS copolymers in aqueous solution was determined with the hydrophobic dye 1,6-diphenyl-1,3,5hexatriene (DPH) as a probe.24 A certain amount of copolymer sample was directly dissolved in Milli-Q water to produce a high-concentration aqueous solution, and the solution was diluted to the desired concentration (ranging from 10-3 to 10-6 g/mL). Then, 25 µL of DPH solution (0.4 mM in methanol) was injected into 2.5 mL of aqueous copolymer solution in a cuvette. The cuvette was sealed and kept in a dark place for 24 h before measurement. The absorption of the samples was recorded over a range of 200-500 nm using a scanning multiwell enzyme-linked immunosorbent assay (ELISA) reader (SpectraMax M2e, Molecular Devices Corporation, U.S.A.). The absorbance at 356 nm of the DPH probe was selected to explore the CAC.
Drug Release Experiment. Release of doxorubicin from the micelles was studied at 37 °C using a dialysis bag diffusion technique.25 Briefly, 2 mg of drug-loaded PLA-TPGS nanoparticles were dispersed in 10 mL of phosphate buffer saline (PBS) and sealed in a dialysis bag (molecular weight cutoff ) 5000). The dialysis bag was submerged in 30 mL of PBS and incubated at 37 °C for 24 h. The released doxorubicin in the buffer was collected at predetermined time intervals and frozen for further quantitative analysis of absorbance at 480 nm with a Cary 50 spectrophotometer.26 All experiments were carried out in triplicate. Cell Culture and Incubation Conditions. Doxorubicin-sensitive (MCF-7) and doxorubicin-resistant (MCF-7/ADR) human breast adenocarcinoma cells were grown in 75T culture flasks in Dulbecco’s modified Eagle medium (DMEM) or MEM culture medium supplemented with 10% fetal bovine serum (FBS; all from Gibco-BRL, U.S.A.) and 1% penicillin-streptomycin-neomycin solution (SigmaAldrich, U.S.A.) at 37 °C in a 5% CO2 incubator. The cells were subcultured 2-3 times per week with 0.25% trypsin-EDTA. The MCF7/ADR cells were maintained continuously in 0.5 µM doxorubicin. Uptake of Calcein-AM in MDR Cells. The activity of P-gp in the MCF-7 or MCF-7/ADR cells was characterized with the Vybrant Multidrug Resistance Assay Kit (V-13180, Molecular Probes, U.S.A.), performed according to the manufacturer’s instructions with a multiwell reader (SpectraMax M2e, Molecular Devices Corporation, U.S.A.).27 MCF-7/ADR cells (5 × 103) seeded into the 96-well culture plates and incubated for 24 h were then treated with either TPGS or PLATPGS (10 µg/mL) in PBS for 15 min. Calcein AM (final concentration 0.25 µM) was then added to each well, and the incubation was continued for another 15 min. The cells were then washed three times with PBS, and the fluorescence was measured using a multiwell ELISA reader at λex 488 nm. Uptake of Doxorubicin or Doxorubicin-Loaded Nanoparticles by Drug-Resistant Cells. MCF-7/ADR cells were seeded onto sixwell culture plates (1 × 105/well) and incubated for 24 h. The cells were pretreated with 10 µM doxorubicin or doxorubicin-loaded PLATPGS nanoparticles for 1, 3, 6, and 12 h. The cells were then washed three times with PBS, trypsinized, and transferred to centrifuge tubes. The cells were fixed in 10% formalin for 15 min on ice, washed, and resuspended with FACS Flow PBS (Becton-Dickinson, U.S.A.) and then analyzed using the FACS Calibur flow cytometer (BectonDickinson, U.S.A.) at λex 488 nm. A total of 10000 events were counted in each sample. Intracellular Localization of Doxorubicin. MCF-7/ADR cells (1 × 105) were seeded onto glass coverslips with 1 mL culture medium in 35 mm dishes and incubated for 24 h at 37 °C. To study the effects of PLA-TPGS formulation on the intracellular distribution of free doxorubicin, two groups were compared. Doxorubicin or doxorubicinloaded PLA-TPGS nanoparticle was added for each group to a final concentration at 10 µM. The cells were incubated for various times, the medium was removed by washing twice with PBS, and then the
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Table 1. Preparation of PLA-TPGS Catalyzed by [(MPTHQ)ZnEt]2 (0.05 mmol) with TPGS as Initiator in Toluene (20 mL)a entry
ROH
[M]0/[Zn]/[ROH]
time (h)
PDIb
Mnb (GPC)
Mnc (calcd)
Mnd (NMR)
convd (%)
PLA30-TPGS PLA40-TPGS
TPGS TPGS
120/1/4 160/1/4
3.5 3.5
1.11 1.12
11600 14100
5700 7000
5800 7800
97 96
a The molecular weight of TPGS is 1542 g/mol. b Obtained from GPC analysis and calibrated by a polystyrene standard. c As determined via integration of the methine resonances of LA and poly(L-lactide) (CDCl3, 400 MHz). d Calculated from the molecular weight of L-lactide times [LA]0/[ROH]0 times the conversion yield plus the molecular weight of ROH. [M]0/[LA]0; PLA-TPGS: poly(L-lactide)-D-R-tocopheryl polyethylene glycol 1000 succinate (the subscript indicates the ratio of PLA to TPGS, e.g., PLA40-TPGS indicates [LA]0/[TPGS] ) 40).
Table 2. Characterization of PLA-TPGS Nanoparticlesa
cells were observed under the confocal scanning microscope (Leica TCS SP5 Spectral Confocal System, Germany) at a λex of 488 nm. Cytotoxicity Studies. To investigate the cytotoxicity of doxorubicin with or without encapsulation into PLA-TPGS, drug-sensitive MCF-7 and drug-resistant MCF-7/ADR cells were first seeded into 96-well plates at a density of 5000 cells per well and cultured for 24 h. The free doxorubicin or doxorubicin-loaded PLA-TPGS nanoparticles were then added to give final concentrations of 20-0.1 µM in a total volume of 0.1 mL at 37 °C. Cytotoxicity was determined over 24 and 48 h with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma-Aldrich, U.S.A.) assay measured by an ELISA reader (SpectraMax M2e, Molecular Devices Corporation, U.S.A.). Statistical Analysis. The P-gp activities, intracellular accumulation of doxorubicin and cell viability are represented as mean ( standard deviation (SD). Statistical analysis was performed with the Student’s t-test and P < 0.05 was considered significant.
Results and Discussion Synthesis and Characteristics of PLA-TPGS Nanoparticles. The PLA-TPGS diblock copolymer was prepared by ROP of L-lactide with the hydroxyl group of TPGS under [(MPTHQ)ZnEt]2 catalysis in toluene at room temperature, and the results are shown in Table 1. The structure of the synthesized PLA-TPGS copolymer was characterized by 1H NMR in CDCl3 (Figure S2). The signals at 5.15 and 1.57 ppm were assigned to the methine protons and methyl protons of the PLA segment, respectively. The peak at 3.64 ppm was assigned to the methylene protons of the polyethylene glycol part of TPGS, and the lower signals in the aliphatic zone belong to various moieties of the vitamin E tails.23 The molecular weight of the PLA-TPGS was determined according to the ratio of the peak areas at 5.15 and 3.64 ppm. The molecular weight distribution of the prepared copolymers was measured by GPC. In the GPC analysis, no physical mixture of TPGS with L-lactide was observed. The peak for TPGS appeared at 8.38 min, and the peak of the copolymer was shifted to 7.41 min without the TPGS peak (data not shown). It was noted that the polymerization was well controlled, with the expected molecular weight and a low polydispersity index (PDI) ranging from 1.1 to 1.12. This catalyst showed a shorter reaction time at room temperature, higher conversion rate (>96%), and narrower molecular weight distribution (∼1.1) for ROP of L-lactide compared to the general catalyst stannous octoate.28 Properties of PLA-TPGS Nanoparticles. In previous reports, drug-loaded PLA-TPGS nanoparticles were fabricated by a solvent extraction/evaporation method with methylene chloride as an organic solvent; and a narrow size distribution could be achieved with an appropriate sonication strength during the formulation process.17,28 In this study, the PLA-TPGS nanoparticles with or without doxorubicin loading were prepared by the solvent-evaporation method without sonication, and THF was used as an organic solvent. The hydrodynamic diameter and critical aggregation concentrations (CACs) of the PLATPGS nanoparticles are shown in Table 2. The PLA-TPGS nanoparticles exhibited a uniform diameter around 130 nm with
polymer
particle sizeb (nm)
polydispersityb
CACc (g/mL)
PLA30-TPGS PLA40-TPGS
133.0 127.1
0.060 0.059
7.29 × 10-5 2.06 × 10-5
a The concentration of polymers was 2 × 10-4 g/mL. b Determined by DLS. c CAC ) critical aggregation concentration.
narrow polydispersity (
