AS1411 Aptamer and Folic Acid Functionalized pH ... - ACS Publications

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AS1411 Aptamer and Folic Acid Functionalized pH-Responsive ATRP Fabricated pPEGMA−PCL−pPEGMA Polymeric Nanoparticles for Targeted Drug Delivery in Cancer Therapy Shantanu V. Lale,† Aswathy R. G.,‡ Athulya Aravind,§ D. Sakthi Kumar,‡ and Veena Koul*,† †

Centre for Biomedical Engineering, Indian Institute of Technology Delhi, New Delhi 110016, India Bio-Nano Electronics Research Centre, Toyo University, Kawagoe, Saitama 350-8585, Japan § Indian Institute of Technology Delhi as a Joint Bio-Nano Research Collaboration Program, Toyo University and IIT Delhi, New Delhi 110016, India ‡

S Supporting Information *

ABSTRACT: Nonspecificity and cardiotoxicity are the primary limitations of current doxorubicin chemotherapy. To minimize side effects and to enhance bioavailability of doxorubicin to cancer cells, a dual-targeted pH-sensitive biocompatible polymeric nanosystem was designed and developed. An ATRP-based biodegradable triblock copolymer, poly(poly(ethylene glycol) methacrylate)−poly(caprolactone)−poly(poly(ethylene glycol) methacrylate) (pPEGMA−PCL−pPEGMA), conjugated with doxorubicin via an acid-labile hydrazone bond was synthesized and characterized. Dual targeting was achieved by attaching folic acid and the AS1411 aptamer through EDC−NHS coupling. Nanoparticles of the functionalized triblock copolymer were prepared using the nanoprecipitation method, resulting in an average particle size of ∼140 nm. The biocompatibility of the nanoparticles was evaluated using MTT cytotoxicity assays, blood compatibility studies, and protein adsorption studies. In vitro drug release studies showed a higher cumulative doxorubicin release at pH 5.0 (∼70%) compared to pH 7.4 (∼25%) owing to the presence of the acid-sensitive hydrazone linkage. Dual targeting with folate and the AS1411 aptamer increased the cancertargeting efficiency of the nanoparticles, resulting in enhanced cellular uptake (10- and 100-fold increase in uptake compared to single-targeted NPs and non-targeted NPs, respectively) and a higher payload of doxorubicin in epithelial cancer cell lines (MCF7 and PANC-1), with subsequent higher apoptosis, whereas a normal (noncancerous) cell line (L929) was spared from the adverse effects of doxorubicin. The results indicate that the dual-targeted pH-sensitive biocompatible polymeric nanosystem can act as a potential drug delivery vehicle against various epithelial cancers such as those of the breast, ovary, pancreas, lung, and others.

1. INTRODUCTION Chemotherapy has become an integral part of cancer treatment because of its effective cancer cell-killing potential. Chemotherapeutic agents like doxorubicin act on rapidly dividing cells and show cytotoxicity by blocking critical cellular pathways during mitosis and by inducing apoptosis. The major drawback of conventional doxorubicin therapy is its nonspecificity, which leads to cardiotoxicity and P-glycoprotein-mediated multidrug resistance.1−3 To overcome these lacunae of conventional chemotherapy, nanotechnological platforms like polymeric © 2014 American Chemical Society

nanoparticles, liposomes, metal nanoparticles, dendrimers, and others have been developed. Polymeric nanoparticles have emerged as a robust and promising drug delivery vehicle in targeted cancer therapy. Drug delivery using polymeric nanoparticles imparts unique advantages such as the passive targeting of solid tumors via an enhanced permeation and Received: January 25, 2014 Revised: April 1, 2014 Published: April 1, 2014 1737

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retention (EPR) effect, which results in a higher bioavailability of the drug in the tumor, controlled release of the drug, protection of labile drugs during circulation, longer circulation times (with a hydrophilic surface/coating), and overcoming multidrug resistance.4−6 Various polymerization techniques have evolved over a period of time, with atom transfer radical polymerization (ATRP) being one of the finest methods for the synthesis of polymers. Polymers of various geometries with predetermined molecular weight, and narrow molecular weight distribution of polymer chains can be synthesized by ATRP. Because ATRP is a living polymerization method, any number of polymer blocks can be added to the polymer chains even after the polymerization.7,8 Fabricating polymeric nanoparticles with a high drug payload is one of the major challenges in polymer nanotechnology. Chemical conjugation of doxorubicin molecules with polymer chains increases the total drug payload in nanoparticles. Additionally, unlike doxorubicin encapsulation in polymeric nanoparticles where doxorubicin can leak/release from nanoparticles during circulation, causing toxicity to normal healthy cells, chemical conjugation of doxorubicin via acid-sensitive linkage (e.g., acetal, ketal, hydrazone, β-propionate, cis-aconityl, etc.) would prevent its release from nanoparticles during circulation in the blood (pH 7.4), thus sparing normal healthy cells from the adverse effects of doxorubicin. After entering the cancer cell, the acid-labile linkage will break in the acidic environment of endolysosomes (pH 4.5−6.0), releasing the doxorubicin, which will cause apoptosis and subsequent cancer cell death.9−11 Passive targeting of tumors by nanoparticles via the EPR effect was initially employed for drug delivery, but now, active targeting has become a necessity for the therapeutic efficacy of nanoparticulate drug delivery systems. Active targeting of nanoparticles to cancer cells can be achieved using ligands that bind specifically to certain biomolecules that are overexpressed on the surface of cancer cells. Various ligands, such as folate, antibodies, growth factors, lipoproteins, sugars, hormones, aptamers, and others, have been studied for cancer targeting.12,13 Because of their specificity, these ligands act as a homing device and direct the nanoparticles to specific cancer cells. These nanoparticles are taken up by the cancer cells through receptor-mediated endocytosis, resulting in delivery of the chemotherapeutic drug inside the cancer cells. Folic acid has been widely used as a ligand for cancer targeting. Because folic acid is an essential ingredient for biosynthesis of purines and pyrimidines, and thus DNA synthesis, rapidly dividing cancer cells overexpress folate receptors in order to maintain the increased requirement of folate for DNA synthesis and rapid multiplication. Thus, folic acid makes an excellent choice as a homing device for targeting cancer cells.14,15 Aptamers have garnered a lot of interest as targeting agents in cancer therapy because of their small size, nonimmunogenicity, robust synthesis process, and chemical stability. They are short DNA/ RNA nucleotides or peptides that can bind to specific biomolecules on a cancer cell’s surface with a high affinity and precision that are similar to antibodies.16 The AS1411 aptamer is a 26-nucleotide guanosine-rich DNA aptamer that binds specifically to a cellular protein called nucleolin, which is overexpressed in most cancers. Various studies have shown the efficacy of the AS1411 aptamer as a cellular uptake enhancer in cancer cells, making AS1411 a suitable ligand for precisely targeting cancer cells.17−20

The concept of targeting nanoparticles (NPs) to cancer cells using two or more ligands is a recent trend in cancer drug delivery systems and is currently gaining wide acceptance in cancer research because of its high effectiveness in therapeutic applications. Many researchers have reported the enhanced efficacy of dual-targeted nanosystems in targeting cancer cells. The ability of nanoparticles to bind cancer cells is significantly enhanced by multivalent targeting.21−25 Because dual-targeted nanoparticles target two different molecular targets on the surface of cancer cells, one ligand on the NPs can still target the nanoparticles to the cancer cell even if the cancer cells do not express both of the target moieties on its surface, thus counteracting the defense mechanism of the cancer cell. In the present work, a dual-functionalized ATRP-based biocompatible polymeric nanosystem was designed and developed for targeted delivery of doxorubicin to cancer cells (Scheme 1). A simple ATRP-based poly(poly(ethylene glycol) Scheme 1. Schematic Illustration of Dual-Targeted (AS1411 Aptamer and Folic Acid) pH-Sensitive pPEGMA−PCL− pPEGMA Nanoparticles for Targeted Doxorubicin Delivery in Cancer Therapy

methacrylate)−poly(caprolactone)−poly(poly(ethylene glycol) methacrylate) (pPEGMA−PCL−pPEGMA) triblock copolymer with the desired molecular weight and narrow molecular weight distribution was synthesized. Its multiple poly(ethylene glycol) (PEG) chains impart a stealth nature to the nanoparticles, which helps them to escape macrophage recognition, resulting in the nanoparticles having an enhanced circulation half-life. To improve the doxorubicin payload in the nanoparticles and to prevent its leakage/release during circulation, doxorubicin molecules were conjugated on multiple PEG chains of pPEGMA−PCL−pPEGMA via an acid-labile 1738

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and a saturated solution of NaHCO3. After extraction, the DCM layer was dried over Na2SO4 for 2 h and evaporated under vacuum. The viscous solution obtained was then precipitated three times in cold methanol. The PCL macroinitiator thus obtained was characterized by 1 H NMR (Brukers, USA) and ATR−FTIR (PerkinElmer, USA). Yield = 84%. 1 H NMR (300 MHz, CDCl3) δ (ppm): 1.38 (m, -CO-CH2-CH2CH2-CH2-CH2-O-), 1.63 (m, -CO-CH2-CH2-CH2-CH2-CH2-O-), 1.92 (s, geminal CH3- from α-bromoisobutyryl group), 2.29 (t, -CO-CH2CH2-CH2-CH2-CH2-O-), 3.69 (t,-CO-O-CH2-CH2-O-CH2-CH2-OCO-), 4.04 (t, -CO-CH2-CH2-CH2-CH2-CH2-O-), 4.17 (t, -COCH2-CH2-CH2-CH2-CH2-OH), 4.22 (m, -CO-O-CH2-CH2-O-CH2CH2-O-CO-). 2.2.2. Synthesis of pPEGMA−PCL−pPEGMA Triblock Copolymer by ATRP. Copper(I) bromide (CuBr) (0.29 g; 2 mM) and PMDETA (0.42 mL; 2 mM) were added to dry toluene (20 mL) in a Schlenk flask and allowed to stir for 1 h at 90 ± 1 °C under a N2 atmosphere. To this mixture were added PEGMA (Mn ∼360 Da, 4.904 mL; 15 mM) and PCL macroinitiator (2.3 g; 1 mM), and the reaction mixture was allowed to stir at 90 ± 1 °C for 24 h under a N2 atmosphere. The reaction was then terminated by introducing air, and the reaction mixture was passed through an activated neutral alumina column to remove copper complex. The eluate was concentrated on a rotary evaporator, precipitated three times in cold diethyl ether, and dried overnight in vacuum oven at 40 ± 1 °C. The pPEGMA−PCL− pPEGMA triblock copolymer thus obtained was characterized by 1H NMR, ATR−FTIR, and GPC (Waters, USA). Yield = 71%. 1 H NMR (300 MHz, CDCl3) δ (ppm): 0.90 (m, -CH3 from PEGMA), 1.05 (m, geminal -CH3 groups), 1.41 (m, -CO-CH2-CH2CH2-CH2-CH2-O-), 1.63 (m, -CO-CH2-CH2-CH2-CH2-CH2-O-), 1.96 (m, -C-CH2-C- from PEGMA), 2.32 (m, -CO-CH2-CH2-CH2-CH2CH2-O-), 3.67 (m, -O-CH2-CH2-O from PEGMA), 4.07 (m, -COCH2-CH2-CH2-CH2-CH2-O-). 2.3. Conjugation of Folic Acid and Doxorubicin to the pPEGMA−PCL−pPEGMA Triblock Polymer. 2.3.1. Functionalizing Hydroxyl Groups of Triblock Polymer to −CONHNH2 with the Help of Succinic Anhydride and Hydrazine. Triblock polymer (Mn (NMR) ∼ 7000 Da, 5.5 g; 10.2 mM corresponding to hydroxyl groups) was dissolved in dry THF (50 mL) in a Schlenk flask. Succinic anhydride (2.04 g; 20.4 mM) and DMAP (0.62 g; 5.1 mM) were added to the flask, and the reaction was continued at 30 ± 1 °C for 24 h under a N2 atmosphere. The product was then precipitated three times in diethyl ether. The triblock polymer containing −COOH functionality (Tri-COOH) thus obtained was characterized by 1H NMR and ATR−FTIR. Yield = 82%. 1 H NMR (300 MHz, CDCl3) δ (ppm): 0.90 (m, -CH3 from PEGMA), 1.05 (m, geminal -CH3 groups), 1.41 (m, -CO-CH2-CH2CH2-CH2-CH2-O-), 1.63 (m, -CO-CH2-CH2-CH2-CH2-CH2-O-), 1.96 (m, -C-CH2-C- from PEGMA), 2.32 (m, -CO-CH2-CH2-CH2-CH2CH2-O-), 2.67 (m, -CO-CH2-CH2-COOH from succinic anhydride), 3.67 (m, -O-CH2-CH2-O from PEGMA), 4.07 (m, -CO-CH2-CH2CH2-CH2-CH2-O-). In the next step, Tri-COOH (3.0 g; 2.57 mM corresponding to six carboxyl groups) was dissolved in dry THF (50 mL). DCC (0.53 g; 2.57 mM) and NHS (0.3 g; 2.57 mM) were added to the flask subsequently, and the reaction mixture was allowed to stir at 30 ± 1 °C overnight under a N2 atmosphere. Hydrazine hydrate (0.126 mL; 2.57 mM) was then added to the flask, and the reaction was allowed to continue for another 12 h. The reaction mixture was then filtered to remove DCC byproduct, and the product was precipitated three times in diethyl ether. The triblock polymer containing −CONHNH2 functionality (Tri-CONHNH2) thus obtained was characterized by ATR−FTIR. Yield = 79%. 2.3.2. Conjugation of Folic Acid to Triblock Polymer. Folic acid (0.31 g; 0.714 mM) was dissolved in dry DMSO (50 mL). DCC (0.295 g; 1.428 mM) and NHS (0.165 g; 1.428 mM) were added to the solution subsequently, and the reaction mixture was allowed to stir overnight at 30 ± 1 °C under a N2 atmosphere. Tri-CONHNH2 (2.5 g; 0.714 mM corresponding to two −CONHNH2 groups) was then added to the flask, and the reaction was continued at 30 ± 1 °C for 24

hydrazone linkage. To improve the uptake of the nanoparticles and to deliver a higher payload of doxorubicin into the cancer cells, dual targeting using folic acid and the AS1411 aptamer was achieved. The biocompatibility of the polymeric nanosystem was evaluated using MTT cytotoxicity assays, blood compatibility studies, and protein adsorption studies. In vitro drug release studies were carried out at pH 7.4 (physiological pH) and pH 5.0 (endolysosomal pH). Cellular uptake and apoptosis studies as well as nuclear internalization of doxorubicin were carried out using confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) to evaluate the polymeric nanosystem as a potential therapeutic agent for targeted drug delivery in cancer therapy.

2. MATERIALS AND METHODS 2.1. Materials. Polycaprolactone diol (Mn ∼2000 Da), αbromoisobutyryl bromide, 4-dimethylaminopyridine (DMAP), folic acid, N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA), 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT), bovine serum albumin (BSA), trehalose, Pluronic F68, and Annexin V−FITC assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used as received without further purification. Poly(ethylene glycol) methacrylate (PEGMA) (Mn ∼360 Da) (Sigma-Aldrich) was passed through a column of basic alumina before use to remove the polymerization inhibitor monomethyl ether hydroquinone (MEHQ). Copper(I) bromide (Sigma-Aldrich) was purified by sequential washing with glacial acetic acid, ethanol, and diethyl ether followed by vacuum drying. Amine-modified AS1411 aptamer was obtained from Eurofins Genomics (Bangalore, India). Doxorubicin was purchased from Biochem Pharmaceuticals (Mumbai, India). N,N′-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), triethylamine, succinic anhydride, hydrazine hydrate, sodium bicarbonate (NaHCO3), sodium sulfate (Na2SO4), sodium acetate, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, diethyl ether, neutral alumina, 4A molecular sieves, dimethyl sulfoxide (DMSO), sodium chloride (NaCl), calcium chloride, tetrahydrofuran (THF), dichloromethane (DCM), hydrochloric acid (HCl), and Triton X-100 were obtained from Merck Millipore (Mumbai, India) and were used as received without further purification. Dialysis membrane (3.5 kDa) was purchased from Himedia Pvt. Ltd. (Mumbai, India). Amicon ultracentrifugal filters (30 kDa) were obtained from Merck Millipore (Billerica, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and a penicillin−streptomycin solution were obtained from Gibco, Life Technologies. Hoechst 33258, LysoTracker Green DND189, and DNase/RNase-free water were purchased from Invitrogen, Life Technologies. All reagents used were of analytical grade. Ultrapure water with resistivity of 18 MΩ cm was obtained using a Milli-Q system (Merck Millipore, Billerica, MA, USA). 2.2. Synthesis of Poly(poly(ethylene glycol) methacrylate)− poly(caprolactone)−poly(poly(ethylene glycol) methacrylate) (pPEGMA−PCL−pPEGMA) Triblock Copolymer. Triblock copolymer pPEGMA−PCL−pPEGMA was synthesized in two steps. In the first step, polycaprolactone diol was converted to polycaprolactone macroinitiator. In the next step, polycaprolactone macroinitiator initiated the polymerization of PEGMA to synthesize pPEGMA− PCL−pPEGMA using ATRP. 2.2.1. Synthesis of Polycaprolactone Macroinitiator from Polycaprolactone Diol (PCL Diol). PCL diol (Mn ∼2000 Da, 10.0 g; 5 mM) was dissolved in dry THF (50 mL) in a Schlenk flask followed by dry triethylamine (3.48 mL; 25 mM), and the resulting solution was stirred in an ice bath for 30 min. α-Bromoisobutyryl bromide (3.09 mL; 25 mM) was added dropwise to the solution, and the temperature of the system was slowly increased to 30 °C. The reaction was allowed to stir for 24 h at 30 ± 1 °C under a N2 atmosphere. The mixture was then filtered to remove quaternary ammonium salts, and the filtrate was evaporated under vacuum in a rotary evaporator (HB 10, Ika, Germany). The resulting viscous solution was extracted with DCM 1739

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Figure 1. Schematic representation of the synthesis of doxorubicin-conjugated triblock copolymers. h under a N2 atmosphere. The reaction mixture was then filtered to remove DCC byproduct, dialyzed, and lyophilized. The yellow, folate-

conjugated triblock polymer (Tri-FA) thus obtained was characterized by differential scanning calorimetry (DSC). Yield = 85%. 1740

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2.3.3. Doxorubicin Conjugation to Triblock Polymer. Tri-FA (1.5 g; 0.21 mM) was dissolved in dry DMSO (50 mL) in a Schlenk flask. Acetic acid (0.1 mL) and doxorubicin hydrochloride (0.1 g; 0.17 mM) were added to the flask, and the reaction was allowed to stir in dark for 48 h at 30 ± 1 °C under a N2 atmosphere. The solution was then dialyzed to remove unconjugated doxorubicin and lyophilized. The red folate and doxorubicin-conjugated polymer (Tri-Dox-FA) thus obtained was characterized by DSC. Yield = 82%. Similarly, Tri-CONHNH2 polymer was treated with doxorubicin hydrochloride to obtain the doxorubicin-conjugated polymer (TriDox) that was devoid of folic acid (non-targeted polymer). Yield = 80%. The synthesis scheme is shown in Figure 1. 2.4. DSC Studies for the Determination of Doxorubicin and Folate Conjugation. Differential scanning calorimetry was used to confirm conjugation of doxorubicin on PEG chains of the polymer. The thermal behavior of the triblock polymer, doxorubicin hydrochloride, doxorubicin-conjugated polymer, and a physical mixture of doxorubicin hydrochloride with triblock polymer was analyzed by DSC (Pyris 6, PerkinElmer, USA) over a range of 0−300 °C at a scan rate of 10 °C/min. An empty aluminum pan was used as the reference. Similarly, to confirm the conjugation of folic acid with the polymer, the thermal behavior of the triblock polymer, folic acid, folateconjugated polymer, and a physical mixture of folic acid with triblock polymer was analyzed by DSC over a range of 0−300 °C at a scan rate of 10 °C/min. 2.5. CHN Analysis for Folic Acid Content Determination. The folic acid content in the Tri-FA polymer was determined by CHN analysis using an element analyzer (Vario EL III, Elementar Analysensysteme GmbH, Germany). The folic acid content was determined on the basis of the C/N ratio of the polymer sample obtained from this analysis. 2.6. Doxorubicin Content of the Conjugated Polymers. The doxorubicin content of the conjugated polymers (Tri-Dox-FA and TriDox) was determined using a UV−vis spectrophotometer. A weighed amount of doxorubicin-conjugated polymer was suspended in 5 mL of 0.1 N HCl and sonicated for 10 min followed by stirring in the dark for 48 h at 25 ± 1 °C. It was then centrifuged at 3000 rpm for 2 min to remove insoluble particles, and the supernatant was analyzed at 481 nm using a UV−vis spectrophotometer (PerkinElmer, USA). Doxorubicin content was determined using a doxorubicin standard calibration curve equation. 2.7. Preparation and Characterization of Nanoparticles (NPs). A weighed amount of polymer (10 mg) was dissolved in 2 mL of DMSO and was added dropwise using a syringe to 10 mL of water containing 1% Pluronic F68 under mild stirring at 25 ± 1 °C. The resulting nanoparticle suspension was washed three times with water at 4500 rpm for 30 min using an Amicon ultracentrifugal filter (30 kDa). Purified nanoparticles were then lyophilized with 5% trehalose as a cryoprotectant. Prepared nanoparticles were characterized using dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd., UK), high-resolution transmission electron microscopy (HRTEM) (Technai G2, 200 kV, FEI, USA), and scanning electron microscopy (SEM) (Zeiss EVO 50, Carl Zeiss Microscopy GmbH, Germany). The reconstitution time for the nanoparticles was also determined by addition of 10 mL of water to the lyophilized vial. 2.8. In Vitro Stability Studies of Nanoparticles. The stability of the nanoparticles was evaluated in normal saline (0.9% w/v NaCl) and DMEM by analyzing their mean diameter. Briefly, 0.1 mL of a 5 mg/ mL nanoparticle suspension was resuspended in 3 mL of normal saline or 3 mL of DMEM at 37 ± 1 °C. The particle size of the nanoparticles was determined periodically after 1, 3, 6, 9, and 24 h by DLS using a Zetasizer. 2.9. Drug Release Studies. A weighed amount of Tri-Dox-FA NPs (2.5 mg) was dispersed in 2 mL of water in a dialysis membrane and placed in 10 mL of a phosphate buffer solution (pH 7.4) in an incubator at 37 ± 1 °C for 48 h at 120 rpm. Periodic sampling was carried out, and samples were analyzed using a UV−vis spectrophotometer at 481 nm. The concentration of released doxorubicin was

measured using a doxorubicin standard calibration curve equation. A similar procedure was carried out for drug release studies in acetate buffer at pH 5.0. 2.10. Protein Adsorption Study. A weighed amount of nanoparticles (5 mg) was incubated with 10 mL of a 4% bovine serum albumin (BSA) solution in a phosphate buffer solution (pH 7.4) at 37 ± 1 °C for 24 h at 120 rpm. The solution was then centrifuged at 26 200 rpm (60 628g) for 30 min, and the supernatant was analyzed for BSA using the Biuret test at 540 nm in a UV−vis spectrophotometer. A standard calibration curve of known concentrations of BSA (500−2500 μg/mL) was prepared using the Biuret test. Briefly, 4 mL of Biuret reagent was incubated with 1 mL of a BSA solution at 37 ± 1 °C for 20 min at 120 rpm. The absorbance of the solution was then measured at 540 nm using a UV−vis spectrophotometer.26 2.11. Blood Compatibility. The blood compatibility of the nanoparticles was determined using hemolysis and coagulation studies. Blood samples were collected from healthy volunteers from IIT Delhi Hospital, New Delhi. Blood was collected in evacuated glass tubes containing disodium citrate as an anticoagulant. 2.11.1. Hemolysis Study. RBCs were separated from whole blood by centrifuging 2 mL of whole blood at 1500 rpm for 10 min. The supernatant was removed, and 400 μL of concentrated RBCs was washed three times with phosphate buffered saline (PBS), pH 7.4. A stock solution was prepared by diluting 50 μL of RBCs with 10 mL of PBS. One-hundred microliters of polymer nanoparticles with concentration ranging from 0.25 to 5 mg/mL in PBS was incubated with 100 μL of RBC stock solution at 37 ± 1 °C for 1 h at 120 rpm, resulting in final concentrations in the range 0.125−2.5 mg/mL. After 1 h, the mixture was centrifuged at 1500 rpm for 5 min, and the supernatant was analyzed for the amount of released hemoglobin using a UV−vis spectrophotometer at 540 nm. The percent of hemolysis was calculated relative to hemolysis caused by positive (1% Triton X-100) and negative (PBS) controls as follows ⎛ ⎞ sample540nm − negative control540nm ⎟⎟100 hemolysis (%) = ⎜⎜ ⎝ positive control540nm − negative control540nm ⎠

2.11.2. Coagulation Studies. One-hundred microliters of nanoparticles with various concentrations (0.1, 1, and 10 mg/mL) was incubated with 900 μL of blood at 37 ± 1 °C for 1 h, resulting in final concentrations of 0.01, 0.1, and 1 mg/mL. Blood was then centrifuged at 4000 rpm for 10 min to obtain platelet-poor plasma, which was incubated at 37 ± 1 °C. For prothrombin time (PT) determination, tissue factor (containing phospholipid) was added to platelet-poor plasma at 37 ± 1 °C followed by addition of an excess of a calcium chloride solution (25 mM). The time taken for the formation of fibrin clots was measured using an automated coagulation analyzer (Diagnostica Stago, Germany). For activated partial thromboplastin time (aPTT) determination, micronized silica and cephalin were added to platelet-poor plasma at 37 ± 1 °C followed by the addition of a calcium chloride solution (25 mM). The time taken for the formation of fibrin clots was measured using an automated coagulation analyzer.27 2.12. Preparation of Aptamer-Conjugated Nanoparticles. Polymeric nanoparticles (Tri-Dox-FA NPs and Tri-Dox NPs) were suspended in DNase/RNase-free water at a 2 mg/mL concentration and were incubated with EDC (200 μL; 400 mM) and NHS (200 μL; 100 mM) at 25 ± 1 °C for 30 min with gentle shaking. Aminemodified AS1411 aptamer (100 μL; 10 μM) was added to the NHSactivated nanoparticles, and the sample was incubated at 25 ± 1 °C for 3 h with gentle stirring. The sample was then washed three times with DNase/RNase-free water through an Amicon ultracentrifugal filter. The resulting aptamer nanoparticle conjugates (Tri-Dox-FA-A and Tri-Dox-A, respectively) were resuspended in DNase/RNase-free water and kept at 4 °C in a refrigerator. 2.13. Characterization of Aptamer-Conjugated Nanoparticles. Aptamer conjugation on the surface of the nanoparticles was confirmed by studying the surface chemistry using X-ray photoelectron spectroscopy (XPS) (AXIS His 165 Ultra, Kratos Analytical, Shimadzu 1741

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Corporation, Japan). Twenty microliters of sample was applied on a silicon substrate and dried in a desiccator. The binding energy spectrum was recorded from 0 to 1100 eV with a pass energy of 80 eV using the fixed transmission mode.28 Data was analyzed using CasaXPS software. Aptamer conjugation on the surface of nanoparticles was also confirmed by measuring the zeta potential of the aptamer-conjugated nanoparticles using DLS. 2.14. Cell Line Experiments. MCF-7 (breast cancer cells), PANC-1 (pancreatic cancer cells), and L929 cell lines (mouse fibroblast cells) were obtained from Riken Bioresourse Centre, Japan. Cells were cultured in 25 cm2 tissue culture flasks with DMEM supplemented with 10% fetal bovine serum and a 1% penicillin− streptomycin solution in a 5% CO2 atmosphere at 37 °C. 2.14.1. Cell Viability Studies. MTT assay was performed on MCF-7 and L929 cell lines according to a published protocol.29 At about 80% cell confluence, cells were trypsinized and counted with a hemocytometer. Ten thousand cells/well were seeded in a 96-well plate and incubated at 37 °C for 24 h in a CO2 incubator. After incubation, 50 μL of a nanoparticle suspension of different concentrations (0.5−8 mg/mL) was added to 150 μL of media in each well, resulting in final concentrations of 0.125, 0.25, 0.5, 1, and 2 mg/mL. The plate was incubated at 37 °C for 24 h. Ten microliters of a 5% MTT solution was then added to the wells, and the plate was incubated for 4 h. Two-hundred microliters of DMSO was then added to each well, and the absorbance was measured at 540 nm on a microplate spectrophotometer (PowerWave XS2, BioTek Instruments, USA). The percent cell viability was calculated relative to positive (1% Triton X-100) and negative (PBS) controls using the following equation

Hoechst stain for 10 min followed by PBS washing and observed under a confocal laser scanning microscope at 100× magnification. 2.14.5. Annexin V−FITC Apoptosis Assay. Cellular apoptosis and necrosis in MCF-7, PANC-1, and L929 cells after nanoparticle treatment was investigated using an Annexin V−FITC assay. Cells were seeded in a 6-well plate at a density of 2 × 105 cells/well and incubated at 37 °C for 24 h. Polymeric nanoparticles at a concentration equivalent to 10 μg of doxorubicin were then incubated with the cells for 8 h. Cells were then rinsed with PBS, trypsinized, treated with 5 μL of Annexin V−FITC and 10 μL of a propidium iodide solution, and incubated for 10 min in the dark. The fluorescence of the cells was then measured by flow cytometry, and on the basis of the differential staining, the percent of early apoptotic cells (stained with Annexin V−FITC), necrotic cells (stained by both Annexin V−FITC and propidium iodide), and live cells (no staining) was determined. 2.15. Statistical Analysis. Data are presented as the mean ± standard deviation. Experiments were carried out in triplicate. Statistical analysis was performed using Student’s t test (SigmaStat 3.5, Systat Software Inc., San Jose, CA, USA).

3. RESULTS AND DISCUSSION 3.1. Synthesis of the pPEGMA−PCL−pPEGMA Triblock Copolymer. Synthesis of the pPEGMA−PCL− pPEGMA triblock copolymer was carried out as per the synthesis scheme shown in Figure 1. First, PCL macroinitiator was synthesized by reacting terminal hydroxyl groups of PCL diol with α-bromoisobutyryl bromide in the presence of triethylamine in dry THF. The synthesized macroinitiator was characterized using 1H NMR and ATR−FTIR (Supporting Information Figures S1 and S2, respectively). The 1H NMR spectrum of PCL macroinitiator showed the appearance of a new peak at δ 1.92 ppm (peak i), which can be attributed to the geminal methyl groups of α-bromoisobutyryl bromide, thus indicating the conversion of PCL diol to PCL macroinitiator. The shifting of peak d (corresponding to methylenic protons adjacent to terminal hydroxyl groups in PCL diol) from δ 3.68 to 4.17 ppm resulting from the carboxyl group of αbromoisobutyryl bromide also confirmed the conversion of PCL diol to PCL macroinitiator. According to the FTIR spectrum of PCL macroinitiator (Supporting Information Figure S2B), the presence of a vibration band at 647 cm−1 (corresponding to C−Br stretching vibrations) and the disappearance of a vibration band at 3435 cm−1 (corresponding to O−H stretching vibrations of PCL diol) confirmed the formation of PCL macroinitiator. In the next step, triblock polymer pPEGMA−PCL− pPEGMA was synthesized using ATRP. PCL macroinitiator, in the presence of catalysts CuBr and PMDETA, initiates freeradical-catalyzed polymerization of PEGMA in dry toluene under an inert atmosphere. The ratio of monomer/initiator was kept at 15:1 to synthesize polymer with a molecular weight of ∼7500 Da. The ratio of CuBr/PMDETA was kept at 1:1 to achieve a higher activation rate constant for the ATRP reaction.30 The ratio of PCL macroinitiator/CuBr/PMDETA was kept as 1:2:2 because PCL macroinitiator has two isobutyryl bromide groups on both ends and hence the molar equivalents of the catalysts (CuBr and PMDETA) were double that of the macroinitiator. Synthesis of the triblock polymer was confirmed by 1 H NMR and ATR−FTIR (Supporting Information Figures S1 and S2, respectively). 1 H NMR shows a peak at δ 3.67 ppm (peak e), which corresponds to −OCH2 groups from PEGMA chains in the triblock polymer. Polycaprolactone methylenic peaks were retained in the spectrum (peaks a, b, c, and f). The FTIR

⎛ ⎞ sample540nm − positive control540nm ⎟⎟100 cell viability (%) = ⎜⎜ ⎝ negative control540nm − positive control540nm ⎠

2.14.2. In Vitro Cellular Uptake Studies Using Confocal Microscopy. To visualize the cellular uptake of the nanoparticles, confocal laser scanning microscopy was employed. MCF-7, PANC-1, and L929 cells were seeded in Petri plates with a glass bottom at a density of 1 × 104 cells/plate, and the plates were incubated at 37 °C for 24 h in a CO2 incubator. Cells were then treated with polymeric nanoparticles at a concentration equivalent to 10 μg of doxorubicin and incubated at 37 °C for 2 h. At the end of incubation period, the cells were rinsed three times with PBS to remove excess nanoparticles. Cells were then stained with 150 μL of LysoTracker Green for 30 min followed by 100 μL of Hoechst stain for 10 min with intermediate washing with PBS and observed under the confocal laser scanning microscope (Olympus IX81 with DU897 mode). Images were obtained at 100× magnification from the fluorescence emitted by Hoechst (460 nm), LysoTracker Green (505 nm), and doxorubicin (560 nm). 2.14.3. Cellular Uptake Studies Using Fluorescence Activated Cell Sorting (FACS). MCF-7, PANC-1, and L929 cells were seeded at a density of 1 × 105 cells/well in a 6-well plate and incubated at 37 °C for 24 h in a CO2 incubator. Cells were then treated with polymeric nanoparticles at a concentration equivalent to 10 μg of doxorubicin and incubated at 37 °C for 2 h. Cells were then washed three times with PBS to remove excess nanoparticles, trypsinized, and centrifuged at 3500 rpm for 3 min. The cell pellet thus obtained was washed with PBS, and cells were fixed with a 3% paraformaldehyde solution. After PBS washing, cells were treated with ice-cold methanol for 10 min followed by PBS washing. Cells were then resuspended in 0.5 mL of PBS, and the cellular uptake of nanoparticles was investigated by flow cytometry (FACScan, Becton Dickinson, USA). 2.14.4. Nuclear Localization of Doxorubicin. MCF-7, PANC-1, and L929 cells were seeded in Petri plates with a glass bottom at a density of 1 × 104 cells/plate and incubated at 37 °C for 24 h. Cells were then treated with polymeric nanoparticles at a concentration equivalent to 10 μg of doxorubicin and incubated at 37 °C for 24 h. At the end of incubation period, cells were rinsed three times with PBS to remove excess nanoparticles. Cells were then stained with 100 μL of 1742

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polymer sample, which can be used to calculate the number of folic acid molecules attached to the polymer. The C/N ratio of the Tri-FA polymer was found to be 15.33. By calculating the total number of carbon and nitrogen atoms in each unconjugated polymer and folic acid, the number of folic acid molecules in a polymer chain was calculated. According to the calculations, it was found that 1.86 molecules of folic acid were conjugated in a single polymer chain (i.e., about two molecules of folic acid per molecule of Tri-FA polymer). Doxorubicin was conjugated to the Tri-FA polymer via an acid-labile hydrazone linkage by reacting the remaining four −CONHNH2 end groups of the polymer with doxorubicin for 24 h in acidified DMSO in the dark. Unreacted doxorubicin was removed by dialysis, and red colored folate and doxorubicinconjugated triblock polymer (Tri-Dox-FA) was obtained after lyophilization. Doxorubicin conjugation to the triblock polymer was characterized using DSC. Similarly, Tri-CONHNH 2 polymer was treated with doxorubicin hydrochloride to synthesize doxorubicin-conjugated polymer (Tri-Dox) that is devoid of folic acid (nontargeted); it will serve as a control in the cell culture studies for determining the efficiency of folic acid as a targeting agent for enhanced cellular uptake of NPs in cancer cells. Doxorubicin content in the conjugated polymers was determined by treating the polymer with 0.1 N HCl at 25 ± 1 °C for 48 h to cleave the acid-labile hydrazone bond completely, releasing the conjugated doxorubicin. Released doxorubicin was quantified using a standard calibration curve equation and was found to be 37.22 μg/mg of Tri-Dox-FA polymer and 36.43 μg/mg of Tri-Dox polymer. 3.3. DSC Studies for the Determination of Doxorubicin and Folic Acid Conjugation. DSC was used to confirm the conjugation of doxorubicin with the triblock polymer and to address whether doxorubicin is conjugated to the polymer or is adsorbed on the surface. Supporting Information Figure S5A shows the thermograms of triblock polymer, doxorubicin hydrochloride, doxorubicin-conjugated polymer, and a physical mixture of the triblock polymer and doxorubicin hydrochloride. The DSC thermogram of the triblock polymer [Supporting Information Figure S5A (c)] showed three endothermic peaks: a sharp peak at 45.89 °C and broad peaks at 129.87 and 157.14 °C. Doxorubicin hydrochloride [Supporting Information Figure S5A (d)] also showed one sharp endothermic peak at 197.22 °C, corresponding to the melting peak of doxorubicin hydrochloride, and three small endothermic peaks at 211.52, 227.80, and 270.20 °C. Endothermic peaks of a physical mixture of the triblock polymer and doxorubicin hydrochloride [Supporting Information Figure S5A (b)] were observed in regions similar to those of the individual triblock polymer and doxorubicin hydrochloride, whereas in the case of the doxorubicin-conjugated polymer [Supporting Information Figure S5A (a)], the melting peak of doxorubicin hydrochloride disappeared and three prominent endothermic peaks at 45.30, 56.41, and 132.05 °C were observed. Thus, compared to individual thermograms of the triblock polymer, doxorubicin hydrochloride, and a physical mixture of the two, a significant difference was observed in the thermal behavior of the doxorubicin-conjugated polymer, which indicates a strong interaction between doxorubicin and the triblock polymer, suggesting a successful chemical conjugation of doxorubicin with the triblock polymer. Similarly, DSC was used to confirm the conjugation of folic acid with the triblock polymer. Supporting Information Figure

spectrum (Figure S2C) shows a broad band in the region of 3400−3500 cm−1, corresponding to hydroxyl groups of PEGMA units, and an intense vibration band at 1100 cm−1, which can be assigned to C−O−C (aliphatic ethers) of PEGMA. This confirms the presence of PEGMA units in the triblock polymer. The molecular weight of the triblock polymer was determined using gel-permeation chromatography (GPC) (Waters, USA) with a refractive index (RI) detector using THF as the eluting solvent. According to GPC, Mn and Mw of the synthesized polymer were found to be 5538 and 6543 Da, respectively, with a polydispersity index of 1.18 (Supporting Information Figure S3). According to 1H NMR, the molecular weight was found to be ∼7000 Da. Thus, polymers of the desired molecular weight with a narrow molecular weight distribution were successfully synthesized using ATRP. 3.2. Conjugation of Folic Acid and Doxorubicin to the pPEGMA−PCL−pPEGMA Triblock Polymer. To conjugate folic acid and doxorubicin, the end groups of the triblock polymer were functionalized with succinic anhydride and hydrazine in a two-step reaction to confer −CONHNH2 functionality to the triblock polymer. In the first step, the triblock polymer was treated with succinic anhydride in dry THF for 24 h to convert hydroxyl groups of the triblock polymer into carboxylic groups. The formation of the carboxylated triblock polymer (Tri-COOH) was confirmed by 1H NMR and ATR−FTIR. The FTIR spectrum (Supporting Information Figure S2D) depicts a broad vibration band in the region 2600−3400 cm−1, which can be attributed to O−H stretching vibrations of carboxylic functional groups of TriCOOH. 1H NMR shows a peak at δ 2.67, which corresponds to -CH2-CH2 group of succinic acid attached to the polymer (Supporting Information Figure S4). From the integration of peaks, it was observed that eight succinic anhydride molecules were conjugated to the polymer, resulting in eight −COOH end groups in the triblock copolymer chain. Out of eight −COOH end groups in the polymer chain, six were converted to -CONHNH2 for folate and doxorubicin conjugation. The remaining two -COOH end groups were reacted later with EDC−NHS for aptamer conjugation after nanoparticle preparation. In the next step, Tri-COOH was reacted with hydrazine (6 equiv of -COOH groups) to convert six -COOH end groups to six -CONHNH2 functionalities in the polymer chain (TriCONHNH2 polymer). The FTIR spectrum (Supporting Information Figure S2E) depicts the presence of a broad band at 3326 cm−1, confirming the presence of aliphatic amines in the polymer chain. The absence of a broad vibration band in the region 2600−3400 cm−1 (corresponding to carboxyl groups present in Tri-COOH) also confirms the conversion of −COOH end groups to −CONHNH2 end groups. Folic acid was then conjugated to the polymer using DCC− NHS chemistry. The γ-carboxylic acid of folic acid was first activated by DCC−NHS, and then activated folate (2 equiv) was further reacted with −CONHNH2 end groups of the triblock polymer to conjugate two molecules of folic acid per polymer chain. Unreacted folic acid was removed by dialysis, and yellow folate-conjugated triblock polymer (Tri-FA) was obtained after lyophilization. Conjugation of folic acid to the triblock polymer was characterized using DSC. CHN analysis was carried out to determine the number of folate groups attached per polymer chain in the Tri-FA polymer. CHN analysis determines the C/N ratio of the 1743

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Table 1. Optimization of Pluronic F68 Concentration for Nanoparticle Preparationa before lyophilization Pluronic F68 (%) 0.0 0.1 0.5 1.0 2.0 4.0 a

particle size (nm) 86.96 88.49 89.90 86.85 101.33 106.27

± ± ± ± ± ±

3.07 3.82 4.49 8.81 3.06 1.96

after lyophilization

polydispersity index 0.306 0.428 0.407 0.391 0.498 0.713

± ± ± ± ± ±

0.043 0.033 0.023 0.069 0.056 0.042

particle size (nm) 198.57 188.97 172.33 137.70 131.93 136.27

± ± ± ± ± ±

3.18 3.27 3.69 2.21 2.65 2.58

polydispersity index 0.241 0.363 0.417 0.242 0.240 0.591

± ± ± ± ± ±

0.035 0.036 0.052 0.024 0.055 0.040

Mean ± SD, n = 3.

Figure 2. Characterization of Tri-Dox-FA NPs using various techniques. (A) DLS, (B, C) TEM, and (D) SEM of Tri-Dox-FA NPs showing monodispersed matrix-structured solid nanoparticles with an apparent spherical morphology.

nm in the case of NPs prepared without Pluronic F68 as well as for NPs with Pluronic F68 concentrations of 0.1 and 0.5%. With 1% Pluronic F68, the average particle size obtained was 137.7 ± 2.2 nm (PDI 0.24 ± 0.01), which remained relatively constant at higher concentrations of Pluronic F68 (2 and 4%). Thus, the Pluronic F68 concentration was optimized at 1% and was used in the nanoparticle formulations for further studies. Nanoparticles of the Tri-Dox-FA polymer prepared with 1% Pluronic F68 showed a narrow size distribution and a low standard deviation, displaying low batch-to-batch variation. The average particle size of optimized NPs before and after lyophilization was 86.8 ± 8.8 (PDI 0.39 ± 0.07) and 137.7 ± 2.2 nm (PDI 0.24 ± 0.01), respectively. The reconstitution time of lyophilized nanoparticles was determined by the addition of 10 mL of water to the lyophilized vial and was found to be ∼1 min. Nanoparticles were readily dispersible, showing uniform dispersion and no aggregation. The lyophilization process generates a variety of stresses, such as freezing stress, phase separation at the ice−water interface, increased ionic strength, and others, which cause a destabilization of the colloidal nanoparticle suspension. To protect the nanoparticles during lyophilization and to increase their stability during storage, it is recommended that cryoprotectants are used in the nanoparticle formulation. Trehalose (5%) was used as a cryoprotectant for lyophilization of nanoparticles in the current study. Trehalose is a widely used cryoprotectant for the stabilization of nanoparticles during lyophilization. It offers many advantages compared to other cryoprotectants, such as chemical inertness, higher glasstransition temperature, less hygroscopicity, and the absence of internal hydrogen bonds that helps to create hydrogen bonds with nanoparticles for stabilization during lyophilization. Trehalose forms a glassy matrix, causing immobilization of the nanoparticles and preventing their aggregation.34−36 TEM and SEM of the polymeric nanoparticles were carried out to determine the absolute particle size of the nanoparticles because the DLS technique gives the hydrodynamic diameter of the nanoparticles. As shown in Figure 2, TEM images showed

S5B shows the thermograms of the triblock polymer, folic acid, folate-conjugated polymer, and a physical mixture of polymer and folic acid. The folic acid thermogram [Supporting Information Figure S5B (d)] showed two endothermic peaks at 133.79 and 195.68 °C. Endothermic peaks of the physical mixture of triblock polymer and folic acid [Supporting Information Figure S5B (b)] were observed in regions similar to those of the individual triblock polymer and folic acid, whereas in the case of the folate-conjugated polymer [Supporting Information Figure S5B (a)], one prominent endothermic peak at 111.40 °C with one small peak at 49.51 °C was observed. Thus, compared to the individual thermograms of the triblock polymer, folic acid, and a physical mixture of the two, there was an apparent difference in the thermal behavior of the folate-conjugated polymer, which suggests a strong interaction between folic acid and the triblock polymer, indicating a successful chemical conjugation of folic acid with the triblock polymer. 3.4. Preparation and Characterization of Nanoparticles. The nanoprecipitation method developed by Fessi et al.31 was used for the preparation of nanoparticles. Nanoparticle formation during nanoprecipitation is primarily governed by the diffusion stranding phenomenon and the Marangoni effect.32,33 The average particle size of Tri-Dox-FA polymeric nanoparticles prepared without surfactant was 86.96 ± 3.1 nm with a polydispersity index (PDI) of 0.31 ± 0.04, but after lyophilization, they aggregated and were difficult to redisperse. To prevent the aggregation and to stabilize the nanoparticle suspension, Pluronic F68 was used as a surfactant. The Pluronic F68 surfactant concentration was optimized by preparing nanoparticles with varying concentrations of Pluronic F68 (0.1, 0.5, 1, 2, and 4%). The particle size of the resulting nanoparticles was determined before and after lyophilization using DLS. As shown in Table 1, before lyophilization, nanoparticles were in the range of 85−120 nm for all concentrations of Pluronic F68 used. After lyophilization, however, the average particle size obtained was higher than 150 1744

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Table 2. In Vitro Stability Studies of Nanoparticles in Normal Saline and DMEMa normal saline (0.9% w/v NaCl solution) time (h) triblock NPs 1 3 6 9 24 Tri-Dox-FA NPs 1 3 6 9 24 a

particle size (nm)

polydispersity index

DMEM particle size (nm)

polydispersity index

101.07 95.91 115.92 106.80 114.22

± ± ± ± ±

2.38 0.69 15.98 0.87 14.09

0.451 0.428 0.478 0.596 0.340

± ± ± ± ±

0.038 0.020 0.142 0.050 0.055

120.93 122.43 111.97 115.10 119.87

± ± ± ± ±

3.00 0.50 2.50 0.89 2.14

0.436 0.437 0.440 0.424 0.631

± ± ± ± ±

0.010 0.006 0.050 0.078 0.058

83.39 78.41 97.70 105.43 117.50

± ± ± ± ±

1.83 0.51 1.68 11.92 3.90

0.389 0.374 0.511 0.492 0.338

± ± ± ± ±

0.010 0.013 0.066 0.068 0.014

99.20 97.95 97.15 110.13 125.03

± ± ± ± ±

2.45 1.80 1.07 9.11 1.70

0.542 0.550 0.533 0.480 0.563

± ± ± ± ±

0.043 0.038 0.097 0.014 0.144

Mean ± SD, n = 3.

electrophilicity of carbon (−CN−NH2), which makes it highly susceptible to hydrolysis.37,38 This results in regeneration of a carbonyl group, thus releasing the drug in the acidic environment. After 48 h, ∼70.30% of doxorubicin was released at pH 5.0, whereas only small amount of doxorubicin was released at pH 7.4 (∼25.09%), as desired. The initial rapid release of doxorubicin at pH 5.0 can be attributed to the breaking of the hydrazone bond to release doxorubicin. The acid-labile hydrazone bond prevents doxorubicin leakage/ release in the blood during circulation, thus minimizing the side effects of doxorubicin on normal healthy cells. 3.7. Protein Adsorption Study. Evaluating protein adsorption on the nanoparticle surface is important to determine nanoparticle stability in the blood and to determine the circulation half-life of the nanoparticles. In the case of nanoparticles with a hydrophobic surface, they tend to adsorb blood proteins on their surface, resulting in enhanced macrophage recognition and opsonization and leading to the elimination of NPs from circulation and thus limiting their efficiency. One approach to improve the circulation time of NPs and to decrease protein adsorption on their surfaces is to have hydrophilic molecules on the surface of NPs. The presence of hydrophilic poly(ethylene glycol) chains in the triblock polymer imparts a stealth nature to the nanoparticles, which helps to avoid macrophage recognition, opsonization, and protein adsorption on the nanoparticle surface. This results in an enhanced circulation half-life of the nanoparticles and an increased probability of accumulation at the target site.39−41 Nanoparticles were incubated with 4% BSA to simulate the physiological albumin concentration in blood. After a 24 h incubation with 4% BSA, unconjugated triblock polymeric nanoparticles showed 5.00 ± 1.07% BSA adsorption, whereas doxorubicin-conjugated polymeric nanoparticles (Tri-Dox-FA NPs) showed 8.99 ± 1.41% BSA adsorption. Lower protein adsorption on the surface of the NPs can be attributed to the PEG chains present in the polymeric structure of the NPs. PEG chains render hydrophilicity to the surface of the NPs as well as steric repulsion because of their length and thus they prevent proteins from being adsorbed on the nanoparticle surface. The higher relative protein adsorption in the case of the doxorubicin-conjugated polymer compared to the unconjugated polymer can be attributed to the higher hydrophobicity imparted by the attached doxorubicin and folic acid, which are both hydrophobic molecules.

solid-matrix-structured nanoparticles with an apparent spherical morphology and an average particle size in the range ∼80−120 nm, which is consistent with the DLS results. The SEM image is consistent with the TEM results, showing matrix-structured nanoparticles. 3.5. In Vitro Stability Studies of the Nanoparticles. Nanoparticle stability is an important factor for the clinical use of Tri-Dox-FA NPs because nanoparticles could aggregate and clog blood vessels, resulting in severe complications. The stability of triblock polymer NPs and Tri-Dox-FA NPs was studied in normal saline (0.9% w/v NaCl) and DMEM at 37 °C to simulate nanoparticle stability in blood and in cell culture experiments, respectively. As shown in Table 2, the nanoparticles did not show any substantial increase in particle size in normal saline and DMEM, with an average particle size remained below 125 nm up to 24 h, indicating the high stability of the nanoparticles. 3.6. Drug Release Studies. In vitro release studies of doxorubicin from the conjugated nanosystem (Tri-Dox-FA) were carried out at 37 ± 1 °C in two buffer systems: phosphate buffer, pH 7.4, which simulates physiological pH (pH 7.4), and acetate buffer, pH 5.0, which simulates endolysosomal pH (pH 4.5−6.0). As shown in Figure 3, doxorubicin release was significantly higher in acetate buffer, pH 5.0, compared to that in phosphate buffer, pH 7.4, owing to the acid-labile hydrazone linkage through which doxorubicin is conjugated to the triblock polymer. The acid-labile nature of hydrazone is because of the acid-catalyzed hydrolysis of the CN bond. Protonation of nitrogen (−CN−NH2) in hydrazone leads to the enhanced

Figure 3. Cumulative doxorubicin release profile from Tri-Dox-FA NPs in acetate buffer, pH 5.0, and phosphate buffer, pH 7.4, at 37 ± 1 °C. (Mean ± SD, n = 3.) 1745

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3.8. Blood Compatibility. Blood compatibility of nanoparticles was determined using hemolysis and coagulation studies. 3.8.1. Hemolysis Study. Hemocompatibility is an essential criterion for the safety and nontoxicity of nanoparticles when they are to be administered intravenously. Nanoparticle interaction with erythrocytes (RBCs) may cause erythrocyte damage, resulting in the release of hemoglobin from the erythrocytes. The released hemoglobin can be determined spectrophotometrically at 540 nm and is inversely proportional to the hemocompatibility of the nanoparticles. Some studies consider a hemolysis value of less than 10% to be nonhemolytic and a value greater than 25% to be hemolytic,42,43 whereas according to other studies, a hemolysis value of less than 20% is considered acceptable for hemocompatibility.44−46 Polymer nanoparticles with concentrations ranging from 0.125 to 2.5 mg/mL were incubated with an RBC stock solution for 1 h at 37 °C. As shown in Figure 4, nanoparticles at

Table 3. Coagulation Studies of the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) To Determine the Hemocompatibility of Tri-Dox-FA Nanoparticlesa Tri-Dox-FA NPs (mg/mL) control (PBS) 0.01 0.10 1.00 a

PT (s) 13.2 13.3 13.1 13.4

± ± ± ±

0.1 0.3 0.1 0.4

aPTT (s) 30.1 33.2 29.6 30.2

± ± ± ±

0.1 0.4 0.2 1.0

Mean ± SD, n = 3.

activated NPs, which were then reacted with amine-modified AS1411 aptamer to form aptamer-conjugated NPs: Tri-DoxFA-A NPs (Folate and AS1411 aptamer-targeted doxorubicinconjugated NPs) and Tri-Dox-A NPs (AS1411 aptamertargeted doxorubicin-conjugated NPs). The composition of each polymer is given in Table 4. Table 4. Composition of Targeted Polymers polymer

composition

Tri-Dox Tri-Dox-FA Tri-Dox-A Tri-Dox-FA-A

doxorubicin-conjugated triblock polymer (without any ligand) doxorubicin-conjugated triblock polymer with folic acid doxorubicin-conjugated triblock polymer with AS1411 aptamer doxorubicin-conjugated triblock polymer with folic acid and AS1411 aptamer

AS1411 aptamer conjugation on the surface of nanoparticles was confirmed using zeta potential measurement and XPS. As shown in Table 5, the zeta potential of Tri-Dox-FA NPs and

Figure 4. Percent hemolysis with different concentrations of triblock polymeric nanoparticles. (Mean ± SD, n = 3.)

a maximum concentration of 2.5 mg/mL showed 1.32 ± 0.54% hemolysis, whereas lower concentrations showed a similar amount of hemolysis as that of negative control (PBS), indicating the hemocompatibility of the polymer, with no apparent toxicity to RBCs in the concentration range 0.125−2.5 mg/mL. 3.8.2. Coagulation Studies. After entering the circulation, nanoparticles may interact with coagulation factors in the blood and can induce alterations in their functions, causing severe lifethreatening toxicities, termed nanoparticle-induced coagulopathies (e.g., intravascular thrombosis, deep vein thrombosis, etc.) leading to multiple organ failure and death.47,48 PT is related to the activity of the extrinsic coagulation pathway, whereas aPTT is related to the activity of the intrinsic coagulation pathway. Any changes in PT and aPTT are direct indicators of the impact of nanoparticles on the coagulation system. Polymeric nanoparticles in the concentration range 0.01−1 mg/mL were incubated with blood for 1 h, and PT and aPTT were calculated. The normal range for PT is 11−14 s, whereas for aPTT, it is 27−40 s.49 As shown in Table 3, at nanoparticle concentrations ranging from 0.01 to 1 mg/mL, no apparent change in PT and aPTT was observed. All results were within the normal range, which indicates that the polymeric nanoparticles did not cause activation of the coagulation system and are thus biocompatible with respect to coagulation studies in the concentration range 0.01−1 mg/ mL. 3.9. Aptamer Conjugation on Nanoparticles. AS1411 aptamer was conjugated to Tri-Dox-FA NPs and Tri-Dox NPs in two steps. In the first step, carboxylic groups of polymeric NPs were activated using EDC−NHS chemistry to form NHS-

Table 5. Determination of AS1411 Aptamer Conjugation on Nanoparticles Using Zeta Potential Measurementsa polymer NPs

zeta potential (mV)

Before Aptamer Conjugation Tri-Dox-FA NPs −23.1 Tri-Dox NPs −11.5 After Aptamer Conjugation Tri-Dox-FA-A NPs −36.1 Tri-Dox-A NPs −24.3 a

± 0.9 ± 0.6 ± 0.4 ± 2.6

Mean ± SD, n = 3.

Tri-Dox NPs before aptamer conjugation was −23.1 ± 0.9 and −11.5 ± 0.6 mV, respectively. After aptamer conjugation, the zeta potential of the resulting Tri-Dox-FA-A NPs and Tri-DoxA NPs was −36.1 ± 0.4 and −24.3 ± 2.6 mV, respectively. The negative shift in the zeta potential of NPs after aptamer conjugation can be attributed to the negatively charged phosphate groups of AS1411 (a DNA aptamer), indicating successful conjugation of negatively charged AS1411 aptamer on the surface of the NPs. The surface elemental composition of the aptamerconjugated nanoparticles was determined using XPS, as shown in Table 6. Before aptamer conjugation, the elemental composition of Tri-Dox-FA NPs and Tri-Dox NPs consisted of C, O, and N, but no Phosphorus (P) was detected. After aptamer conjugation, P was detected in both the Tri-Dox-FA-A and Tri-Dox-A samples, which can be ascribed only to P from the AS1411 aptamer, confirming the decoration of the AS1411 aptamer on the nanoparticle surface. Also, the N content of 1746

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Table 6. Determination of AS1411 Aptamer Conjugation on Nanoparticles Using XPSa elemental composition polymer NPs

a

C (%)

Tri-Dox-FA NPs Tri-Dox NPs

78.77 ± 2.35 80.18 ± 3.76

Tri-Dox-FA-A NPs Tri-Dox-A NPs

74.26 ± 3.07 71.16 ± 4.45

O (%) Before Aptamer Conjugation 20.18 ± 1.96 19.71 ± 2.43 After Aptamer Conjugation 23.65 ± 2.67 27.26 ± 2.59

N (%)

P (%)

1.05 ± 0.25 0.10 ± 0.07

0.00 ± 0.00 0.00 ± 0.00

1.94 ± 0.87 1.34 ± 0.21

0.16 ± 0.09 0.24 ± 0.07

Mean ± SD, n = 3.

Figure 5. MTT assay depicting the effect of triblock polymeric NPs (Tri NPs) and doxorubicin-conjugated polymeric nanoparticles (Tri-Dox-FA NPs) on the viability of L929 and MCF-7 cells (mean ± SD, n = 3). The effect of Tri NPs on both noncancerous cells (L929, left) and cancerous cells (MCF-7, right) were similar at all concentrations (0.125−2 mg/mL), with no statistical difference between two cell types (cancerous and noncancerous) (**p > 0.5 and *p > 0.2, respectively).

folate-targeted NPs (Tri-Dox-FA NPs), aptamer-targeted NPs (Tri-Dox-A NPs), and dual-targeted folate−aptamer-targeted NPs (Tri-Dox-FA-A NPs)] in the MCF-7, L929, and PANC-1 cell lines were examined to evaluate the targeting efficiency of nanoparticles conjugated with folate and/or aptamer. The folate receptor, mainly the RFα isoform of the folate receptor, is minimally expressed in normal cells but is overexpressed in several tumors of epithelial origin because rapidly dividing cancer cells require folic acid for the synthesis of nucleotides and DNA. RFα receptor binds and internalizes folic acid in the cells via an endocytosis pathway. RFα folate receptor has a high binding affinity toward folic acid, with a dissociation constant (a constant representing the binding affinity of RFα toward folic acid) Kd < 1 nM, whereas reduced folate carrier (RFC), a folate binding protein present ubiquitously in normal cells, is involved in the majority of the folate transport across the cell membrane. It has a high affinity for reduced folates, with a Michaelis constant (a constant representing the binding affinity as well as the transport of folates by RFC) Km = 1−10 μM, but it has relatively low affinity toward folic acid with a Km = 200−400 μM.15 Thus, folic acid makes a good targeting ligand for specifically targeting cancer cells. Nucleolin receptors have also been reported to be overexpressed in most cancer cells, making the AS1411 aptamer a good homing agent for cancer cells. The cellular uptake of NPs and their localization within the cells was observed in MCF-7, L929, and PANC-1 cell lines after a 2 h incubation using confocal laser scanning microscopy, as shown in Figure 6. LysoTracker Green was used for colocalization study and to determine the intracellular localization of NPs, whereas Hoechst stain was used for nuclear staining. As shown in Figure 6, in the case of MCF-7 and PANC-1 cells (cancer cell lines) treated with folate-targeted NPs (TriDox-FA NPs), a higher doxorubicin fluorescence intensity was observed, indicating a higher cellular uptake compared to cells

both of the polymeric NPs increased after aptamer conjugation because of N from the AS1411 aptamer.50 3.10. Cell Viability Studies. The cytotoxicity of the polymeric nanoparticles was determined in the MCF-7 and L929 cell lines using the MTT cytotoxicity assay. The MTT assay measures cellular oxidative metabolic activity based on the activity of NAD(P)H-dependent dehydrogenase enzymes.51 The effect of various concentrations (0.125−2 mg/mL) of triblock polymeric NPs (Tri NPs) and doxorubicin-conjugated polymeric NPs (Tri-Dox-FA NPs) on the viability of MCF-7 and L929 (cancerous and noncancerous cell lines, respectively) cells was evaluated using MTT assay, as shown in Figure 5. In the case of the L929 noncancerous mouse fibroblast cell line, incubation with triblock polymeric NPs showed cell viability above ∼93% in the concentration range 0.125−2 mg/ mL, indicating the biocompatibility and nontoxicity of the triblock polymer toward normal cells. Tri-Dox-FA NPs, however, showed slight toxicity toward L929 cells (∼80% cell viability in the concentration range 0.125−2 mg/mL). This can be attributed to the nonspecific cellular uptake of Tri-Dox-FA NPs by L929 cells. In the case of the MCF-7 breast cancer cell line, after treatment with Tri NPs in the concentrations range 0.125−2 mg/mL, the viability of the cells was above ∼92%, indicating the biocompatibility of the triblock polymer, whereas in the case of Tri-Dox-FA NPs, because of folate-mediated cellular uptake of NPs, the viability of MCF-7 cells gradually decreased as the concentration of Tri-Dox-FA NPs increased from 0.125 to 2 mg/mL. These results indicate that the triblock polymeric nanoparticles are biocompatible in the concentration range 0.125−2 mg/mL with respect to cytotoxicity and thus are appropriate as drug carriers. 3.11. In Vitro Cellular Uptake. The cellular uptake of polymeric nanoparticles [non-targeted NPs (Tri-Dox NPs), 1747

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Figure 6. Cellular uptake of nanoparticles in MCF-7 (top left), L929 (top right), and PANC-1 (bottom) cells lines, as observed by confocal laser scanning microscopy (100× magnification). (A) Tri-Dox NPs, (B) Tri-Dox-FA NPs, (C) Tri-Dox-A NPs, and (D) Tri-Dox-FA-A NPs.

Figure 7. Cellular uptake of nanoparticles in cell lines observed by FACS. (A) MCF-7, (B) L929, and (C) PANC-1 cells.

treated with Tri-Dox NPs, which are devoid of any targeting moiety. The higher cellular uptake with Tri-Dox-FA NPs can be

attributed to folate-mediated endocytosis of NPs, resulting in a higher accumulation of NPs in the cells. Similarly, cells treated 1748

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Figure 8. Doxorubicin internalization in the nucleus of MCF-7 (top left), L929 (top right), and PANC-1 (bottom) cells observed by confocal microscopy (100× magnification). (A) Tri-Dox NPs, (B) Tri-Dox-FA NPs, (C) Tri-Dox-A NPs, and (D) Tri-Dox-FA-A NPs.

3.12. Cellular Uptake Studies Using Fluorescence Activated Cell Sorting (FACS). To verify the enhanced uptake of dual-targeted nanosystems in MCF-7 and PANC-1 cells, as observed in confocal studies, cellular uptake was studied using FACS. FACS separates cells based on the fluorescence intensity of cells treated with doxorubicinconjugated NPs. As shown in Figure 7, MCF-7 and PANC-1 cells treated with folate-targeted NPs (Tri-Dox-FA NPs) and aptamer-targeted NPs (Tri-Dox-A NPs), showed a 10-fold increase in mean fluorescence intensity compared to cells treated with nontargeted NPs (Tri-Dox NPs). This effect can be attributed to folate/aptamer-mediated endocytosis of NPs into the cells. In the case of the dual-targeted NPs (Tri-Dox-FA-A NPs), the cells showed a 100-fold increase in mean fluorescence intensity compared to cells treated with non-targeted NPs and a 10-fold increase in mean fluorescence intensity compared to to cells treated with single-targeted NPs (Tri-Dox-FA NPs and TriDox-A NPs), indicating the superior targeting efficiency of the dual-targeted nanosystem compared to the single-targeted

with aptamer-targeted NPs (Tri-Dox-A NPs) showed a higher cellular uptake similar to Tri-Dox-FA NPs compared to cells treated with non-targeted Tri-Dox NPs owing to nucleolinmediated endocytosis of NPs. In the case of cells treated with dual-targeted NPs (i.e., both folate and aptamer-targeted NPs (Tri-Dox-FA-A NPs)), doxorubicin intensity was found to be higher than that in cells treated with single-targeted NPs (TriDox-FA and Tri-Dox-A), indicating a higher efficiency and effectiveness of the dual-targeted NPs for targeting and delivering the drug to cancer cells compared to the singletargeted nanosystems. L929 cells are normal cells (noncancerous) and hence they do not overexpress folic acid or nucleolin receptors. As shown in Figure 6, significantly less doxorubicin fluorescence was observed in the cells treated with NPs. The low doxorubicin fluorescence signal was similar in all cells treated with all four polymeric nanoparticles irrespective of the targeting moieties attached, indicating some nonspecific uptake of nanoparticles by L929 cells. 1749

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Figure 9. Annexin V−FITC apoptosis assay for the determination of apoptotic/necrotic cells in MCF-7 (top), L929 (middle), and PANC-1 (bottom) cells using flow cytometry.

condensation was observed by confocal microscopy, as shown in Figure 8.56 3.14. Annexin V−FITC Apoptosis Assay. The annexin V−FITC assay is a relatively simple, rapid, and reliable method to distinguish apoptotic cells and necrotic cells from live cells. Cells undergoing apoptosis exhibit changes in the membrane phospholipids, resulting in the exposure of phosphatidylserine on the outer cell surface. Annexin V is a calcium-dependent phospholipid-binding protein that selectively binds to exposed phosphatidylserine and is thus able to detect apoptotic cells. Propidium iodide nonspecifically intercalates the DNA of cells whose plasma membrane has been compromised. Necrotic cells generally lose their membrane integrity and hence become stained with both Annexin V−FITC as well as propidium iodide. Apoptotic cells, however, because their membrane is intact, do not take up propidium iodide dye and hence can be differentiated from necrotic cells. Healthy live cells do not become stained by either of the dyes and remain unaffected in their fluorescence spectrum.57,58 As shown in Figure 9, the fluorescence intensity of the cells was plotted with Annexin V−FITC fluorescence on the x axis and propidium iodide fluorescence on the y axis to distinguish viable cells (lower left quadrant, i.e., negative for both Annexin V and propidium iodide), early apoptotic cells (lower right quadrant, i.e., Annexin V-positive, propidium iodide-negative), late apoptotic and necrotic cells (upper right quadrant, i.e., positive for both Annexin V and propidium iodide), and damaged necrotic cells (upper left quadrant, i.e., Annexin Vnegative and propidium iodide-positive).59,60 In the case of MCF-7 and PANC-1 cells, untreated control cells were devoid of any apoptotic or necrotic cells. Singletargeted NPs (Tri-Dox-FA NPs and Tri-Dox-A NPs) resulted in a higher percent of apoptotic cells (14.33 and 10.30% in MCF-7; 18.81 and 21.58% in PANC-1, respectively) and necrotic cells (37.56 and 27.29% in MCF-7; 34.20 and 37.64% in PANC-1 respectively) compared to non-targeted NPs (TriDox NPs) (5.01% apoptotic cells and 25.44% necrotic cells in MCF-7; 0.66% apoptotic cells and 9.05% necrotic cells in PANC-1 cells). The higher percentage of apoptotic and necrotic cells in Tri-Dox-FA NPs- and Tri-Dox-A NPs-treated

nanosystems. In the case of the L929 cells, a low mean fluorescence intensity was observed in all cells treated with all four polymeric nanoparticles irrespective of the targeting ligand attached, similar to the results obtained in the confocal studies. This study clearly indicates the higher efficiency of the dualtargeted nanosystems for the delivery of doxorubicinconjugated NPs to cancer cells. 3.13. Nuclear Localization of Doxorubicin. It is hypothesized that after receptor-mediated endocytosis, nanoparticles travel inside the cell through endosomes and endolysosomes. The acidic pH in endolysosomes breaks the acid-labile hydrazone bond, releasing the doxorubicin. Doxorubicin freely diffuses through the endolysosomal membrane and enters the nucleus. It then intercalates the DNA, resulting in intranuclear accumulation of doxorubicin.52 To investigate the nuclear localization of doxorubicin, cells were incubated with NPs for 24 h and observed under a confocal laser scanning microscope. As shown in Figure 8, in the case of MCF-7 and PANC-1 cells treated with dual-targeted NPs (Tri-Dox-FA-A NPs), the doxorubicin fluorescence intensity inside the nucleus was higher compared to cells treated with single-targeted NPs (TriDox-FA and Tri-Dox-A NPs) and non-targeted NPs (Tri-Dox NPs) (very low doxorubicin fluorescence in the nucleus). In the case of L929 cells, very low doxorubicin fluorescence was observed, as expected, in all cells treated with all four polymeric nanoparticles irrespective of the targeting ligand. Changes in cell morphology resulting in the rounding of cells is one of the prominent hallmarks of apoptosis. In addition, a change in nuclear morphology associated with chromatin condensation/clumping is another distinct hallmark of apoptosis.53−55 Cells treated with NPs showed a rounding of the cells in all cases, indicating that cells were undergoing apoptosis because of the action of doxorubicin in the nucleus. As observed in the cellular uptake studies, after a 2 h incubation with NPs (Figure 6), the nuclei from MCF-7 and PANC-1 cells showed condensed chromatin, indicating preapoptotic cells or cells at an early apoptosis stage. After a 24 h incubation, doxorubicin was able to enter the nucleus, and because of its intercalating ability with DNA, complete nuclear chromatin 1750

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Biomacromolecules cells can be attributed to receptor-mediated endocytosis and accumulation of the NPs in the cells. Dual-targeted NPs with folate as well as aptamer (Tri-Dox-FA-A NPs) resulted in a higher percent of apoptotic and necrotic cells (55.08 and 7.59% in MCF-7; 63.89 and 25.15% in PANC-1, respectively) compared to single-targeted NPs, indicating the higher efficiency of the dual-targeted systems for the cellular uptake of NPs, resulting in the higher apoptosis of the cancer cells. In the case of L929 cells, the majority of treated cells were healthy and did not take up Annexin V or propidium iodide. Treated cells showed similar results as those of control (untreated) cells, indicating the low induction of apoptosis and low toxicity of the nanoparticles to the normal cell line that lacks overexpressed folate and/or nucleolin receptors on its cell surface. The results obtained showed a significant increase in early apoptotic cells (Annexin V-positive cells) and late apoptotic and necrotic cells (both Annexin V- and propidium iodidepositive cells) after treatment with dual-targeted NPs. This effect can be attributed to folate as well as nucleolin receptormediated endocytosis of NPs in MCF-7 and PANC-1 cells, resulting in the enhanced uptake of NPs and leading to a higher doxorubicin payload inside the nucleus, which initiated apoptosis.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of polycaprolactone diol, polycaprolactone macroinitiator, pPEGMA−PCL−pPEGMA triblock copolymer, and tri-COOH polymer; FTIR spectra of polycaprolactone diol, polycaprolactone macroinitiator, pPEGMA−PCL−pPEGMA triblock copolymer, tri-COOH polymer, and tri-CONHNH2 polymer; GPC spectra of pPEGMA−PCL−pPEGMA triblock copolymer; DSC studies for the determination of doxorubicin and folic acid conjugation; and spectrophotometric standard calibration curve of doxorubicin. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

Shantanu Lale is thankful to the Department of Biotechnology (DBT), India for research funding (project no. BT/PR13341/ NNT/28/467/2009) and the Indian Institute of Technology, Delhi for awarding an Institute Fellowship. He is also thankful to Dr. Aby C. P. for help with the XPS studies at Toyo University, Japan and to Prof. Renu Saxena from the All India Institute of Medical Sciences (AIIMS), New Delhi for help with the coagulation studies.

4. CONCLUSIONS Dual-functionalized pH-sensitive biocompatible polymeric nanoparticles for the targeted delivery of doxorubicin to cancer cells were developed. Dual targeting with folate and the AS1411 aptamer increased the cancer cell-targeting efficiency of the nanoparticles, which resulted in the enhanced cellular uptake and higher payload of doxorubicin in the cancer cells with subsequent higher induction of apoptosis, while at the same time sparing normal cells from the toxic effects of doxorubicin. The pH-sensitive hydrazone linkage controlled the release of doxorubicin by allowing its release mainly in the acidic environment, thus reducing doxorubicin toxicity to normal healthy cells. The results indicate that the dual-targeted pHsensitive biocompatible polymeric nanosystem can act as a promising weapon in the war against cancer. This simple nanosystem possesses high biocompatibility and shows promise for its ability to be translated to preclinical and clinical settings.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91 1126591041; E-mail: [email protected]. Notes

The authors declare no competing financial interest. 1751

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NOTE ADDED AFTER ASAP PUBLICATION This article posted ASAP on April 15, 2014. In the Results and Discussion section, paragraph 3, sentence 1, paragraph 5, sentence 6, and Figure 6 caption have been revised. The correct version posted on April 18, 2014.

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