Article pubs.acs.org/molecularpharmaceutics
Reduced Folate Carrier Independent Internalization of PEGylated Pemetrexed: A Potential Nanomedicinal Approach for Breast Cancer Therapy Mallaredy Vandana and Sanjeeb K. Sahoo* Laboratory of Nanomedicine, Institute of Life Sciences, Chandrasekarpur, Bhubaneswar 751023, India ABSTRACT: Pemetrexed has been widely used as an effective chemotherapeutic agent for the treatment of a variety of cancers including breast cancer. It is a multitargeted antifolate that gets transported to cells primarily by reduced folate carrier (RFC) and exerts its action by disrupting folate-dependent metabolic processes essential for cell replication. The loss of RFC leads to impaired transport of pemetrexed, which in turn decreases its intracellular concentration and reduces its cytotoxic effect on cancer cells. Furthermore, the multidrug resistance (MDR) related proteins (MRPs) contribute to pemetrexed efflux from the cancer cells. These observations prompted us to develop PEGylated pemetrexed that follows an efficient cellular internalization route independent of RFC and simultaneously bypasses the MRP efflux mechanism for acting as an efficient chemotherapeutic agent. Thus, the present study focuses on PEGylation of pemetrexed for its superior therapeutic efficiency by evaluating its cellular uptake and retention by flow cytometry, confocal microscopy, and reversed-phase high-performance liquid chromatography (RP-HPLC) in breast cancer cell lines having RFC expression and lacking RFC expression, that is, MCF7 and MDA MB231, respectively. In addition, the treatment of PEGylated pemetrexed lead to enhanced cytotoxicity due to S-phase arrest and apoptosis in the above mentioned cell lines. Interestingly, the longer circulation time of PEGylated pemetrexed in animal model concomitant with the RFC independent uptake and enhanced cytotoxicity suggests it to be a potential candidate for cancer therapy in a clinical setting. KEYWORDS: pemetrexed, reduced folate carrier, multidrug resistance, PEGylated pemetrexed, breast cancer, endocytosis
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gastric cancers, in addition to breast cancer.5−7 The commonly used anthracyclines and taxanes as chemotherapeutic agents for metastatic breast cancer are currently under scanner as the enhanced employment of these agents at an early stage of cancer often makes the tumors resistant to these drugs when the cancer relapses.8 Thereby, the strategies for noncrossresistant treatment options for metastatic breast cancer patients previously exposed to anthracyclines are clinically important.9 In this milieu, pemetrexed with its multitargeted activity demonstrated promising single-agent activity in metastatic breast cancer patients pretreated with anthracyclines, including those patients pretreated with anthracyclines and taxanes during phase II trials.8,10 Hence, pemetrexed can be considered as a most prospective candidate for metastatic breast cancer therapy. Additionally, the progress in prominent molecular action of pemetrexed is essentially governed by its efficient cellular internalization and retention.11,12 Hence, membrane transport of pemetrexed is a critical determinant for its pharmacological
INTRODUCTION Antimetabolites have been in use for the treatment of cancer for many years. They exert their anticancer action either as folate antagonists by interfering with the metabolic processes necessary for DNA synthesis or as nucleoside analogues that disrupt cellular replication and division by directly incorporating into the DNA.1 In this regard, during recent years, pemetrexed (Alimta; Eli Lilly and Company), a novel pyrrolo[2,3-d]pyrimidine-based folate analogue, has emerged as a new generation antimetabolite.2 It has multitargeted activity on the enzymes such as thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyl transferase (GARFT), and aminoimidazole carboxamide ribonucleotide formyltransferase (AICARFT), unlike other classical antimetabolites, such as methotrexate, which selectively target a single enzyme critical in purine and pyrimidine biosynthetic pathways.1,3 The multiple enzyme−inhibitory properties of pemetrexed creates a combinatorial effect wherein inhibition of three enzymes at multiple sites gives an advantage in overcoming acquired or intrinsic resistance associated with overexpression or mutation of any one of the enzyme.4 Thus, the multiple approaches of pemetrexed render it as a prospective therapeutic candidate for the treatment of a variety of cancers like nonsmall-cell lung, head and neck, bladder, and © 2012 American Chemical Society
Received: Revised: Accepted: Published: 2828
March 7, 2012 May 29, 2012 August 15, 2012 August 15, 2012 dx.doi.org/10.1021/mp300131t | Mol. Pharmaceutics 2012, 9, 2828−2843
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PEGylated pemetrexed in the animal model is reported to provide the clinical benefits of PEGylated pemetrexed as a potential therapeutic anticancer agent.
activity. Pemetrexed enters the cell through the reduced folate carrier (RFC), a bidirectional transporter that also acts as a major cellular transport system for folates.12 This lead to the crucial role of RFCs for the therapeutic action of pemetrexed, as loss of RFC or its impaired functioning can hamper its cellular internalization and lead to acquired resistance.12,13 In addition, various multidrug resistance (MDR) proteins in cancer cells acting as efflux transporters contribute to the depletion of the intracellular pool of the pemetrexed. Several studies have reported the active participation of multidrug resistance proteins (MRPs) for antifolates efflux as one of the causes for acquired resistance.14,15 Thus, the putative function of transporters and efflux proteins limits the effectiveness of pemetrexed in chemotherapy. Besides, a large number of other limitations such as hepatic degradation, immunogenicity, and high interstitial fluid pressure that is typically seen in tumor physiology add to the woes of pemetrexed.16,17 Equally, the physicochemical characteristics also act as a limiting factor in the pharmacokinetic behavior of pemetrexed. In the form of free acid, pemetrexed is poorly water-soluble, sensitive to light, heat, and moisture, and also has a strong tendency of degradation.18,19 In addition, the administration of pemetrexed in the form of disodium salt fails to provide high aqueous solubility and more stable storage.18 Thus, restraining the administration of the drug in a high dose along with a frequent dosage schedule, that results in greater systemic toxicity. Hence, efforts to enhance and safeguard the therapeutic activity of pemetrexed by furnishing an efficient delivery vehicle that masks its limitations during breast cancer therapy are the major goals. “Polymer therapeutics”, a technological podium in the field of nanomedicine, has emerged as one of the most propitious platforms for the efficient delivery of anticancer agents for the treatment of cancer.20,21 Conjugation to polymers saves the fate of many promising low molecular weight anticancer drugs, since a specific water-soluble polymer is linked by a biodegradable polymer−drug linker to these agents to increase their hydrodynamic size for prevention of rapid renal clearance.22,23 Thus, these drugs differ from controlled drug delivery systems as they now behave as a new chemical entity.20,22 Not only is their pharmacokinetic profile distinct from that of the parent drug, but the route of cellular uptake may also differ, as the polymer−drug can only enter cells by the endocytic route, leading to lysomotropic drug delivery.24 Hence, this strategy proves to be highly beneficial in overcoming the limitations associated with the membrane transporters and MDR efflux during pemetrexed treatment. Thus, in the new era of cancer therapeutics, we have developed PEGylated pemetrexed that exhibits its potential therapeutic activity through efficient cellular uptake and intracellular retention by overcoming the resistance because of lack of RFC or its impairment and MDR effect in breast cancer. In this regard, we report the detailed assessment of the transport or uptake mechanism of PEGylated pemetrexed through confocal microscopy, flow cytometry, and reversed phase high-performance liquid chromatography (RP-HPLC) using breast cancer cell lines, that is, MCF 7 (having RFC expression) and MDA MB231 (lacking RFC expression) cell lines. Besides, the enhanced retention leading to cytotoxicity due to S-phase arrest and apoptosis by PEGylated pemetrexed treatment is studied by MTT assay, PI/RNase analysis, Annexin-V-FITC/PI analysis and Western blot in the above mentioned cell lines. Furthermore, the bioavailability study of
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EXPERIMENTAL SECTION Materials. Pemetrexed disodium (Alimta) was purchased from Eli Lilly (Indianapolis, IN). NH2−PEG (MW ≈ 5 kDa) and NH2−PEG−COOH (MW ≈ 5 kDa) were obtained from JenKem Technology USA Inc. (Allen, TX). Verapamil, Nhydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide (DCC), fluorescein isothiocyanate (FITC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Annexin V-FITC, RNase A, propidium iodide (PI), Bradford reagent, dimethyl sulfoxide (DMSO), triethylamine (TEA), ethylene diamine, potassium bromide (KBr), and acetonitrile of HPLC grade were obtained from Sigma Aldrich Chemicals (Munich, Germany). All other inorganic salts used were obtained from Qualigens Fine Chemicals (Mumbai, India). A dialysis membrane (molecular weight cutoff = 3.5 kDa) was purchased from Spectra/Por 6 (Spectrum Laboratories, Inc., Rancho Dominguez, CA). MCF 7 and MDA MB231 cell lines were purchased from American Type Culture Collection (Rockville, MD). Cells were cultured in DMEM (Invitrogen, Grand Island, NY) with 1% L-glutamine, 10% fetal bovine serum (Gibco, Grand Island, NY), 10000 units/mL penicillin, and streptomycin. Maintenance of cells was done at 37 °C in an incubator (Hera Cell, Thermo Scientific, Waltham, MA) with an atmosphere of 5% carbon dioxide (CO2) and routinely passaged by treatment with trypsin (0.25%)−EDTA (0.1%) solution in Hank's balanced salt solution (Himedia Laboratories Pvt. Ltd., Mumbai, India). Preparation of PEGylated Pemetrexed. The PEGylated pemetrexed was synthesized using a method described by Yoo et al. with minor modifications.25 The carboxylate group of pemetrexed was activated using NHS and DCC. Briefly, 0.02 mM pemetrexed dissolved in 5 mL of DMSO was reacted with 0.04 mM NHS and 0.04 mM DCC under nitrogen atmosphere at room temperature for 12 h. The activated drug was then reacted with 0.05 mM NH2−PEG dissolved in 5 mL of DMSO. The reaction was performed under nitrogen atmosphere at room temperature for 4 h. The resultant solution was further diluted with deionized water for dialysis. The dialysis was carried out by taking the diluted polymer−drug solution of around 12 mL in a dialysis bag (Spectra/Por 6, molecular weight cutoff = 3.5 kDa) against deionized water in a 2 L beaker for a period of 24 h with frequent change of dialysate in every 2 h. Then, the dialyzed solution was freeze-dried at a temperature of −48 °C and 0.05 mbar using a lyophilizer (Labconco Corporation, Kansas City, MO) to obtain the powdered form of the PEGylated pemetrexed. For the cellular uptake study, FITC−pemetrexed and FITC−PEGylated pemetrexed were synthesized. Preparation of Fluorescence Labeling of PEGylated Pemetrexed and Native Pemetrexed. For the cellular uptake study, FITC−pemetrexed and FITC−PEGylated pemetrexed were synthesized. Briefly, 100 μg from 10 mg/ mL stock solution of FITC prepared using anhydrous DMSO was added dropwise to carbonate buffer (150 mM, pH 9.5) containing 2 mg/mL of pemetrexed, and the mixture was gently stirred at room temperature for 4 h. Then, the solution was freeze-dried to obtain the powdered form of the FITC− pemetrexed. The FITC−PEGylated pemetrexed was synthesized by slightly modifying protocol mentioned by Kim et al.26 2829
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3 mg/mL of polymer conjugate in PBS (0.1 M, pH 7.4). The samples were kept in a shaker at 37 °C and 150 rpm (Wadegati Orbit Shaking Incubator, Wadegati Labequip, India). At scheduled time intervals, 20 μL was withdrawn from the samples and analyzed by the RP-HPLC method, as mentioned previously. The exact volume of supernatant removed was replaced by the same amount of fresh PBS (0.1 M, pH 7.4). Experiments were carried out up to 96 h in triplicates. In Vivo Pharmacokinetic Study. Pharmacokinetic studies were carried out to find the bioavailability of the native pemetrexed and PEGylated pemetrexed in animal model. The experiment on animals was performed with the permission of Institutional Animal Ethics Committee of Institute of Life Sciences, Bhubaneswar, India. The in vivo pharmacokinetic study was performed on female Balb/C (25−28 g) mice divided into two groups (n = 3). Group 1 received native pemetrexed, and group 2 received PEGylated pemetrexed. Each mouse was administered with either native pemetrexed or PEGylated pemetrexed dissolved in aqueous solution via tail vein, at a dose of 10 mg/kg of pemetrexed. Blood samples for the analysis of pemetrexed in plasma were collected from the retro-orbital plexus at various time periods and processed by the method, as described by Malepati et al. with slight modifications.28 Plasma samples (0.1 mL) were precipitated with ice-cold methanol (0.2 mL) for deproteinization. After vortex mixing for 15 s, samples were incubated on ice for 15 min. This was followed by centrifugation at 14000 rpm for 4 min at 4 °C using Sigma 1-15K centrifuge. The aqueous methanol supernatant (∼250 μL) obtained after centrifugation was then analyzed by the RP-HPLC method as described in the Materials section, for determining the concentration of pemetrexed in plasma. Cellular Uptake Analysis by Confocal Microscopy. Cellular uptake experiments were performed to study the endocytosismediated uptake of PEGylated pemetrexed with minor modifications.29 Briefly, 2 × 105 cells of MCF 7 and MDA MB231 were separately seeded onto coverslips placed in sixwell flat-bottom tissue-culture plates (Corning Life Sciences, Corning, NY). After 24 h, medium was changed with 1 mL of either FITC−PEGylated pemetrexed or FITC−pemetrexed having a concentration of 25 ng/mL pemetrexed and incubated for different time periods at 37 °C. After the stipulated time period, the cells were washed with PBS (0.1 M, pH 7.4) twice to remove uninternalized FITC−PEGylated pemetrexed or FITC−pemetrexed and then stained with LysoTracker Red DND-99 (Molecular Probes Inc., Eugene, OR) for 20 min to visualize early endosome/lysosomes. Following the incubation period, the cells were washed twice with PBS (0.1 M, pH 7.4) and fixed with 4% (v/v) formaldehyde for 15 min. Then the cells were washed twice with PBS (0.1 M, pH 7.4) and stained with DAPI for 20 min to visualize nuclei. After specific incubation time, the cells were carefully washed thrice with PBS (0.1 M, pH 7.4) and the coverslips containing the cells were mounted onto a glass slide for viewing. The imaging was done with confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems GmbH, Germany) using the 63 X oil immersion lens using UV, Helium−Neon and Argon lasers for the detection of the three flourescent dyes at 370 nm excitation/ 470 nm emission (blue, DAPI), 490/525 nm (green, FITC) and 577/592 nm (red, Lysotracker Red) respectively. Cellular Uptake Analysis by Flow Cytometry. The cellular uptake study was further extended by flow cytometry analysis with slight modifications.30 Briefly, MCF 7 and MDA MB231
Briefly, heterobifuctional PEG, that is, COOH−PEG−NH2 (MW ≈ 5 kDa), was used, and the PEGylated pemetrexed was synthesized in the above-mentioned procedure. The carboxylic acid end group of COOH−PEG−pemetrexed (0.01 mM) was activated to the succinimidyl ester by DCC/ NHS chemistry and conjugated to the amine group of ethylene diamine (0.1 mM) in 10 mL of anhydrous DMSO. After 2 h of reaction, the resulting product was diluted with deionized water and subjected to dialysis using a dialysis bag (Spectra/Por 6, molecular weight cutoff = 3.5 kDa) against deionized water in a 2 L beaker for a period of 24 h with frequent change of dialysate in every 2 h. Following this, the dialyzed solution was freeze-dried. The lyophilized, aminated PEGylated pemetrexed was made to react with FITC (3 μM dissolved in 5 mL of anhydrous DMSO) at room temperature for 16 h. The resultant solution was diluted with deionized water and dialyzed against the same to remove unreacted FITC. The dialysis was carried out by taking the FITC−PEGylated pemetrexed solution of around 10 mL in a dialysis bag (Spectra/Por 6, molecular weight cutoff = 3.5 kDa) against deionized water in a 2 L beaker for a period of 24 h with a frequent change of water in every 2 h. Then, the dialyzed solution was freeze-dried to get the powdered product. Characterization of PEGylated Pemetrexed. 1H NMR Spectroscopy. NMR spectra were recorded for native pemetrexed, PEG−NH2, and PEGylated pemetrexed in FT NMR spectrometer (JEOL 400 MHz, Japan) using DMSO as the solvent. Gel Permeation Chromatography (GPC) Study. The GPC analysis was performed using OmniSEC-Viscotek with VE 3580 RI detectors and A600 M General mixed aqueous columns (30 mm × 8 mm). The column was maintained at 22 °C with a flow rate of 1 mL/min, and the detector was maintained at 35 °C. The analysis was done for 4 mg/mL of aqueous sample (i.e., PEG−NH2, PEGylated pemetrexed, H2N−PEG−COOH, or FITC−PEGylated pemetrexed) solution with an injection volume of 100 μL. Fourier Transform Infrared (FTIR) Spectroscopy Study. The FTIR spectra for native pemetrexed, PEG−NH 2 , and PEGylated pemetrexed were done by FTIR spectrometer [Spectrum RX I (Perkin-Elmer, Waltham, MA)] for characterizing PEGylated pemetrexed as described previously.27 Briefly, 7 mg of native pemetrexed, PEG−NH2, and PEGylated pemetrexed were pressed into a KBr pellet before obtaining their IR absorption spectra, and the spectra were detected over a range of 4400−393 cm−1. RP-HPLC Study. The amount of pemetrexed conjugated to PEGylated pemetrexed was analyzed by a RP-HPLC system of Waters 600 (Waters Co., Milford, MA) having a C18 column (Nova-Pak C18, 3.9 mm × 300 mm; Waters Associates) operated at 25 °C with Waters 2489 UV/visible detector (Waters Co.) at a wavelength of 230 nm. The mobile phase was composed of water with 0.1% phosphoric acid/acetonitrile (86:14) at a flow rate of 1 mL/min. The amount of pemetrexed in PEGylated pemetrexed was determined from the peak area correlated with the standard curve. The standard curve of pemetrexed was prepared under identical conditions mentioned above. All analysis was performed in triplicates with an injection volume of 20 μL in RP-HPLC. In Vitro Release Kinetic Study. The rate of pemetrexed release from the polymer conjugate was examined in an aqueous buffer solution at physiological pH. The release of the drug from PEGylated pemetrexed was carried out by dissolving 2830
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cells were seeded separately at a density of 2 × 105 cells per well in 6-well flat-bottom tissue-culture plates (Corning Life Sciences) overnight. Next day, cells were incubated with FITC- PEGylated pemetrexed or FITC−pemetrexed having 1 μg/mL concentration of pemetrexed and incubated at 37 °C for different time periods. In addition, both the cell lines are known to express MDR proteins which are responsible for pemetrexed efflux or a barrier to pemetrexed uptake.14,31,32 Thus, the uptake mechanism of FITC−pemetrexed and FITC−PEGylated pemetrexed in presence of Verapamil (MDR protein inhibitor) was evaluated in MCF 7 and MDA MB231 cells.33 In this regard, 2 × 105 cells per well were seeded in 6-well flat-bottom tissue-culture plates (Corning Life Sciences) overnight. Then, FITC−PEGylated pemetrexed with 10 μM verapamil or FITC−pemetrexed with 10 μM verapamil having 1 μg/mL concentration of pemetrexed were added to the cells and incubated for different time periods. At the end of the incubation period, the cells were harvested by trypsinization and centrifugation using SIGMA 1−15K at 3500 rpm for 5 min. The pelleted cells were washed twice with PBS (0.1 M, pH 7.4) and analyzed directly by flow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA) using the Cell Quest program (Becton Dickinson). Intracellular Retention Studies. The intracellular retention of PEGylated pemetrexed in comparison to native pemetrexed was assessed by RP-HPLC method, as mentioned above with slight modifications.34 Briefly, MCF 7 and MDA MB231 cells were plated separately in 6-well flat-bottom tissue-culture plates (Corning Life Sciences) at a density of 2 × 105 cells/well in appropriate growth medium at 37 °C overnight. The cells were then treated with native pemetrexed, PEGylated pemetrexed, native pemetrexed with verapamil (10 μM), and PEGylated pemetrexed with verapamil (10 μM), wherein the concentration of pemetrexed was 5 μg/mL, and were incubated for different time periods. After the particular time point, the cells were harvested by trypsinization followed by centrifugation using Sigma 1-15K at 3500 rpm for 5 min. Furthermore, the cell pellets were washed thrice with PBS (0.1 M, pH 7.4) and incubated with 0.1% Triton-X 100 at 37 °C for 20 min for cell lysis. An equal volume of methanol was added to the cell lysate for precipitating the protein. Next, the precipitated cell lysate was centrifuged at 13000 rpm for 5 min, and the supernatant was used for the detection of pemetrexed by the RP-HPLC method as mentioned previously. Cells Cytotoxicity Assay. The cytotoxic effect of native pemetrexed and PEGylated pemetrexed was assayed colorimetrically by the MTT assay with slight modifications.35 MCF 7 and MDA MB231 cells were plated separately and kept overnight in 96-well plates (Corning Life Sciences) at a density of 2000 cells per well in appropriate growth medium at 37 °C. The next day, different concentrations of native pemetrexed and PEGylated pemetrexed were added to the above cells. Then, the cells were incubated for 5 days at 37 °C. No further drug was added, and the media were changed every alternative day. Media-treated cells served as control for the experiment. Each test was performed in n = 6 wells. After incubation, 10 μL of 5 mg/mL of MTT was added and incubated for 3 h. The extent of cell viability was indicated by conversion of MTT into purple formazon by metabolically active cells. The crystals of formazon were dissolved with 100 μL of DMSO, and the optical density was measured at 570 nm using the enzymelinked immunosorbent assay plate Reader (Synergy HT,
BioTek Instruments Inc., Winooski, VT). The drug concentration that caused a 50% inhibition of the control growth rate (IC50) was calculated from a sigmoid plot by nonlinear regression analysis using the equation.36 Cell Cycle Analysis. Cell cycle alterations induced by treatment of native pemetrexed and PEGylated pemetrexed were studied by flow cytometry analysis in MCF 7 and MDA MB231 cell lines.37 The cells were plated at a density of 1 × 106 cells in 25 cm2 culture flasks (Corning Life Sciences) and allowed to attach for 24 h. The cells were then treated with native pemetrexed and PEGylated pemetrexed with a concentration of 1 μg and incubated for 3 days. After the particular time period, the cells were harvested by trypsinization and centrifugation using Sigma 1-15K at 3500 rpm for 5 min. The pelleted cells were washed thrice with PBS (0.1 M, pH 7.4) and fixed with 70% ethanol at 4 °C for 30 min. The DNA of the fixed cells was stained with 2 μL of PI (1 μg/μL), 2 μL of RNase A (1 μg/μL), and 0.5% Tween 20 in 500 μL of PBS (0.1 M, pH 7.4) and incubated for 30 min at room temperature in the dark before analysis. The cell cycle distribution was determined by analyzing 10000 gated cells with flow cytometer (FACSCalibur; Becton Dickinson) using Cell Quest software (Becton Dickinson). Apoptosis Measurement. The measurement of apoptosis was conducted by the Annexin V-FITC/PI apoptosis detection method in MCF 7 and MDA MB231 cell lines.38 Annexin VFITC and PI labeling was done using Annexin V apoptosis kit (Sigma) according to the prescribed protocol. Briefly, 1 × 106 cells were plated in 25 cm2 culture flasks (Corning Life Sciences) and incubated overnight for attachment. After drug treatment with native pemetrexed or PEGylated pemetrexed (1 μg/mL) for 3 days, cells were harvested by trypsinization and centrifugation using Sigma 1-15K at 3500 rpm for 5 min. Furthermore, the cells were washed twice with PBS (0.1 M, pH 7.4). After they were washed, 500 μL of Annexin V binding buffer was added to the cells followed by the addition of 5 μL of Annexin V-FITC (1 μg/μL) and 5 μL of PI (10 μg/μL). Cells were then incubated at room temperature in the dark for 10 min. The apoptosis was determined by analyzing 10000 gated cells directly by flow cytometry (FACSCalibur; Becton Dickinson) using the Cell Quest program (Becton Dickinson). Cells displaying phosphatidyl serine (PS) on their surface (positive Annexin-V fluorescence) were considered to be apoptotic, regardless of viability (PI staining). Cells staining positive for PI uptake were considered dead, regardless of Annexin-V staining. All experiments were performed in triplicates. Western Blotting Analysis. The expression of RFC and effect of PEGylated pemetrexed on the enzymes of the folate metabolism that lead to cell cycle arrest and apoptosis at the molecular level was demonstrated by Western blot analysis.27 In brief, MCF 7 and MDA MB231 cells at a density of 1 × 106 cells/mL were seeded separately and incubated overnight at 37 °C in 25 cm2 culture flasks (Corning Life Sciences) containing 10 mL of media. The next day, 5 mL of media containing pemetrexed or PEGylated pemetrexed with a 1 μg/mL concentration was added to the culture flasks and incubated for 3 days at 37 °C. Medium-treated cells served as the control for the experiment. After the incubation time, cell extracts were obtained by scraping the cells and washing once with ice-cold PBS (0.1 M, pH 7.4). Whole cell lysates were prepared with lysis buffer having 50 mM/L Tris-hydrochloride (Tris-HCl) (pH 7.5), 150 mM/L sodium chloride (NaCl), 1% NP40, 0.5% 2831
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Scheme 1. Schematic Illustration of the Reaction Involved in the Synthesis of the PEGylated Pemetrexed
Figure 1. 1H NMR characterization of PEG, native pemetrexed, and PEGylated pemetrexed. (a) NH2−PEG, (b) native pemetrexed, and (c) PEGylated pemetrexed.
Furthermore, the membrane was washed twice with rinsing buffer, and the protein of interest was detected with Enhanced Chemiluminescence Plus reagent (Amersham Biosciences, LittleChalfont, United Kingdom) and then exposed to X-ray film. All experiments were performed in triplicates. Quantitative densitometry analysis using ImageJ 1.45s [National Institutes of Health (NIH), Bethesda, MD] of Western blot assays for proteins was obtained from three independent experiments, and all results were normalized over β-actin. Statistical Analysis. Student's t test (two-tailed) was used to conduct statistical analysis. Data are expressed as means ± standard deviations and (*) p values
