Journal of Controlled Release 209 (2015) 88–100

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In vitro and in vivo evaluation of therapy targeting epithelial-cell adhesion-molecule aptamers for non-small cell lung cancer Mona Alibolandi a, Mohammad Ramezani b,c, Khalil Abnous b, Fatemeh Sadeghi d,e, Fatemeh Atyabi f, Mohsen Asouri g, Ali Asghar Ahmadi g, Farzin Hadizadeh a,⁎ a

Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Nanothechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran d Targeted Drug Delivery Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran e Department of Pharmaceutics, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran f Nanothechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran g North Research Center, Pasteur Institute of Iran, Amol, Iran b c

a r t i c l e

i n f o

Article history: Received 17 February 2015 Received in revised form 17 April 2015 Accepted 21 April 2015 Available online 23 April 2015 Keywords: Epithelial cell adhesion molecule Doxorubicin Nanopolymersome PEG–PLGA Non-small cell lung cancer SK-MES-1

a b s t r a c t Targeted, disease-specific delivery of therapeutic nanoparticles shows wonderful promise for transmitting highly cytotoxic anti-cancer agents. Using the reaction of non-small cell lung cancer (SK-MES-1 and A549 cell lines) as representative of other cancer types', the present study examines the effects of EpCAM-fluoropyrimidine RNA aptamer-decorated, DOX-loaded, PLGA-b-PEG nanopolymersomes that bond specifically to the extracellular domain of epithelial-cell adhesion molecules. Results demonstrate that EpCAM aptamer-conjugated DOX-NPs (Apt-DOX-NP) significantly enhance cellular nanoparticle uptake in SK-MES-1 and A549 cell lines and increase the cytotoxicity of the DOX payload as compared with non-targeted DOX-NP (P b 0.05). Additionally, Apt-DOX-NP exhibits greater tumor inhibition in nude mice bearing SK-MES-1 non-small cell lungcancer xenografts and reduces toxicity, as determined by loss of body weight, cardiac histopathology and animal survival rate in vivo. After a single intravenous injection of Apt-DOX-NP and DOX-NPs, tumor volume decreased 60.9% and 31.4%, respectively, in SK-MES-1-xenograft nude mice compared with members of a saline-injected control group. This study proves the potential utility of Apt-DOX-NP for therapeutic application in non-small cell lung cancer. In the future, EpCAM-targeted therapies might play a key role in treating non-small cell lung cancer, the most common type of lung cancer. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide and is particularly prevalent in economically developing countries because of smoking and ambient air pollution [1]. Most lung cancer patients are active tobacco smokers. Based on its histologic appearance, lung cancer can generally be classified into two major types: small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [2]. In comparison with the small-cell type, non-small cell lung cancer is relatively insensitive to chemotherapy and accounts for between 85% and 90% of lung-cancer deaths [3,4]. During its first stage, NSCLC is treated using surgical resection with curative intent, while chemotherapy is generally used both pre⁎ Corresponding author at: Biotechnology Research Center, School of Pharmacy, Mashhad, University of Medical Sciences, P.O. Box 9196773117, Mashhad, Iran. E-mail address: [email protected] (F. Hadizadeh).

http://dx.doi.org/10.1016/j.jconrel.2015.04.026 0168-3659/© 2015 Elsevier B.V. All rights reserved.

operatively (neo-adjuvant chemotherapy) and post-operatively (adjuvant chemotherapy) [5]. Due to a lack of capable diagnostic tools for detecting lung cancer early and for effectively administering therapy, lung cancer is considered a serious illness and is a major cause of death [6,7]. Over the past several decades, numerous attempts have been made to address aforementioned issues, and now targeted nanotherapeutic systems are being examined as a possible solution [8–10]. The physiochemical characteristics of such nanoscale systems, especially polymeric nanoparticles, align favorably with developing a versatile cancer celltargeted nanotherapeutic system [11–13]. The nanoscale dimension of these therapeutic systems confers the advantage of passive tumor-site targeting through enhanced permeation and retention (EPR) effects, which increase the encapsulated drugs' therapeutic potency [14–16]. In solid tumors, such as lung cancer, the EPR effect plays a crucial role in therapeutic efficacy of the nanoparticle-based chemotherapy [17]. The existence of a highly fenestrated blood vasculature in the tumor

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tissues promotes the EPR effect and enhances uptake of nano-scale vehicles (less than 200 nm in size) into the tumor. Moreover, the poor lymphatic flow in the tumor tissue increases EPR effect and enhances retention of nanoparticles within the tumor tissue. On the other hand, vehicles less than 50 nm in size extravasate from the tumor tissue through the fenestrated vessels of the tumor blood vascular system and consequently, their retention time in the tumor site is decreased [18]. Previous studies have proved that the poorly developed lymphatic drainage of shielded or pegylated nanoparticles also contributes indirectly to nanoparticle accumulation at the tumor site [19,20]. Additionally, research has shown that polymeric nanoparticles achieve kidney clearance, consequently allowing the therapeutics they carry good blood-circulation time [21]. However, the most crucial feature of polymeric nanoparticles is their ability to load and carry high quantities of anticancer agents [22]. Current therapeutic strategies, such as routine chemotherapy regimens and radiation therapy, are only effective in the treatment of stage I SCLC, whereas NSCLC is less sensitive to these kinds of treatments, leaving nanoscale therapeutics and resection as versatile, alternative NSCLC treatments [23]. In this regard, lung-cancer treatments that use polymeric nanoparticles are attracting increased attention for their novel intrinsic physical properties and their ability to functionalize and target specific tissues, thereby minimizing patients' suffering the severe side effects of anti-cancer agents. Research into available anti-cancer formulations based on polymeric nanoparticles, including nano-sprays and nanogels (both of which are intratracheally administered to the lungs), have yielded promising results [24–28]. Recent studies suggest that intratracheal drug delivery for lung-cancer therapy is preferable to the parenteral route, but the concept of treating lung cancer through inhalation delivery of chemotherapeutics is not safe enough because of anti-cancer agents' severe cardiac toxicity [29,30]. Hence, intravenous administration of anticancer agents is still the main chemotherapy strategy in lung-cancer treatment, and consequently, there is serious demand for a high-tech, safe lung cancer-treatment strategy. To meet this demand, Dai et al. [31] developed pegylated dihydroartemisinin nanoparticles (PEG-DHA) for lung-cancer treatment. The obtained results proved that their PEG-DHA nanoparticles have a significant therapeutic effect on inhibiting NSCLC-tumor growth in vivo. Another innovative concept for treating lung cancer, meanwhile, is to deliver anti-cancer agents to cancer stem cells using targeted drugdelivery systems, which have received only cursory attention thus far. This cancer stem-cell (CSC) theory assumes the existence of undifferentiated cells responsible for tumor initiation and relapse [32,33]. Therefore, targeting CSCs could be a versatile strategy for combating non-small cell lung cancer, which is more resistant to radiotherapy and routine chemotherapeutics; indeed, the complete eradication of lung cancer would require an entirely new approach, such as the utilization of CSC-targeted nanoscale therapeutics. Contributing to the development of such an approach, one previous study performed a precise characterization of putative NSCLC-CSC markers [34]. In particular, the study found epithelial cell-adhesion molecules (EpCAMs) to be CSC markers overexpressed in non-small cell lung cancers with A549 and SK-MES-1 adenocarcinoma lung-cancer cell lines [35]. The EpCAM is a glycosylated transmembrane protein that has oncogenic potential because of its capacity to upregulate cmyc, e-fabp, and cyclins A and E [36]. Moreover, EpCAM mediates Ca2+-independent, homotypic cell-to-cell adhesions in the epithelia, is highly expressed in many adenocarcinomas, and exhibits a low level of expression in normal tissues [37]. In lung cancer, EpCAM overexpression is related to overall unfavorable survival rates for NSCLC patients [38]. In fact, mounting evidence suggests EpCAM-positive tumor cells are tumor-initiating cells with stem- or progenitor-cell features in colorectal, breast, lung, and pancreatic cancers [39]. Currently, numerous Ep-CAM-

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targeted immunotherapies are in phase I and II clinical trials, including anti-Ep-CAM antibodies adecatumumab [40,41] edrecolomab [42], ING-1 [43], and an immunotoxin [44,45]. Aptamers are small strands of DNA or RNA that have unique structural characteristics and bind exclusively to the target molecule with a high affinity. In addition to their elegant structural and molecular properties, then, aptamers are easily modified by chemical reactions, and compared with antibodies, induce fewer immune responses in vivo [46]. To date, none of the relevant literature has reported using EpCAM aptamer-guided nanoparticles in the delivery of therapeutic quantities of DOX in vivo to combat SK-MES-1-model lung cancer. For this reason, in this study an EpCAM-fluoropyrimidine RNA aptamer-conjugated, DOX-loaded, PLGA-b-PEG nanopolymersome formulation was developed [47]. This targeted, polymeric drug-delivery system was characterized by cellular uptake, internalization, and cytotoxicity for SK-MES-1 and A549 cell lines in vitro. The extent of the antitumor effect of EpCAM-targeted, DOX-loaded PEG-PLGA nanopolymersomes on SKMES-1-model lung cancer was evaluated.

2. Materials and methods 2.1. Materials Doxorubicin hydrochloride (DOX.HCl) for the present study was purchased from Euroasian Chemicals Pvt. Ltd. (Delhi, India). A heterofunctional PEG polymer with terminal amine and carboxylic-acid functional groups (HCl. NH2-PEG-COOH, Mw: 3500) was purchased from JenKem Technology USA Inc. (Beijing, China). Poly(lactic-co-glycolic acid) (PLGA) (Mw ~ 10,000 Da; lactic acid:glycolic acid = 75:25), Nhydroxysulfosuccinimide (NHS), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) were obtained from SigmaAldrich (Schnelldorf, Germany). Dulbecco's Modified Eagle's Medium (DMEM), fetal-bovine serum (FBS), penicillin–streptomycin, and trypsin were purchased from Gibco (Darmstadt, Germany). Other solvent and chemical reagents were procured from Merck & Co. (Darmstadt, Germany) and were not purified further. Finally, the above-mentioned anti-EpCAM RNA aptamer [48] was employed as the targeting ligand. The 19-mer EpCAM RNA aptamer (sequence: 5′-amino-C6 linker—A[2′-F-C]G[2′-F-U]A[2′-F-U] [2′-F-C] [2′-FC] [2′-F-C] [2′-F-U] [2′-F-U] [2′-F-U] [2′-F-U] [2′-F-C]G[2′-F-C] G[2′-FU]-3′; where F = 2′-fluoro, molecular weight: 6,345) with and without 3′-fluorescein modification was custom-synthesized by Microsynth AG (Balgach, Switzerland).

2.2. Cell line Non-small cell lung-cancer adenocarcinoma cell lines SK-MES-1 (C562) and A549 (C137) were obtained from the National Cell Bank of Iran at the Pasteur Institute of Iran. The cells were maintained in GlutaMAX DMEM (high glucose), supplemented with 10% (v/v) heatinactivated FBS and penicillin/streptomycin (100 U/mL, 100 units/mL) at 37 °C in a humidified atmosphere (95%) containing 5% CO2. 2.3. Synthesis and characterization of PEG–PLGA copolymer PEG–PLGA copolymer was synthesized as described in a recent report [47]. The synthesis of the PEG–PLGA copolymer proceeded through two steps. In the first step, 1 g of PLGA-COOH (MW ~ 10,000 Da, Sigma Aldrich) in 4 mL dichloromethane was gently stirred at room temperature in the presence of N-hydroxysuccinimide (1:8 PLGA:NHS molar ratio) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (1:8 PLGA:EDC molar ratio) to prepare PLGA-NHS.

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PLGA–NHS was precipitated with cold diethyl ether and washed three times with cold freezing solution containing 80% diethyl ether and 20% methanol to remove residual NHS and EDC. In the second step, after drying under a vacuum, PLGA–NHS was dissolved in chloroform (5 mL) followed by the addition of HCl.NH2-PEGCOOH (1:1.2 PLGA:PEG molar ratio) and N,N-diisopropylethylamine (0.2 mmol). The co-polymer was precipitated with cold diethyl ether after 24 h and washed with methanol:diethyl ether solution (30%:70%) to remove unreacted PEG. The final PLGA–PEG block co-polymer was freeze dried for 48 h and stored at −20 °C until use. The 1H-NMR spectra of the PEG–PLGA copolymer in deuterated chloroform was recorded at room temperature using a Bruker Avance 400 MHz NMR spectrometer (Rheinstetten, Germany) to analyze the presence of any intermediary products and to quantify the extent of PEG conjugation to PLGA [49]. Molecular weight and polydispersity of PEG–PLGA polymer was determined using the Agilent GPC-Addon system and RID-A refractive index signal detector recording at 212 nm coupled to the PLgel columns and operated at temperature 25 °C. The molecular weights were calibrated with polystyrene standards. Tetrahydrofuran was used as eluent (flow 1 mL/min), and the sample injection volume was 10 μL.

2.4. Preparation of DOX loaded nanopolymersomes DOX loaded polymersomes were prepared using the ammonium sulfate gradient method [50] as described in a recent report by the present study's authors [47]. For this purpose, the thin dried polymer film of copolymer (20 mg) was hydrated using 2 mL aqueous ammonium sulfate 250 mM at 55 °C (above Tg of the copolymer) under 1250 rpm continuous stirring overnight. The polymersome dispersion was extruded 15 times at 55 ± 2 °C through polycarbonate membranes of 400 nm and 100 nm sequentially, using a mini-extruder device (Avantipolar lipids Inc. USA). The extruded polymersomes were kept undisturbed for 4 h in a refrigerator at 4 °C. To establish a transmembrane pH gradient, dialysis against NaCl solution 150 mM was carried out. Briefly, the dialysis tubing (cut off 3.5 kD) was filled with 2 mL ammonium sulfate polymersome suspension. The sac was then immersed in a flask containing 200 mL NaCl solution 150 mM. The contents of the flask were stirred at 400 rpm, and the flask was closed with Parafilm. The dialysis was carried out for 48 h to develop a transmembrane pH gradient. After the establishment of the pH gradient, volumes equivalent to 20 mg of polymersomes (10 mg/mL) were incubated with the 160 μl of DOX solution (10 mg/mL) for 18 h at 55 ± 2 °C in bath oil. At the final stage, ultrafiltration (15 min, 3500 rpm, MWCO: 30 kDa) (Millipore, Hampshire, UK) was used to remove free DOX.

2.5. NP Apt conjugation The blank, or DOX-loaded, PLGA–PEG-COOH NP suspension (10 mg/mL) in DNase RNase-free water was incubated with 400 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 100 mM Nhydroxysuccinimide and gently stirred for 1 h at room temperature. The resulting N-hydroxysuccinimide-activated particles were washed three times with DNase RNase-free water using an ultrafiltration device (15 min., 3500 rpm, MWCO: 30 kDa) (Millipore, Hampshire, U.K.) to remove any remaining NHS or EDC. Then, the activated nanoparticles were covalently linked to 5′-NH2-modified EpCAM Apts (5% molar compared with polymer molarities). The resulting NP-Apts were washed three times with DNase RNase-free water and ultrafiltration to remove any free aptamers (15 min., 3,500 rpm, MWCO: 30 kDa) (Millipore, Hampshire, U.K.).

2.6. Calculation of encapsulation efficiency and loading content The amount of DOX in the NPs was analyzed by fluorescence measurements at λex = 480 nm and λem = 580 nm using a Jasco FP-6200 spectrofluorometer (Tokyo, Japan). The DOX-loaded nanoparticles were dissolved in dimethylsulfoxide (DMSO) at a concentration of 0.2 mg/mL, and the amount of DOX encapsulated in the polymersomes was then calculated from the established standard curve. The drug loading content (LC) and encapsulation efficiency (EE) of the prepared formulation was calculated using Eqs. (1) and (2), respectively. EE% ¼ Mass of drug loaded in NPs=Mass of drug initially used

ð1Þ

LC% ¼ Mass of drug in the formulation=Mass of drug‐loaded NPs: ð2Þ

2.7. Physiochemical properties of self-assembled nanostructure The particle size and size distribution were determined by dynamic light scattering. Briefly, the particle suspensions were diluted 10% in deionized water and then analyzed with a Zetasizer (NANO-ZS, Malvern, UK) at a scattering angle of 90° equipped with a 4-mW He-Ne laser operated at 633 nm through back-scattering detection. All of the measurements were performed at 25 ± 3 °C. The morphologies of the blank nanoparticles were examined with a JEOL-5300 scanning electron microscope (SEM) using an accelerating voltage of 10 kV. Briefly, the freeze-dried NPs were mounted on stubs with colloidal graphite and sputter-coated with gold to an approximate thickness of 200 Å under vacuum for 5 min to prevent charging and distortion prior to SEM analysis. High resolution transmission electron microscopy was performed to investigate the bilayer structure, size and homogeneity of the aptamer conjugated DOX loaded nanopolymersomes using an electron microscope (HR-TEM; JEOL-2100) operated at 200 kV with a Gatan Orius SC600 CCD camera. The sample for TEM observation was prepared as follows: The suspension of NPs (0.5 mg/mL) was dropped onto copper grids coated with an amorphous carbon film and dried thoroughly in an electronic drying cabinet at a temperature of 25 °C and a relative humidity of 45%. 2.8. In vitro release profile of DOX from aptamer-conjugated DOX-NPs The in vitro release of DOX from the Apt-NPs was detected using the membrane-diffusion technique. Suspended (1 mL) DOX-Apt-NPs were introduced into a dialysis bag (MWCO 3500), then immersed in 50 mL phosphate-buffered saline (PBS, pH 7.4) or citrate buffer (pH 5.5) in an incubator shaker set at 80 rpm at 37 °C. Samples (1 mL) were withdrawn at predetermined time intervals and replaced by the addition of 1 mL of fresh release medium. The samples were analyzed in a 96-well bottom black plate using a Synergy H4 Hybrid Multi-Mode Microplate Reader (Biotek, Model: H4MLFPTAD) with λexcitation and λemission values set to 480 nm and 580 nm, respectively. All of the assays were performed in triplicate. 2.9. Hemolysis assay Polymer solutions' hemolytic activity was evaluated as previously described [51]. In short, 5 mL of fresh human blood was immediately added with heparin to prevent coagulation. The erythrocytes were collected by centrifugation at 1300 rpm for 10 min at 4 °C. After being washed three times in ice-cold Dulbecco's phosphate-buffered saline (DPBS), the red blood cells were diluted at a final concentration of 5 × 108 cells/mL in ice-cold DPBS. 1 mL Apt-DOX-NPs, DOX-NPs, blank

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Apt-NPs, or NP solution (1 mg/mL and 0.1 mg/mL) was mixed with 1 mL erythrocyte suspension. DPBS and 1% Triton X-100 in DPBS were used as negative (0% lysis) and positive (100% lysis) controls, respectively. Samples were incubated for one hour at 37 °C in a shaker incubator set to 120 rpm. After centrifugation at 1300 rpm for 10 min at 4 °C, the supernatant was analyzed for hemoglobin release at 540 nm using an Infinite® 200 PRO multimode microplate reader from Tecan Group Ltd. (Männedorf, Switzerland). The percentage of hemolysis was calculated as (ODsample − ODnegative control) / (ODpositive control − ODnegative control) × 100%. Moreover, absorption of the DOX-loaded nanopolymersomes was determined at 540 nm in order to reduce the effect of DOX absorption in calculating hemolysis. Hemolysis was determined using three independent experiments. 2.10. Investigation of epithelial-cell adhesion-molecule expression level To evaluate the EpCAM expression level, cells (SK-MES-1 and A549) were washed two times with phosphate-buffered saline (PBS; 0.1 M) and suspended in PBS 0.1 M that contained 0.02% sodium azide and 0.1% bovine-serum albumin (BSA). The cells (1 × 106 cells) were incubated with a 20 μL anti-EpCAM-fluorescein, isothiocyanate-conjugated monoclonal antibody (Cat No: ab8666, Abcam Plc., Cambridge, U.K.) at 4 °C for 2 h in PBS 0.1 M, containing 0.02% sodium azide and 0.1% BSA. The cells were then washed three times with cold PBS. The fluorescence signal was read using a BD FACSCalibur equipped with a 488 laser in the FL1 channel. The data were analyzed using WinDMI 2.9 analysis software. 2.11. Cellular aptamer binding Aptamer cellular binding was determined by performing a flowcytometry (FCM) analysis of the cells after incubation using a fluorescein-labeled RNA aptamer. SK-MES-1 and A549 cells (1 × 106 cells) were gently washed with PBS twice. The cells were suspended in the binding buffer (100 mM NaCl, 5 mM MgCl2, pH 7.2) and incubated with the EpCAM aptamer at a concentration of 300 nM for 2 h. The FCM analysis was performed to examine the binding of aptamers to the SKMES-1 cell line. 2.12. Cellular uptake using flow cytometry SK-MES-1 and A549 cells were seeded in six-well plates at 1 × 105 cells per well and cultured for 24 h. The following day, the cells were incubated for 2 h with either targeted or non-targeted nanopolymersomes, or with free DOX (DOX concentration: 20 μg/mL). Then, the culture medium was removed, and the cells were trypsinized and centrifuged at 1400 rpm for 10 min. Supernatants were removed and the resultant pellet was washed three times with cold PBS. The cells were then suspended in an FCM buffer (0.1% sodium azide and 2% FBS in PBS), and the intensity of the cells' DOX fluorescence was determined on a BD FACSCalibur equipped with 488 laser in the FL2 channel. The data were analyzed using WinDMI 2.9 analysis software. Additionally, to confirm the findings of the EpCAM receptors' selectively targeting the SK-MES-1 and A549 cells for EpCAM-targeted, DOXloaded nanopolymersomes, a competitive experiment was carried out. In it, excessive amounts (2 μg/well) of free EpCAM aptamer were added to each well for 30 min. before introducing the targeted formulation. 2.13. Cellular uptake using fluorescence microscopy The cellular uptake of NPs or Apt-NPs by SK-MES-1 and A549 was further studied using a CETI inverted-fluorescence microscope (Oxford, U.K.). Collagen-treated cover slips (0.1% collagen in acetic acid) were deposited in the wells of a six-well plate. The cells at a density of

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1 × 105 per well were seeded and allowed to adhere overnight. Next, the cells were incubated for 2 h with either non-targeted or targeted polymersomes or with free DOX (DOX concentration: 20 μg/mL). After a two-hour incubation period, the cells on glass cover slips were washed five times with cold PBS and fixed with 4% formaldehyde for 15 min. The cover slips were then placed on a slide for fluorescencemicroscopy analysis. 2.14. NP cytotoxicity studies SK-MES-1 and A549 cells were cultured in a DMEM medium supplemented with 10% (v/v), heat-inactivated FBS, 1% penicillin–streptomycin at 37 °C, and 5% CO2. The SK-MES-1 and A549 cells were then seeded into 96-well plates at 5 × 103 and incubated for 24 h. DOX at different concentrations was prepared in DMEM containing 10% FBS. Targeted and non-targeted formulations containing equivalent amounts of DOX (0.25, 0.5, 1, 2, 5, 10, 20 and 30 μg/mL) were also dispersed in DMEM containing 10% FBS. The prepared drug concentration was added to each well, followed by a five-hour cell incubation. The medium was then removed, washed with PBS, replaced with fresh, complete medium, and further incubated in a humidified incubator for 48 h at 37 °C. After 48 h of incubation, a 20 μL MTT (5 mg/mL in PBS) solution was added to each well, and the cells incubated for a further 4 h. The MTT solution was then aspirated from the wells using a vacuum and 100 μL DMSO was added to each well. Absorbance at 570 nm, with a reference wavelength of 630 nm, was measured using the aforementioned Infinite® 200 PRO multimode microplate reader from Tecan Group Ltd. (Männedorf, Switzerland). Next, to confirm selective A549 and SK-MES-1-cell EpCAM-receptor targeting by the EpCAM-aptamer conjugated DOX-loaded NPs, a competitive experiment was performed. In this experiment, excessive amounts (0.2 μg/well) of free EpCAM aptamer were added to each well 30 min. before introducing the targeted formulation. 2.15. In vivo efficacy study All animal experiments were conducted with the approval of both the Institutional Ethics and Research Advisory committees for the School of Medical Sciences at Mashhad University. The SK-MES-1 cells (3 × 105 cells/200 μl DMEM media) were subcutaneously inoculated into the right, lateral flanks of C57BL/6 nude mice. After two weeks, mice with a 200–300 mm3 tumor received 0.2 mL of DOX or prepared formulation (equivalent DOX concentration: 5 mg/kg) via a single tail-vein injection. NaCl 0.9% solution was injected as a negative control. Each group consisted of five mice. Tumor volume was calculated using the formula a × b × w/2, where “a” is the tumor's largest diameter, “b” is its smallest diameter, and “w” is its height; the toxicities of the DOX-free and DOX-loaded nanopolymersomes were evaluated by monitoring the mice's body weights and survival rates. In each experiment, follow-ups were performed on the mice either 21 days after tumor inoculation or when one of the following conditions for euthanasia had been met: (1) the mouse's body weight dropped below 20% of its initial weight; (2) the mouse became so ill it was unable to feed; or (3) the mouse was found dead [52]. 2.15.1. Cardiac toxicity of therapeutic DOX dosage An autopsy was performed 18 days after injecting DOX-NPs, AptDOX-NPs, or free DOX (equivalent DOX concentration 5 mg/kg) and samples of heart were collected for microscopic examination. Eighteen days after treatment, all mice were euthanized with an overdose of ether inhalation anesthesia. Their organs were fixed in 10% neutralbuffered formalin solution, and then embedded in paraffin. Embedded organs were then cut to 5 μm thickness and stained with hematoxylin and eosin (HE). Images of the resulting organ sections were prepared

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at 40× magnification. Histopathological analyses were performed at Dr. Moayed's laboratory.

Table 1 Polymer characteristics determined by GPC. Polymer

GPC results Mna

M wb

Mw/Mnc

COOH-PEG–PLGA

7113

13172

1.8

2.16. Statistics A one-way analysis of variance (ANOVA) was used to analyze the present study's data. A probability value of less than 0.05 was considered significant. Results are expressed as mean ± SD unless otherwise noted. 3. Results and discussion 3.1. Synthesis and characterization of PEG–PLGA copolymer The PLGA–PEG-COOH copolymer was synthesized by a one-step EDC/NHS amide coupling reaction between PLGA-COOH and PEG-NH2. The 1HNMR spectrum of the copolymer is shown in Fig. 1. The singlet signal at 3.6 ppm (peak a) is attributed to the methylene group of the PEG block in the copolymer chain. Polylactic acid (PLA) block presents a doublet at 1.4–1.7 ppm (peak d) and a quartet at 5.2–5.4 ppm (peak c) which are attributed to the CH3 and CH protons, respectively. A multiplet of the CH2 protons of polyglycolic acid (PGA) block is detected at 4.6 ppm (peak b). Using the integration of the relative molecular weights and peaks, the conjugation efficiency of NH2-PEG-COOH to PLGA-COOH was estimated to be approximately 86%. Furthermore, Table 1 shows the average molecular weight and average molecular number data resulting from the GPC analysis of the synthesized COOH-PEG–PLGA. 3.2. Morphological characterization of PEG–PLGA self-assembled structure Scanning electron microscopy (SEM) was used to investigate the morphology of the self-assembled structures. Fig. 2A shows SEM image of the PEG–PLGA NPs, which demonstrates the water filled hollow spherical configuration of the self-assembled structures that collapse after water removal under high vacuum of SEM. Obtained results illustrated partial vesicular collapse inward which is in agreement with earlier report as means of vesicular structure verification [53]. Furthermore, the PEG–PLGA NPs were studied by atomic force microscopy (AFM) in a dehydrated state. As reported in Fig. 2B, the polymersomes exhibited “donutlike” structures with centers that are lower than their edges. This result indicates that the spheres observed

Fig. 1. 1HNMR spectrum of the PEG–PLGA copolymer in CDCl3.

a b c

Average molecular number. Average molecular weight. Polydispersity.

by SEM are water-filled vesicles that will collapse into the dehydrated disks detected by AFM. The morphological characteristics of the nanoparticles were identical to those reported previously and confirm the formation of polymeric vesicles (nanopolymersome) [54,55]. Based on evidences provided above, it could be concluded that the vesicle like polymersomes rather than micelles have been formed upon hydration of PEG–PLGA polymeric film.

3.3. DOX-loaded NP preparation Preparation of nanopolymersomes carrying DOX, based on the block copolymer PEG–PLGA, was described in a recent publication by this study's authors [47]. The ammonium sulfate-gradient method [50] was used to encapsulate DOX within PLGA-b-PEG nanopolymersomes and to develop DOXencapsulated, 100% pegylated PLGA NPs (124 ± 0.9 nm [mean ± SD], with a PDI of 0.062; n = 5). The results demonstrated that DOX was entrapped in PEG–PLGA nanopolymersomes with encapsulation efficiency and loading content of 91.25 ± 4.27% and 7.3 ± 0.34%, respectively (n = 10). Nanoparticles with a PEG brush on their surfaces are generally considered to have a “stealth character” due to the PEG molecules' minimization of interfacial free energy and steric repulsion. NP PEGylation was implemented to prevent NP uptake through the mononuclear phagocytic system in vivo and, consequently, to prolong the circulation half-life of the nanoassembly [56]. Each nanopolymersome thusly prepared bore an EpCAM aptamer as a targeting ligand on its surface layer to allow differential uptake by the targeted SK-MES-1 and A549 cells. The morphology, homogeneity, bilayer configuration and size distribution of aptamer-conjugated DOX-NPs were evaluated with high resolution transmission electron microscopy (TEM) (Fig. 3A, B) and DLS (Fig. 3C). Conjugation of the NPs with EpCAM aptamers caused a 10 nm increase in formulation size, and this is possibly attributable to the existence of aptamers on the NPs' surfaces (136 ± 0.21 nm with a PDI of 0.12). The diameters of both the DOX-NPs and Apt-DOX-NPs were less than 200 nm, in keeping with the precept that particles smaller than 200 nm are conducive to drug accumulation at the tumor site through EPR [57]. High zeta potential is another important requirement for nanoparticle stability because high values impede nanoparticle aggregation due to strong repellent forces. In this study, the zeta potentials of both the DOX-NPs and Apt-DOX-NPs were − 23.42 mV and − 36.13 mV, respectively. It is possible that the conjugating the aptamers to the nanoparticles' surfaces increased the Apt-DOX-NPs' negative charges. Thus, due to the presence of carboxylate groups of PEG on the nanopolymersome surfaces, the resultant NPs would have a negative zeta potential. This might minimize the nonspecific interactions between the negatively charged conjugated aptamers and the NP surfaces [58]. Moreover, the NP hydrodynamic properties preserved EpCAM-aptamer conformation and binding characteristics. On the other hand, covalent conjugation of aptamer with NPs and also enhancement of RNA aptamer stability by 2′-fluoro pyrimidine nucleotide modification could guarantee the stability of the targeted formulation in vitro and in vivo.

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Fig. 2. SEM image of PEG–PLGA self-assembled structures (A); AFM image of PEG–PLGA self-assembled structures.

3.4. Profile of DOX in vitro release from Apt-NPs Next, the present study's authors examined how DOX was released in vitro from aptamer-conjugated DOX nanopolymersomes in PBS (pH 7.4) or citrate buffer (pH 5.5) (Fig. 4). The release behavior suggested that the release rate of DOX was higher at pH 5.5 than at pH 7.4. The release rates were accelerated in citrate buffer at pH 5.5 due to the improved solubility of protonated DOX in water. After an initial burst release (6%), a constant DOX release was observed in pH 7.4; after five days, approximately 9% of the loaded drug had been released from the targeted nanopolymersomes, showing a typically sustained and prolonged drug release that depends on drugdiffusion and matrix-erosion mechanisms [59]. In this study, DOX loading with an ammonium-sulfate gradient was driven by DOX protonation and charging within the polymeric vesicle and by DOX precipitation in the vesicle's hydrophilic interior due to the protonated doxorubicin's interaction with SO24 − anions [60]. Fig. 3D illustrates DOX's gel-like structures in the nanopolymersomes'

interior compartments. This loading process was chosen because earlier studies showed that positively charged DOX precipitates and negatively charged sulfate form a gel-like structure within vesicles that don't easily leak. Meanwhile, the system's sustained-release property is a result of both the DOX's gel-like precipitate in the polymersome's hydrophilic interior as well as the polymersome's stability and rigid bilayer. However, nanoparticle behavior in vivo might differ due to the presence of hydrolytic enzymes, which might contribute to polymeric-bilayer degradation and therefore result in a faster drug release. 3.5. Hemolytic test The biocompatibilities of blank PEG–PLGA nanopolymersomes with DOX-loaded PEG–PLGA nanopolymersomes, blank aptamer-conjugated PEG–PLGA nanopolymersomes, and DOX-loaded, aptamer-conjugated PEG–PLGA nanopolymersomes in the presence of erythrocytes were investigated with a hemolytic experiment. As illustrated in Fig. 5, the percentage of hemolysis increased commensurately with the formulation's concentration. The use of blank

Fig. 3. High resolution TEM image of aptamer conjugated DOX loaded nanopolymersomes (A); high resolution TEM image of a PEG–PLGA bilayered vesicle (polymersome) (B); size distribution of aptamer conjugated DOX-loaded nanopolymersomes (C); TEM image of DOX loaded nanopolymersomes. Red arrows demonstrate the gel like structures of DOX in interior compartment of nanopolymersomes.

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Fig. 4. Release profile of aptamer conjugated DOX loaded nanopolymersome formulation in PBS pH 7.4 and citrate buffer pH 5.5 (n = 3, error bars represent standard deviation).

nanopolymersomes at concentrations of 0.1 mg/mL and 1 mg/mL resulted in a slight increase in hemolysis compared with use of the negative control. And yet, DOX-loaded polymersomes exhibited higher hemolytic activity than blank polymersomes. This might be due to the release of DOX within the medium, causing hemolysis. Compared with plain NP aptamer conjugation, the NPs did not increase hemolytic activity. The results also demonstrate that the hemolytic activity of DOX-loaded nanopolymersomes depends on their DOX concentration. 3.6. Specific EpCAM aptamer affinity to the SK-MES-1 cell line EpCAM expression was about 54.82% and 61.34% in SK-MES-1 (Fig. 6A, B) and A549 (Fig. 7A, B) cells respectively, as evaluated with direct immune fluorescence followed by flow cytometry. To test whether the EpCAM aptamer would bind to SK-MES-1 and A549 cells, the binding of a fluorescein labeled, “EpCAM-aptamer to SK-MES-1 and A549 cells” was evaluated by FCM analysis. Previous studies have established that SK-MES-1 and A549 are human non-small cell lung cancer that overexpresses the EpCAM protein on their surface [61]. Consistent with the original report, the EpCAM-aptamer demonstrated a high level of binding to SK-MES-1 (Fig. 5C, D: 46.12%) and A4549 (Fig. 6C, D: 54.27%) cells. 3.7. Cellular uptake experiment by flow cytometry The most important property of a targeted drug-delivery system is its specificity toward target cells. To explore the in vitro cancer targeting of Apt-NPs against EpCAM-overexpressing cells, this study compared

Fig. 5. In vitro hemolysis assay of Apt-DOX-NP, DOX-NP, blank NP and blank Apt-NP compared to Triton X-100 and DPBS as positive and negative controls measured at 541 nm (n = 3, error bars represent standard deviation).

the cellular uptake of SK-MES-1 and A549 using a flow-cytometry analysis (Fig. 8). DOX-loaded NPs, DOX-loaded Apt-NPs, and free DOX were co-cultured with SK-MES-1 and A549. As a negative control, excess amounts (2 μg/well) of free EpCAM aptamer were added to the wells 30 min. before the addition of AptNPs. The fluorescent intensity of SK-MES-1 and A549 cells incubated with NPs was similar in amplitude to that of cells treated with free aptamer prior to the addition of Apt-NPs. The addition of excess aptamer—as a free, competing ligand for targeted NPs—prior to the cellular-uptake experiment reduced the aptamer-targeted NPs' internalization efficiency. However, for Apt-NP-treated SK-MES-1 and A549 cells the fluorescent intensity increased by 83% and 78% respectively, while that of NP-treated cells increased by 52% and 46% respectively. This distinction was presumably caused by the conjugated aptamers' binding to EpCAM proteins on the SK-MES-1 and A549 cells' surfaces, resulting in more nanoparticles' attaching to the cells' surface and penetrating the cells. Still, the fluorescence intensity of SK-MES-1 and A549 cells exposed to free DOX increased by 95% and 89% respectively, demonstrating that cells can take up abundant free DOX. The lower cellular uptake of DOX-loaded polymersomes in comparison with that of free DOX polymersomes is largely due to the stability of the former polymersomes' structures and these systems' sustained-release properties. 3.8. Cellular uptake using fluorescent microscopy The above results show that the Apt-NPs in this study generated enhanced fluorescence intensity in EpCAM-positive SK-MES-1 and A549 cells compared with NPs. However, it was not entirely clear whether the Apt-NPs attached to the cells' surfaces or were internalized into the cells. To further study the interaction between Apt-NPs and the target cells, then, fluorescent microscopy was performed to determine the Apt-NPs' locations. As indicated in Fig. 9, this clearly indicated that the Apt-NPs were mainly accumulated within the cells. It was evident, consequently, that Apt-NPs were internalized into the target cells and could carry anticancer drugs into them. Compared to Apt-NPs, far fewer NPs entered the SK-MES-1 and A549 cells, confirming that EpCAM aptamers facilitate cells' uptake of nanoparticles. 3.9. In vitro cellular cytotoxicity (MTT) assays The in vitro differential cytotoxicity of DOX-loaded, PEG–PLGA nanopolymersome-aptamer bioconjugates (Apt-DOX-NP)—in comparison with DOX-NPs lacking the EpCAM aptamer (DOX-NP) on SK-MES-1 and A549 cells that express the EpCAM protein—was evaluated. To do so, DOX-NP and Apt-DOX-NP groups were incubated with SK-MES-1 and A549 cells for 5 h to allow for specific-particle uptake; then, they were further incubated in a medium for 48 h before being measured. This incubation period was consistent with a previous report published by this study's authors that found Apt-DOX-NP bioconjugates bound to the EpCAM were efficiently taken up by MCF-7 cells [47]. The present data suggests that Apt-DOX-NP bioconjugates are significantly more cytotoxic compared with DOX-NPs (Fig. 10). Moreover, Apt-NPs seem to enhance DOX's cytotoxicity in SK-MES-1 and A549 tumor cells, while showing no efficacy toward free aptamertreated cells. This is consistent with the results shown in Fig. 8, which depicts the EpCAM aptamer increasing Apt-NP uptake for SK-MES-1 and A549 cells and the treatment with free aptamer doing otherwise. These results suggest that the Apt-DOX-NPs' enhanced toxicity was due to the high affinity and selectivity of aptamer-conjugated DOX-NP for SK-MES1 and A549 cells during incubation and before the wash step. To evaluate the possibility that the EpCAM-Apt or blanknanopolymersome formulations were responsible for the perceived cytotoxicity, MTT assays were performed with blank nanopolymersomes and blank nanopolymersome–aptamer bioconjugates. Results confirmed a lack of cellular cytotoxicity when DOX was absent from the formulation.

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Fig. 6. Flow cytometry analysis of SK-MES-1 cells (A) incubated with FITC labeled anti-EpCAM monoclonal antibody (B); flow cytometry analysis of SK-MES-1 cells (C) incubated with fluorescein labeled EpCAM aptamer (D).

According to the cellular toxicity of the formulations it was strongly suggested that different phenomena such as cellular enzymatic degradation might occur compared to in vitro release study that caused faster drug release. Obtained results prove the limitation of in vitro drug release experiment and it demonstrated that it cannot simulate the release behavior of drug delivery systems in live systems.

3.10. In vivo anti-tumor efficacy and toxicity studies using mouse SK-MES-1 xenografts Next, the efficacy of an EpCAM aptamer-conjugated, DOX-loaded nanopolymersome formulation was investigated using xenograft models of NSCLC developed through subcutaneously injecting SKMES-1 cells into the flanks of C57BL/6 nude mice.

Fig. 7. Flow cytometry analysis of A549 cells (A) incubated with FITC labeled anti-EpCAM monoclonal antibody (B); flow cytometry analysis of A549 cells (C) incubated with fluorescein labeled EpCAM aptamer (D).

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Fig. 8. Flow cytometry analysis of SK-MES-1 (A) and A549 (B) cells incubated with targeted and non-targeted DOX loaded nanopolymersome formulations.

After tumors had developed to 200–300 mm3, the efficacies of the prepared targeted and untargeted formulations were evaluated by dividing the tumor-bearing mice into four groups (n = 5) as a way of minimizing weight and tumor-size differences among the groups. According to prior studies, the best-tolerated dose of intravenously inoculated DOX in nude mice is 5–7 mg/kg [62]. In the current study, the following regimens were administered by single, intravenous injections of saline as a control, free DOX (5 mg/kg), DOX-NPS (5 mg/kg), and AptDOX-NPs (5 mg/kg) as treated groups. Tumor growth rate in terms of mean tumor size (mm3) is presented in Fig. 11. The results demonstrated that a single, intravenous injection of either Apt-DOX-NP bioconjugates or DOX-NPs was significantly more efficacious in inhibiting tumor growth compared with the injections received by the control group, and this is likely due to enhanced permeation and retention effects. Moreover, the enhanced tumor-inhibition effects of aptamer-conjugated DOX-NPs compared with non-targeted DOX-NPs might have been due to the targeted particles' binding to EpCAM proteins on the SK-MES-1 cells, thus possibly delaying clearance

from the tumor site. Indeed, if targeted NPs are internalized after binding to EpCAM proteins, subsequent, intracellular DOX deliveries might increase cytotoxicity and enhance this group's efficacy at inhibiting tumor growth. As for the saline-and-DOX control group, the treatment had no apparent efficacy. Overall four of five animals in the DOX group and three of five animals in the saline group died or reached their end points (defined as a tumor load in excess of 10 cm3) during the study's 30-day duration (Fig. 12). The fast tumor-growth rate in the NSCLC xenograft models was consistent with the well-documented rate of NSCLCtumor growth, which is also characteristic of this type of lung cancer in humans [63]. Four of the five animals in the Apt-DOX-NP group and three of five mice in the DOX-NP group survived the entire 30 days. The difference in survival time for the DOX group compared with the DOX-NP and Apt-DOX-NP groups was statistically significant (ANOVA at a 95% confidence interval), and this is attributable to free DOX's high cytotoxicity and dose size. Apt-DOX-NP treatment demonstrated sophisticated efficacy, as the final mean tumor load was significantly smaller for individuals in the

Fig. 9. Fluorescent microscopy images detecting cellular uptake of DOX-NP (A); Apt-DOX-NP (B); DOX (C) for A549 and cellular uptake of DOX-NP (D); Apt-DOX-NP (E); DOX (F) for SK-MES-1.

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Fig. 10. Cytotoxicity of free DOX, DOX-loaded nanopolymersomes; EpCAM targeted DOX-loaded nanopolymersomes on the SK-MES-1 (A) and A549 (B) cell lines after 48 h (n = 4, error bars represent standard deviation).

group treated with it than were the final mean tumor loads of all other groups (DOX-NPs and saline by ANOVA at a 95% confidence interval) (Fig. 11). DOX-NP was also more efficacious than free DOX and the saline control, but it was significantly less efficacious compared with Apt-DOX-NP. Obtained results demonstrate that, after just one intravenous administration, Apt-DOX-NP inoculation was the most successful treatment for fighting SK-MES-1 tumors, resulting in greater survival and tumor growth-inhibition effects than the other treatments. Following comparative treatment testing, then, pathological staining of the animals' excised hearts was performed to evaluate the comparative cardiotoxicities of saline (0.9%), free DOX, DOX-NPs, and Apt-DOXNPs 18 days after treatment (DOX dose: 5 mg/kg). In accordance with one previous study, free DOX caused a myofibrillar dropout consisting of swelling of the sarcoplasmic reticulum and, in more advanced stages of damage, complete loss of the myofibrils [64]. An important observation upon opening the chests of the mice in the DOX group was that these individuals suffered smaller heart sizes and their heart tissue was darker and redder in color than the heart tissues of mice in the other groups. The myocardial pathology of DOX treatment exhibited myofibrillar loss and cytoplasmic vacuolization (Fig. 13). In contrast, no pronounced cardiac changes were observed in the saline-, DOX-NP-, or Apt-DOX-NP-treatment groups; all treatments with DOX-loaded nanopolymersomes resulted in less apparent cardiotoxicity in vivo compared with free-DOX treatments. Each treatment's toxicity was further assessed by analyzing its effects on BWL. The body weight-change curve in Fig. 14 shows that weight loss was less than 7% for mice treated with DOX-NPs. BWL that did occur in the DOX-NP treated group might have been due to the slight toxicity of the formulation dosage size, which was used to ensure that injections were sufficiently therapeutic; the treated mice began recovering their body weights 6 days after receiving their injections.

Fig. 11. Tumor growth inhibitory efficacy of DOX-NP and Apt-DOX-NP groups in comparison with saline group in the subcutaneous mouse model of SK-MES-1 (n = 5, error bars represent standard deviation).

The observed loss of body weight and subsequent recovery after dosing in the DOX-NP group might represent bulk PLGA degradation, resulting in an initial burst, followed by a slower, continuous DOX release over time. This release pattern is characteristic of the PLGA controlledrelease polymer system and, crucially, allows for DOX's presence at the administration site over an extended period. Furthermore, mice treated with the targeted formulation did not exhibit any loss of body weight during the experiment, possibly demonstrating modified and better pharmacokinetic characteristics for the targeted formulation's distribution in mice bodies than for the non-targeted formulations'. In contrast, BWL was observed in the free-DOX group (data not shown) because of free DOX's acute toxicity when circulating throughout the body. All mice in the free-DOX group were dead within 21 days of receiving their injections, and this might be attributable to free DOX's acute cardiotoxicity, as shown by the histopathological analysis. Previously it was proved that in comparison with free DOX; liposomal formulation of DOX exhibited the lowest induction of cardiomycyte apoptosis and also has no deleterious effect on heart rate and cardiac output of mice [65]. However, while liposomal formulation of DOX such as Doxil or Caelix are generally used for clinical administration of DOX, they could not provide sustained release of DOX, which marks a significant inadequacy of liposomal formulations [66]. Polymeric vesicles (polymersomes) can encapsulate high quantity of drug and release it at sustained rates in the desirable concentration, and consequently increase the in vivo therapeutic efficacy and facilitate the implementation of highly toxic anti-cancer agents [67,68]. In this regard, in the current study DOX was encapsulated in selfassembled nano-scale PEG–PLGA polymeric vesicles (polymersomes) for controlled release of DOX. Targeted drug-delivery systems are a proposed solution to the problem that most anticancer therapeutic agents fail to act specifically on cancer cells, instead causing toxicity for normal cells. In this study, for

Fig. 12. Kaplan–Meier survival curves of subcutaneous mouse model of SK-MES-1 treated with DOX, DOX-NP, Apt-DOX-NP and saline.

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A

B

C

D

Fig. 13. Mouse myocardium: treated with free DOX, DOX-NP or Apt-DOX-NP at a DOX concentration of 5 mg/kg body weight (H&E stain, magnification: 40×) (A: Saline; B: Free DOX treated; C: DOX-NP treated; D: Apt-DOX-NP treated).

the first time, a short, 19-mer-RNA EpCAM-aptamer-based, targeted drug-delivery system was implemented to enhance the delivery of DOX to model NSCLC tumors. EpCAM is highly overexpressed in primary and metastatic NSCLC and has been reported as a cancer stem-cell marker. Because of this, the present study's authors reasoned, implementation of EpCAM as a targeting ligand was a potential strategy for delivering targeted chemotherapeutic agents to NSCLC. EpCAM appears to be overexpressed by the majority of human epithelial carcinomas, including colorectal, breast, prostate, head-and-neck, and hepatic carcinomas [69–71]. For this reason, EpCAM has attracted major attention as a target for aptamer-guided drug-delivery systems for combatting a wide array of malignancies. The EpCAM aptamer used in this study exhibited a high EpCAM-binding affinity comparable to that of the anti-EpCAM antibody itself [72]. The targeted nanoparticle-delivery system based on this short EpCAM-RNA aptamer was evaluated for its ability to target SK-MES-1 cells in vitro and in vivo. Similar to drugdelivery systems based on PSMA, MUC-1, and AS1411 aptamers, the system observed in this study exhibited the enhanced uptake, cytotoxicity in vitro, and tumor-inhibition effects of Apt-DOX-NPs on SK-MES-1 cells. Finally, because the EpCAM protein is overexpressed on the surface of NSCLC cells, such a drug-delivery system could potentially improve

DOX delivery to tumor sites. As a result, this study's findings suggest that EpCAM-targeted nanopolymersomes offer an effective strategy for delivering therapeutically effective DOX concentrations into NSCLC cells. 4. Conclusion Recently, this study's authors developed EpCAM aptamer-conjugated, DOX-loaded nanopolymersomes for targeted delivery and uptake by MCF-7 cells [47]. The present study, then, demonstrates the in vitro and in vivo efficacy of these EpCAM aptamer-conjugated, DOX-loaded nanopolymersomes against SK-MES-1 cells as models of NSCL. The targeted formulation is composed of FDA-approved PEG–PLGA copolymer, DOX (a broad-spectrum, versatile anticancer agent), and short 19bp EpCAM-RNA aptamers with high stability and non-immunogenicity. In this study, the targeted, DOX-loaded nanopolymersomes demonstrated promising efficacy at inhibiting NSCLC-tumor growth, based on the use of model SK-MES-1 and A59 cells in vitro and SK-MES-1 tumor model in vivo. Similar approaches might be used to develop targeted therapeutics that improve the overall survival rate for non-small cell lung cancer. Declaration of interest The authors declare that they have no conflicts of interest.

Acknowledgments

Fig. 14. Body weight (g) of SK-MES-1 tumor-bearing nude mice (n = 5, error bars represent standard deviation) treated with DOX-NPs, Apt-DOX-NPs (5 mg/kg).

The authors are grateful for the financial support provided by the Iran National Science Foundation (No. 9000719) and the Mashhad University of Medical Sciences (No. 901051). The authors would also like to thank Prof. Hossein Hosseinkhani from the National University of Taiwan for the valuable assistance provided. Also we really appreciate all assistance provided by Dr. Taghi Ghiasi and Dr. Ahmad Moayed for histopathological analysis. The resuls described in this paper were part of the PhD thesis of Mona Alibolandi.

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