BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 379 – 389
Original Article
nanomedjournal.com
Multifunctional nanoparticle–EpCAM aptamer bioconjugates: A paradigm for targeted drug delivery and imaging in cancer therapy Manasi Das, MS a , Wei Duan, MBBS, PhD b , Sanjeeb K. Sahoo, PhD a,⁎ a Institute of Life Sciences, Nalco Square, Bhubaneswar, India School of Medicine, Deakin University, Waurn Ponds, Victoria, Australia Received 21 May 2014; accepted 9 September 2014
b
Abstract The promising proposition of multifunctional nanoparticles for cancer diagnostics and therapeutics has inspired the development of theranostic approach for improved cancer therapy. Moreover, active targeting of drug carrier to specific target site is crucial for providing efficient delivery of therapeutics and imaging agents. In this regard, the present study investigates the theranostic capabilities of nutlin-3a loaded poly (lactide-co-glycolide) nanoparticles, functionalized with a targeting ligand (EpCAM aptamer) and an imaging agent (quantum dots) for cancer therapy and bioimaging. A wide spectrum of in vitro analysis (cellular uptake study, cytotoxicity assay, cell cycle and apoptosis analysis, apoptosis associated proteins study) revealed superior therapeutic potentiality of targeted NPs over other formulations in EpCAM expressing cells. Moreover, our nanotheranostic system served as a superlative bio-imaging modality both in 2D monolayer culture and tumor spheroid model. Our result suggests that, these aptamer-guided multifunctional NPs may act as indispensable nanotheranostic approach toward cancer therapy. From the Clinical Editor: This study investigated the theranostic capabilities of nutlin-3a loaded poly (lactide-co-glycolide) nanoparticles functionalized with a targeting ligand (EpCAM aptamer) and an imaging agent (quantum dots) for cancer therapy and bioimaging. It was concluded that the studied multifunctional targeted nanoparticle may become a viable and efficient approach in cancer therapy. © 2015 Elsevier Inc. All rights reserved. Key words: Nanotheranostics; Nutlin-3a; EpCAM aptamer; Quantum dots; PLGA NPs
One of the foremost impediments in cancer chemotherapy is the poor aqueous solubility, bioavailability and side effects caused by nonspecific cytotoxicity of widely used anticancer drugs. 1 Novel approaches are needed to site-specifically deliver chemotherapeutic agents to tumor cells by overcoming above limitations to enhance their therapeutic window. Presently, research in diverse direction for upgrading the carrier system to deliver the therapeutic payload at the target site efficiently is ongoing. 2–4 In this setting nanotechnology based nanoformulations have played a very crucial role in enhancing the therapeutic potentiality of the conventional chemotherapeutic agents by increasing their aqueous solubility, bioavailability and by reducing non-specific toxicity. 2,5,6 Apart from these requirements, ability to diagnose the diseased conditions in a early stage and monitoring the therapeutic response are also the prerequisite for better cancer therapy. In this milieu, nanotheranostic Conflict of interest: We do not have any conflict of interest. Manasi Das thanks the Council of Scientific Industrial Research, New Delhi, Government of India, for a Senior Research Fellowship. ⁎Corresponding author at: Laboratory for Nanomedicine, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Orissa, India. E-mail address: [email protected] (S.K. Sahoo).
platform are anticipated to play a vital role and multifunctional nanocarrier systems with multimodal capabilities (i.e. simultaneously provides detection, diagnosis and therapy) represent tremendous potentiality to be used in nanotheranostic approach for improved cancer therapy and diagnosis. 3,7 Chemotherapeutic agents are our main weapon in the treatment of cancer. The recently developed chemotherapeutic agent nutlin-3a is a small molecule inhibitor of the murine double minute 2 (MDM2)–p53 interaction, which is as direct non-genotoxic p53 stabilization thereby, activating cell cycle arrest and apoptosis in cancer cells. 8,9 Recently, Lau et al. 10 have shown that nutlin-3a can disrupt the interaction between p73 and MDM2 and induce apoptosis in cells lacking functional p53. A series of recent studies have strengthened the concept that, selective restoration of the tumor suppressor function of p53 by nutlin-3a in various types of cancer might represent a substitute to the existing chemotherapy that generally causes DNA damage and induces various complications related to growth, infertility, and secondary malignant tumor induction. 8,11,12 In contrary to others, in a recent study, Shen and Maki 13 have pointed out that nutlin induces endoreduplication and therapy resistance in colon cancer and osteosarcoma cells. Though the findings are significantly important for use of nutlin-3a in
http://dx.doi.org/10.1016/j.nano.2014.09.002 1549-9634/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Das M, et al, Multifunctional nanoparticle–EpCAM aptamer bioconjugates: A paradigm for targeted drug delivery and imaging in cancer therapy. Nanomedicine: NBM 2015;11:379-389, http://dx.doi.org/10.1016/j.nano.2014.09.002
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clinical settings, their results indicate that this phenomenon is not apparent in all cell lines studied and thus may not completely mask the therapeutic implications observed with nutlin in a diverse cancer type by many research groups. As nutlin-3a represents a potent anticancer agent in recent scenario, the clinical utility of this novel molecule is limited by its poor water solubility, limited dose that is delivered to tumor site and drug resistance phenotype. 14 In this context polymeric nanoparticles can play a vital role in overcoming the restraints associated with native nutlin-3a. 15,16 Biodegradable NPs formulated from poly(D, L-lactide-co-glycolide) (PLGA) are being extensively investigated for the purpose of sustained and localized administration of different antiproliferative agents. 17,18 The extensive biomedical application of PLGA is because of its biocompatibility, ability to encapsulate various drug molecules and sustained release properties. 17 In this milieu, our group has recently developed ligand targeted nutlin-3a encapsulated PLGA nanoparticles to surmount the shortcomings associated with native nutlin-3a and investigated its therapeutic prospective at cellular and molecular level. 19 Our results corroborated the concept that polymeric nanoparticle platform has potential for improving the curative effect of native nutlin-3a. Targeted delivery offers a significant advantage for the efficient delivery of therapeutic payload and/or imaging agents to a specific target site while minimizing the exposure to normal tissues. 2,20 One such potent surface molecule used for targeted drug delivery purpose is epithelial cell adhesion molecule (EpCAM), which is expressed at low levels in normal epithelial cells (expression pattern is mostly on the basal or bosolateral membrane with sequestration in intercellular boundaries) but is frequently over-expressed (up to 1000-fold) homogeneously on epical surface of the cancer cell (distribution varies depending upon the type of carcinoma). 21–25 This differential expression makes EpCAM an attractive tumor-associated surface molecule for targeted drug delivery. As a targeting ligand nucleic acid aptamer holds great promises and has been exploited against various surface molecules by a number of research groups. 26–29 Nucleic acid aptamers are single stranded DNA or RNA oligonucleotides that fold into specific three dimensional (3D) structures, and bind to target molecules with high specificity and affinity. 30 Thus, selective targeting of therapeutic payload to cancer cells via EpCAM specific aptamer could be effective for molecularly targeted cancer therapy. In this regard, Li et al. 31 have recently shown in vitro the enhanced therapeutic potentiality of EpCAM aptamer functionalized PLGA-lecithinPEG based hybrid nanoformulation of curcumin in colon adenocarcinoma cells and advocate toward the advantage of active targeted nanotechnology-based drug delivery approach for cancer therapy. Further, our group has recently developed an EpCAM specific aptamer using cell SELEX (systematic evolution of ligands by exponential enrichment) based approach which is very stable in nature as it contain 2-fluoropyrimidines and a 3′-inverted deoxythymidine cap, leading to a 3′–3′ linkage that inhibits degradation by 3′ exonucleases. We also evaluated its binding specificity with EpCAM in various EpCAM expressing and nonexpressing cell lines and results suggest significantly higher specificity and binding of EpCAM aptamer (Apt) toward EpCAM expressing cancer cells than that of EpCAM non-expressing cells. 32,33
Semiconductor nanocrystals known as quantum dots (QDs) have been increasingly utilized as biological imaging and labeling probes because of their unique optical properties. 34,35 Nanoteranostic approaches for achieving superior cancer treatment options are now utilizing quantum dots in combination with anticancer drugs to amalgamate cancer therapy and imaging. 36,37 Thus in the present study, we aimed to develop a theranostic nanocarrier system capable of targeted cancer therapy and imaging. The approach taken in this work is to prepare and characterize biodegradable PLGA nanoparticulate systems loaded with nutlin-3a and surface functionalized with a targeting ligand i.e. Apt and an imaging agent i.e. quantum dots. Further, to validate our hypothesis that Apt targeted theranostic nanoparticles may exhibit an augmented therapeutic and imaging capability via EpCAM in diverse cancer, therapeutic and imaging potentiality of our theranostic carrier system was evaluated in several different types of solid cancer cell lines that are non-expressing or expressing high or moderate level of EpCAM. The in vitro study includes uptake, cytotoxicity, apoptosis analysis and imaging in 2D monolayer and 3D spheroid model. The molecular mechanism underlining apoptosis was investigated by studying the induction or inhibition of key proteins involved in the regulation of apoptosis. The results suggest that our theranostic nanocarriers may act as a multimodal vehicle capable of eliciting enhanced cancer therapeutics and functioning in molecule imaging in diverse cancer type.
Methods Materials Nutlin-3a was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Poly(D, L-lactide-co-glycolide) PLGA with a copolymer ratio of 50:50 and inherent viscosity (IV = 0.41) was obtained from Birmingham Polymers, Inc. (Birmingham, AL). All chemicals were obtained from Sigma–Aldrich Co. (St. Louis, MO) unless mentioned. All primary antibodies used were either obtained from Cell Signaling Technology, Inc. (Danvers, MA) or from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies (FITC conjugated or horseradish peroxidase-conjugated) were procured from Santa Cruz Biotechnology (Santa Cruz, CA). Qdot® 605 ITK™ amino (PEG) quantum dots (QD605) was purchased from Invitrogen Corp. (Carlsbad, CA). Cell culture MCF-7 (human breast cancer), SKOV3 (human ovarian cancer), and ZR751 (human breast cancer) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). HEK-293 (human embryonic kidney) cell line was obtained from National Centre for Cell Science, Pune, India. MCF-7, SKOV3, and ZR751 cells express EpCAM and HEK-293 is negative for EpCAM expression. All cells were grown in DMEM (Invitrogen, CA) media supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, 1%
M. Das et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 379–389 L-glutamine
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(PAN-Biotech GmbH, Germany) at 37 °C in an incubator with 5% CO2 supply (Hera Cell, Thermo Scientific, Waltham, MA).
studied over a period of time in phosphate buffer at 37 °C (Supplementary Materials).
Formulation of nutlin-3a nanoparticles PLGA NPs loaded with the drug nutlin-3a were prepared by oil-in-water single emulsion-solvent evaporation method as per our previously reported protocol (Supplementary Materials). 19
Immunostaining for EpCAM expression by laser-scanning confocal imaging. To investigate the expression of EpCAM in various cancer cell lines, immunostaining assay was performed according to the protocol followed by Maaser and Borlak 40 (Supplementary Materials).
Conjugation of Apt and quantum dots to PLGA nanoparticles EpCAM specific RNA aptamer was synthesized as per our previously reported protocol. 32 The Apt has sequence 5′-aminoC12-.GCGACUGGUUACCCGGUCG-idT-3′, containing 2′ fluoropyrimidines and a 3′-inverted deoxythymidine cap, leading to a 3′–3′ linkage that inhibits degradation by 3′ exonucleases. Conjugation of QD and Apt onto the surface of PLGA NPs was performed according to the protocol followed by Savla et al. 26 and Farokhzad et al. 27 (Supplementary Materials). Analysis of surface conjugation Agarose gel electrophoresis. The conjugation of Apt onto PLGA NPs surface was investigated by agarose gel electrophoresis. In brief, 2% agarose gel with 0.5 μg/ml of EtBr was prepared in Tris boric acid EDTA (TBE) buffer, Apt conjugated NPs (with EDC and without EDC), before washing and after washing and free Apt was loaded into well and the gel electrophoresis was performed at 60 V in TBE buffer. 38 Imaging was performed by Chemi Doc™ XRS+ Imaging System (Bio-Rad, CA, USA). FTIR analysis. The Fourier transform infrared (FTIR) spectra for void-PLGA-NPs, Apt-PLGA-NPs were obtained from FTIR spectrophotometer SPECTRUM RX I (Spectrum 1, PerkinElmer, San Jose, CA) for studying the surface modification of PLGA-NPs after Apt conjugation to PLGA NPs surface. Briefly, the samples were pressed with KBr to make a pellet by applying a pressure of 300 kg/cm 2 before obtaining their IR absorption spectra. The spectra were detected in KBr disks over a range of 4500–300 cm − 1. Fluorescence spectrophotometer analysis. Briefly, QDPLGA-NPs, void-PLGA-NPs suspension (0.1 mg/ml) in 0.1 M PBS was placed into a quartz cuvette and emission spectrum was recorded with spectrofluorophotometer (ParkinElmer, Massachusetts, USA) at excitation wavelength of 405 nm and emission wavelength of 605 nm. 36 Physiochemical characterization of nutlin-3a loaded PLGA NPs. Particle size, size distribution (polydispersity index) and surface charge of the nanoformulations were measured by dynamic light scattering (DLS), using Zetasizer. Size was further confirmed by TEM analysis. Surface morphology of NPs was studied by AFM analysis. Entrapment efficiency of nutlin-3a in NPs was estimated by high performance liquid chromatography (RP-HPLC) method following established protocol. 19 In vitro release kinetics of nutlin-3a from Apt-Nut-NPs was performed in serum containing media as per previously published protocol. 4,39 The stability of Apt and QD functionalized NPs was
Cellular uptake study of aptamer conjugated NPs. Qualitative and quantitative cellular uptake of native 6-coumarin, 6coumarin loaded NPs (conjugated or unconjugated) was investigated in different cell lines by confocal microscopy and fluorescence spectrophotometer respectively (Supplementary Materials). 19 In vitro cellular cytotoxicity of Apt functionalized nutlin-3a loaded NPs. The cytotoxic effect of Apt functionalized nutlin3a loaded NPs over unconjugated NPs and native nutlin-3a was investigated by MTT assay (Supplementary Materials). 5 Cell cycle arrest and apoptosis study. Cell cycle arrest and induction of apoptotic cell death following treatment with nutlin3a in native or nanoformulations having drug concentration of 1 μg/ml were performed in ZR751 cell line by flow cytometer (Supplementary Materials). 19,5 Immunoblotting. For Western blot analysis, ZR751 cells at cell density 1 × 10 5/ml were treated with 2 μg/ml concentrations of native nutlin-3a or equivalent concentration of drug loaded in nanoformulations (Nut-NPs and Apt-Nut-NPs) for 48 hours. Following various treatments, cell protein was extracted with lysis buffer and subjected to Western blot analysis with specific primary antibody against various proteins studied (Supplementary Materials). Imaging of cancer cells with Apt-QD-Nut-NPs by confocal laser scanning microscope. To image the cancer cells grown in monolayer or three dimensional (3D) spheroid tumor model, Apt-QD-Nut-NPs and QD-Nut-NPs were used as an imaging probe and imaging was performed by confocal microscope (Supplementary Materials). 41,42 Statistical analysis. Data were represented as means ± standard deviation (SD). Comparison of data in quantum dots conjugation assay was performed using a two tailed Paired Student's test. Uptake analysis was done by one-way analysis of variance (ANOVA) with Tukey post-test and cyototoxicity assay was performed by two-way analysis of variance (ANOVA) with Bonferroni multiple comparisons. P values less than 0.05 were considered statistically significant. Originpro 8 was used to fit dose–response and exponential equations to calculate IC50 values. GraphPad Prism Software was used for all statistical analyses. Results Physiochemical characterization of drug loaded PLGA nanoparticles Nutlin-3a loaded PLGA nanoparticles were formulated by emulsion-solvent evaporation method and result indicates that
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Figure 1. Physiochemical characterization of nanoformulations (A, B, C), Analysis of conjugation of Apt (D) and quantum dots (E) with PLGA nanoparticles and in vitro release kinetics of Apt-Nut-NPs (F). Size of Nut-NPs (A) was measured by DLS. (B) TEM analysis of nutlin-3a loaded NPs. Scale bar is 100 nm. (C) AFM image of Nut-NPs. (D) Conjugation of Apt to PLGA NPs (following EDC–NHS conjugation chemistry) was analyzed by 2% agaroge gel electrophoresis. Lanes 1, 2, 3, 4, and 5 correspond to native Apt, Apt-NPs with EDC and NHS before wash, Apt-NPs without EDC and NHS before wash, Apt-NPs with EDC and NHS after wash, Apt-NPs without EDC and NHS after wash respectively. (E) Conjugation of quantum dots to NPs was confirmed by fluorescence spectrophotometer by measuring the fluorescence intensity of void-PLGA-NPs and QD-PLGA-NPs. Error bar shows mean ± SD, n = 3, **p b 0.001 for void-PLGA-NPs versus QD-PLGA-NPs. (F) Sustained release of nutlin-3a exhibited by Apt-Nut-NPs in 10% FBS containing media. Error bar shows mean ± SD, n = 3.
the formulated NPs are of nanometer size range with negative zeta potential (Figure 1, A, B and Table 1) and having smooth surface morphology (Figure 1, C). The EDC/NHS activation technique enables the conjugation of amine functional group of the Apt/QD with the carboxyl group of PLGA through the formation of an amide bond (Figure 1, D, E; Figures S1 and
S2). 27,43 Further, the nanoformulation depicts a sustain release profile of nutlin-3a from Apt-Nut-NPs, over 72 hours as depicted in Figure 1, F. The in vitro stability study of Apt and QD functionalized Nut-NPs indicates the presence of QD or Apt on nanoparticles surface till 48 hours (Figure S3, A and B). Details are shown in supplementary information.
M. Das et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 379–389 Table 1 Physico-chemical characterization of Nutlin-3a loaded nanoparticle. Zeta potential (mV) b
Formulation Size (nm) a Void-NPs Nut-NPs Apt-Nut-NPs QD-Nut-NPs a b c d
237 253 292 257
± ± ± ±
13 − 10.99 ± 0.5 13 − 16.58 ± 0.95 10 − 20.3 ± 0.66 27 − 8.91 ± 3.1
Polydispersity Entrapment index c efficiency (%) d 0.24 0.18 0.36 0.20
± ± ± ±
0.13 0.03 0.02 0.02
– 65.98 ± 2.98 51.24 ± 6.7 –
Size in nm was measured by Zetasizer. Zeta potential in mV was measured by Zetasizer. Polydispersity index was measured by Zetasizer. Entrapment efficiency of nutlin-3a was measured by HPLC.
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treatment (native or nanoformulation) for 24 hours (Figure 4, A). Figure 4, A, shows that 39.54% of cells were arrested at G2/M phase of cell cycle treated with Apt-Nut-NPs compared to 27.45% in Nut-NPs and 16.42% native nutlin-3a in solution. Execution of enhanced apoptosis mediated by Apt-Nut-NPs Nutlin-3a has been advocated for its effectiveness in inducing apoptosis. In the present study, we have tested the efficacy of Apt functionalized Nut-NPs over native drug and unconjugated NutNPs in induction of apoptosis. Apt-Nut-NPs induced 21.08% early apoptotic cell death in comparison to 8.8% and 14.5% early apoptotic cell death exhibited by free nutlin-3a and unconjugated Nut-NPs (Figure 4, B). Thus, Apt-Nut-NPs elicited more potent activation of apoptosis in ZR751 cells than Nut-NPs or free drug.
Comparative expression study of EpCAM The immunofluoroscence result for EpCAM expression indicates that ZR751, MCF-7 and SKOV3 are positive for EpCAM expression with varying degrees of expression level whereas HEK-293 cell line do not express any detectable level of EpCAM (Figure S4). Cellular uptake analysis The cellular uptake analysis by confocal microscope depicts an augmented uptake of Apt-6-coumarin-NPs in all EpCAM expressing cell lines compared to its unconjugated counterpart (Figure 2, A), whereas no such significant difference in uptake efficiency between Apt-6-coumarin-NPs and 6-coumarin-NPs was evident in HEK-293 cell line (Figure 2, A). The uptake of Apt-6-coumarin-NPs in comparison to unconjugated NPs and native 6-coumarin was further validated by fluorescence spectrophotometer in ZR751 and HEK-293 cell line (Figure 2, B). The result indicates a higher uptake of Apt-6-coumarin-NPs compared to unconjugated counterpart in EpCAM expressing ZR751 cell line (Figure 2, B). However, no significant difference in uptake efficiency was observed between Apt-6-coumarin-NPs and 6-coumarin-NPs in HEK-293 cell line. Cellular cytotoxicity study The cytotoxicity results shows an augmented cytotoxic activity of Apt-Nut-NPs over unconjugated counterpart in EpCAM expressing cell lines (ZR751, MCF-7, SKOV3), however there is no substantial difference in cytotoxic effect in EpCAM non-expressing HEK-293 cell line (Figure 3). As shown in Table 2, the IC50 (inhibitory concentration required to induce fifty percent cytotoxic effect) for Apt-Nut-NP is 4-fold in ZR751, 2-fold in SKOV3 and 3-fold in MCF-7 cells lower than that for Nut-NPs, thus substantiating the above findings and indicating that Apt-Nut-NPs is more efficient in causing cell death in a targeted manner compared to unconjugated NPs. Void NPs (without any drug) equivalent to the doses of drug concentration are non-toxic in nature. Cell cycle analysis Nutlin-3a induces cell cycle arrest in G1 and/or G2 phase of cell cycle. To explore the cell cycle regulatory effect of nutlin-3a in ZR751 cells, cell cycle analysis was performed following drug
Modulation of proteins involved in the regulation of apoptosis Effect of Apt-Nut-NPs on p53 activation was studied by Western blot analysis. The result indicates enhanced expression of p53 proteins following treatment with Apt-Nut-NPs compared to native nutlin-3a and unconjugated counterpart (Figure 5). Activation of p53 modulates many signaling molecules involved in apoptotic events. Proapoptotic proteins like BAX and antiapoptotic proteins like bcl2 are the downstream targets of p53. Further, cleavage of caspase 3 also acts as an event during apoptosis process. Thus, we investigated the expression of key proteins like BAX, bcl2, and caspase 3 by Western blotting. The result reveals over-expression of BAX, cleavage of caspase 3 and down-regulation of bcl2 following drug treatment (Figure 5) and compared to Nut-NPs and native nutlin-3a, targeted Apt-NutNPs exhibited enhanced modulation of proteins involved in apoptosis pathway. In vitro bio-imaging To investigate the potentiality of QD conjugated Apt-Nut-NPs as targeted imaging modality; we carried out an in vitro imaging study in a 2D monolayer culture and 3D tumor spheroid model. Confocal microscope image of ZR751 and HEK-293 cell lines grown in monolayer indicates that, the fluorescence intensity of Apt-QD-Nut-NPs exposed to EpCAM over-expressing ZR751 cell line was higher compared to non-targeted counterpart (QD-NutNPs) (Figure 6, A). However, no significant difference in fluorescence intensity was evident in EpCAM negative HEK293 cell line treated with QD-Nut-NPs and Apt-QD-Nut-NPs depicting the potentiality of targeted imaging in vitro (Figure 6, A). To further validate the concept of targeted in vivo imaging by QD functionalized Apt-Nut-NPs, we tested its penetrating ability (a prerequisite for in vivo imaging) in ZR751 cells grown as a tumor spheroid model. Spheroids provide an in vitro avascular 3dimensional model that mimics the in vivo tumor and its interaction with tumor microenvironment. The Z-stack laser scanning confocal images at stack 8 (at the center of the tumor spheroid) exhibited higher fluorescence intensity than that of stack 0 or stack 16 located at the top and bottom, respectively for ZR751 tumor spheroid treated with Apt-QD-Nut-NPs in comparison to QD-NutNPs (Figure 6, B), indicating the ability of the targeted system to penetrate deep into a solid tumor.
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Figure 2. Analysis of cellular uptake of nanoformulations by confocal microscopy and fluorescence spectrophotometer. MCF-7, ZR751, and SKOV3 cell lines positive for EpCAM expression and HEK-293 cell line negative for EpCAM expression were treated with 90 ng/ml of native 6-coumarin, equivalent concentration of 6-coumarin loaded nanoparticles (6-coumarin-NPs) and Apt conjugated 6-coumarin NPs (Apt-6-coumarin-NPs) for 2 hours and uptake of 6coumarin was observed by confocal microscope (A). Quantitative uptake of above formulations was further investigated in ZR751 and HEK-293 cells by fluorescence spectrophotometer (B). Values are shown as mean ± SD, n = 5, significance level was set at p b 0.05.
Discussion In spite of the ability of the contemporary chemotherapeutic treatment regimens to achieve relatively high rates of remission induction and survival, still there are challenges for successful cancer therapy. In this setting to overcome the challenges associated with cancer chemotherapy there has been progressively heightened interest in the development of a combinational approach for targeted drug delivery and controlled release technology which may pave the road to more effective yet safer chemotherapeutic options for cancer therapy. Apart from developing a targeted sustained drug delivery technology, monitoring the effect of chemotherapeutic agents on tumor progression is very much essential. In this setting, molecularly targeted theranostic approach i.e. fusion of therapeutic and diagnostic for simultaneous cancer detection, inhibition and therapeutic response assessment has emerged as a promising therapeutic strategy against cancer. 44 Noteworthy, advances in
nanobiotechnology have enabled the development of such targeted theranostic nanoplatforms. 7,26,37,45 To this end, with an aim to achieve a therapeutic strategy enabling delivery of high therapeutic payload at target site in a controlled fashion and facilitating imaging, in the present investigation, we have surface functionalized nutlin-3a loaded nanocarrier system with a targeting ligand (aptamer specific for targeting EpCAM over-expressed in most of cancer) and an imaging agent (quantum dots) and tested its therapeutic and imaging prospective. In the present work, we have developed nanoparticles of nanometer size range with negative zeta potential (Figure 1, A, B, C and Table 1). Developing a multifunctional nanoformulation capable of both targeted drug delivery and imaging is a challenging task. By implementing the EDC-NHS chemistry, we performed simultaneous conjugation of both targeting (Apt) and imaging modalities (quantum dot) onto nutlin-3a loaded PLGA nanoparticle surface in an efficient way (Figure 1, D and E) to formulate a
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Figure 3. Cytotoxicity assay. Briefly, 3000 cells were seeded per well in 96 well plates for overnight and then incubated with 0.005–6 μg/ml drug concentration (of native nutlin-3a, Nut-NPs, Apt-Nut-NPs) and corresponding amount of void NPs. Cells left untreated serve as control. The extent of growth inhibition was measured at the 5th day using MTT assay and inhibition was calculated with respect to untreated control cells. Data are expressed as mean ± SD., n = 4, *p b 0.05, **p b 0.01, ***p b 0.001, for native nutlin-3a vs Nut-NPs or Nut-NPs vs Apt-Nut-NPs.
Table 2 Cytotoxicity study by MTT assay. Cell line
Treatments
IC50 (μg/ml) a
MCF-7
Native Nutlin-3a Nut-NPs Apt-Nut-NPs Native Nutlin-3a Nut-NPs Apt-Nut-NPs Native Nutlin-3a Nut-NPs Apt-Nut-NPs Native Nutlin-3a Nut-NPs Apt-Nut-NPs
2.46 0.99 0.36 1.02 0.33 0.08 5.30 1.60 0.68 4.08 1.08 1.15
ZR 751
SKOV3
HEK-293
± ± ± ± ± ± ± ± ± ± ± ±
0.40 0.13*** 0.06* 0.15 0.13*** 0.02* 1.01 0.39*** 0.15* 0.61 0.09*** 0.38
a
Inhibitory concentration causing 50% cell death. Data as mean ± SD, n = 4. *p b 0.05, **p b 0.01, ***p b 0.001 for Native Nutlin-3a vs Nut-NPs or NutNPs vs Apt-Nut-NPs.
nanotheranostic system. Further our Apt functionalized NPs exhibited a sustained drug release profile which may facilitate a prolonged cytotoxic effect at tumor site for better cancer remission.
Various site-specific targeting strategies have been adopted in recent years to augment the cellular interaction and internalization of nanoformulations. 2 Thus, to authenticate our concept that, Apt functionalization to drug loaded NPs will result in enhanced cellular internalization of the therapeutic payload, cellular uptake efficiency of the Apt targeted carrier system was assessed by confocal microscopy. Results demonstrate that, uptake of Apt-6-coumarin-NPs was significantly higher than that of the unconjugated nanoparticle in EpCAM expressing cell lines but no significant difference in cellular uptake pattern was evident in EpCAM non-expressing HEK293 cell line (Figure 2, A). In a recent study, Hussain et al. 21 have documented a higher cellular binding and uptake of EpCAM-targeted immunoliposomes compared to non-targeted control liposome in EpCAM expressing MCF-7 and SW2 cell lines, however no remarkable difference was evident in EpCAM negative RL cells. In accordance with above findings, in our cellular uptake study the greater uptake of Apt-6coumarin-NPs in EpCAM positive cells indicates that, EpCAM expression may be playing a crucial role in receptor mediated binding and uptake of targeted NPs. EpCAM mediated internalization of Apt targeted NPs was further validated by
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Figure 4. Cell cycle arrest analysis and apoptosis assay by flow cytometry. In brief, 2 × 10 5 ZR751 cells were seeded overnight in 6-well plates and then treated with 1 μg/ml of native nutlin-3a, Nut-NPs, Apt-Nut-NPs for 24 hours and 36 hours for cell cycle analysis and apoptosis assay respectively. Cell cycle distribution was analyzed by PI staining (A). Content of DNA is represented on the x-axis; number of cells counted is represented on the y-axis. The regions marked M1, M2, M3 and M4 represent G1, S, G2 and G0 phases respectively of the cell cycle. Experiment was performed in duplicates and representative image has been given. Apoptosis percentage was analyzed using standard annexin V assay by FACS (B). The experiment has been performed three times and representative image has been provided. Data represented as mean ± SD.
fluorescence spectrophotometer analysis in ZR751 and HEK293 cell lines (Figure 2, B). To validate the therapeutic potentiality of targeted nanoformulations upon internalization for sustained killing of cancer
cells, MTT based cytotoxicity assay was performed. The enhanced cytotoxic effect of Apt-Nut-NPs than unconjugated NPs in EpCAM over-expressing cell lines and a similar pattern of cytotoxicity in EpCAM non-expressing cell line, stress the key
Figure 5. Study of signaling proteins by Western blot analysis. ZR751 cells were incubated with native nutlin-3a, Nut-NPs, and Apt-Nut-NPs (2 μg/ml) for 48 hours, and various apoptosis associated proteins were analyzed by Western blotting. β-Actin serves as internal control. The experiment has been performed two times and representative image of an experiment has been provided.
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Figure 6. Confocal bio-imaging. (A) HEK-293 and ZR751 cells were grown in 2D monolayer and exposed to 5 μg/ml of Apt-QD-Nut-NPs or QD-Nut-NPs for 4 hours and images were captured by confocal microscope. (B) ZR751 cells were grown as 3D spheroid and treated with 20 μg/ml of Apt-QD-Nut-NPs or QDNut-NPs for 4 hours and Z-stack laser scanning fluorescence images (Z-0 to Z-17) were captured by confocal microscope. Z-0, Z-8, and Z16 images for all samples were given for presentation. Experiment has been performed twice and representative image of an independent experiment has been provided.
role of receptor mediated NPs binding and enhanced internalization in enhancement of cytotoxic activity (Figure 3, Table 2). In a recent study Subramanian et al. 46 have documented the enhanced cytotoxic effect of chimeric EpCAM aptamer doxorubicine conjugate in EpCAM expressing retinoblastoma cells, while no significant cytotoxicity was observed in EpCAM negative non-cancerous Muller-glial cells following higher cellular uptake by EpCAM receptor mediated endocytosis. The above study clearly suggests the key role of EpCAM in mediating targeted cytotoxic effect, thus substantiating our finding. Most of the chemotherapeutic agents exert their effect either by arresting the cell cycle progression or through induction of apoptosis. In the present context, treatment of ZR751 cells with Apt-Nut-NPs resulted in a greater proportion of cells in G2 phase (Figure 4, A) and higher apoptotic cell death (Figure 4, B) compared to unconjugated counterpart and native nutlin-3a. Enhanced efficiency of Apt-Nut-NPs compared to Nut-NPs in arresting more number of cells and inducing enhanced apoptotic cell death may have resulted due to higher intracellular drug levels at the site of action (due to enhanced cellular uptake
following receptor mediated endocytosis) for a longer period of time (following sustain release). 47,48 As loss of proapoptotic signals and gain of anti-apoptotic mechanisms lead to resistance of cancer cells toward apoptosis, most of the current chemotherapeutic agents exert their antitumor effect by modulating these signal transduction pathways. 49,50 We have studied the expression of few signaling molecules playing a role in apoptosis by Western blot analysis. In the present work, we observed enhanced expression of p53, BAX, cleavage of caspase 3, and down-regulation in expression of antiapoptotic protein bcl2 in cells treated with native drug or nanoformulation (Figure 5). Notably, in all cases Apt functionalized nanopaticles showed greater efficacy in modulating the expression of above mentioned proteins compared to native nutlin-3a and Nut-NPs following site specific drug delivery. In relation to cancer, bio-imaging of tumor presents a unique challenge because of the urgent necessity for highly specific and sensitive imaging agents. Among various imaging modalities, semiconductor QDs have received much attention as a new generation imaging agent for molecular, cellular and in vitro/in vivo
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imaging. However, lack of site specific imaging ability may hamper the use of QD in clinical setting. Thus in this study, we have surface functionalized Apt and QD605 quantum dots on to PLGA NPs surface for site specific imaging application and the in vitro results of imaging in tumor spheroid and 2D monolayer suggest that, the Apt targeted theranostic nanosystem is capable of target specific imaging compared to unconjugated counterpart (Figure 6). Recently Gao et al. 45 have shown the higher cancer cell labeling potentiality of prostate specific membrane antigen (PSMA) antibody targeted QD compared to non-targeted QD for PSMA-positive C4-2 cells, however no remarkable difference was observed compared with that in PSMA-negative PC-3 cells. In corroboration with above finding, in our imaging study the greater internalization of Apt-QD-Nut-NPs in EpCAM-positive cells resulting in enhance cellular labeling indicates toward the crucial role of Apt in receptor mediated binding and internalization of conjugated NPs. Further, our Apt-QD-Nut-NPs have also demonstrated deeper penetrating capability compared to unconjugated counterpart as shown from different z-scan confocal images (Figure 6, B). In accordance to above observation Savla et al. 26 have also demonstrated, higher internalization and penetration potentiality of MUC1 aptamer conjugated QD in comparison to unconjugated QD in MUC1 expressing A2780/AD ovarian carcinoma cells grown as 3D tumor spheroids. Thus results suggest that, the above developed multifunctional theranostic nanosystem may be applied for achieving better imaging and therapeutic response in cancer. Extensive investigation by Savla et al. 26 has validated the application of mucin 1 aptamer targeted nanothranostics for their preferential accumulation in the ovarian tumor and tumor imaging in vivo. In relation to this, the preliminary but significantly important findings observed in the present investigation are currently in the process of in vivo validation and may be implemented in clinical settings in the future. In conclusion, current diagnosis and treatment options for cancer must be improved, despite significant advances over the past decades to combat this disease. Extensive preclinical research in cancer nanotechnology holds much potential to generate improved options for diagnosing and treating the disease. In this context, in the present state of art, we have developed a multifunctional nanosystem capable of both cancer therapeutics and imaging. Our results indicate that, Apt targeted nutlin-3a loaded NPs resulted in enhanced cellular accumulation of drug, and greater cytotoxicity in EpCAM overexpressing cancer cells. Further, Apt functionalized nanoparticle induces higher apoptosis by triggering signaling molecules. Moreover, our carrier system has shown its potency as an imaging modality in in vitro tumor spheroid model. Overall, we anticipate that this multifunctional nanosystem may act as a treatment option for cancer, however, for implementing the use of the theranostic nanosystem in clinical settings a detailed investigation on in vivo biodistribution and antitumor effect of the targeted nanoformulations is warranted in the near future. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2014.09.002.
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Original Article
nanomedjournal.com
Multifunctional nanoparticle–EpCAM aptamer bioconjugates: A paradigm for targeted drug delivery and imaging in cancer therapy Manasi Das, MS a , Wei Duan, MBBS, PhD b , Sanjeeb K. Sahoo, PhD a,⁎ a Institute of Life Sciences, Nalco Square, Bhubaneswar, India School of Medicine, Deakin University, Waurn Ponds, Victoria, Australia Received 21 May 2014; accepted 9 September 2014
b
Abstract The promising proposition of multifunctional nanoparticles for cancer diagnostics and therapeutics has inspired the development of theranostic approach for improved cancer therapy. Moreover, active targeting of drug carrier to specific target site is crucial for providing efficient delivery of therapeutics and imaging agents. In this regard, the present study investigates the theranostic capabilities of nutlin-3a loaded poly (lactide-co-glycolide) nanoparticles, functionalized with a targeting ligand (EpCAM aptamer) and an imaging agent (quantum dots) for cancer therapy and bioimaging. A wide spectrum of in vitro analysis (cellular uptake study, cytotoxicity assay, cell cycle and apoptosis analysis, apoptosis associated proteins study) revealed superior therapeutic potentiality of targeted NPs over other formulations in EpCAM expressing cells. Moreover, our nanotheranostic system served as a superlative bio-imaging modality both in 2D monolayer culture and tumor spheroid model. Our result suggests that, these aptamer-guided multifunctional NPs may act as indispensable nanotheranostic approach toward cancer therapy. From the Clinical Editor: This study investigated the theranostic capabilities of nutlin-3a loaded poly (lactide-co-glycolide) nanoparticles functionalized with a targeting ligand (EpCAM aptamer) and an imaging agent (quantum dots) for cancer therapy and bioimaging. It was concluded that the studied multifunctional targeted nanoparticle may become a viable and efficient approach in cancer therapy. © 2015 Elsevier Inc. All rights reserved. Key words: Nanotheranostics; Nutlin-3a; EpCAM aptamer; Quantum dots; PLGA NPs
One of the foremost impediments in cancer chemotherapy is the poor aqueous solubility, bioavailability and side effects caused by nonspecific cytotoxicity of widely used anticancer drugs. 1 Novel approaches are needed to site-specifically deliver chemotherapeutic agents to tumor cells by overcoming above limitations to enhance their therapeutic window. Presently, research in diverse direction for upgrading the carrier system to deliver the therapeutic payload at the target site efficiently is ongoing. 2–4 In this setting nanotechnology based nanoformulations have played a very crucial role in enhancing the therapeutic potentiality of the conventional chemotherapeutic agents by increasing their aqueous solubility, bioavailability and by reducing non-specific toxicity. 2,5,6 Apart from these requirements, ability to diagnose the diseased conditions in a early stage and monitoring the therapeutic response are also the prerequisite for better cancer therapy. In this milieu, nanotheranostic Conflict of interest: We do not have any conflict of interest. Manasi Das thanks the Council of Scientific Industrial Research, New Delhi, Government of India, for a Senior Research Fellowship. ⁎Corresponding author at: Laboratory for Nanomedicine, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Orissa, India. E-mail address: [email protected] (S.K. Sahoo).
platform are anticipated to play a vital role and multifunctional nanocarrier systems with multimodal capabilities (i.e. simultaneously provides detection, diagnosis and therapy) represent tremendous potentiality to be used in nanotheranostic approach for improved cancer therapy and diagnosis. 3,7 Chemotherapeutic agents are our main weapon in the treatment of cancer. The recently developed chemotherapeutic agent nutlin-3a is a small molecule inhibitor of the murine double minute 2 (MDM2)–p53 interaction, which is as direct non-genotoxic p53 stabilization thereby, activating cell cycle arrest and apoptosis in cancer cells. 8,9 Recently, Lau et al. 10 have shown that nutlin-3a can disrupt the interaction between p73 and MDM2 and induce apoptosis in cells lacking functional p53. A series of recent studies have strengthened the concept that, selective restoration of the tumor suppressor function of p53 by nutlin-3a in various types of cancer might represent a substitute to the existing chemotherapy that generally causes DNA damage and induces various complications related to growth, infertility, and secondary malignant tumor induction. 8,11,12 In contrary to others, in a recent study, Shen and Maki 13 have pointed out that nutlin induces endoreduplication and therapy resistance in colon cancer and osteosarcoma cells. Though the findings are significantly important for use of nutlin-3a in
http://dx.doi.org/10.1016/j.nano.2014.09.002 1549-9634/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Das M, et al, Multifunctional nanoparticle–EpCAM aptamer bioconjugates: A paradigm for targeted drug delivery and imaging in cancer therapy. Nanomedicine: NBM 2015;11:379-389, http://dx.doi.org/10.1016/j.nano.2014.09.002
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clinical settings, their results indicate that this phenomenon is not apparent in all cell lines studied and thus may not completely mask the therapeutic implications observed with nutlin in a diverse cancer type by many research groups. As nutlin-3a represents a potent anticancer agent in recent scenario, the clinical utility of this novel molecule is limited by its poor water solubility, limited dose that is delivered to tumor site and drug resistance phenotype. 14 In this context polymeric nanoparticles can play a vital role in overcoming the restraints associated with native nutlin-3a. 15,16 Biodegradable NPs formulated from poly(D, L-lactide-co-glycolide) (PLGA) are being extensively investigated for the purpose of sustained and localized administration of different antiproliferative agents. 17,18 The extensive biomedical application of PLGA is because of its biocompatibility, ability to encapsulate various drug molecules and sustained release properties. 17 In this milieu, our group has recently developed ligand targeted nutlin-3a encapsulated PLGA nanoparticles to surmount the shortcomings associated with native nutlin-3a and investigated its therapeutic prospective at cellular and molecular level. 19 Our results corroborated the concept that polymeric nanoparticle platform has potential for improving the curative effect of native nutlin-3a. Targeted delivery offers a significant advantage for the efficient delivery of therapeutic payload and/or imaging agents to a specific target site while minimizing the exposure to normal tissues. 2,20 One such potent surface molecule used for targeted drug delivery purpose is epithelial cell adhesion molecule (EpCAM), which is expressed at low levels in normal epithelial cells (expression pattern is mostly on the basal or bosolateral membrane with sequestration in intercellular boundaries) but is frequently over-expressed (up to 1000-fold) homogeneously on epical surface of the cancer cell (distribution varies depending upon the type of carcinoma). 21–25 This differential expression makes EpCAM an attractive tumor-associated surface molecule for targeted drug delivery. As a targeting ligand nucleic acid aptamer holds great promises and has been exploited against various surface molecules by a number of research groups. 26–29 Nucleic acid aptamers are single stranded DNA or RNA oligonucleotides that fold into specific three dimensional (3D) structures, and bind to target molecules with high specificity and affinity. 30 Thus, selective targeting of therapeutic payload to cancer cells via EpCAM specific aptamer could be effective for molecularly targeted cancer therapy. In this regard, Li et al. 31 have recently shown in vitro the enhanced therapeutic potentiality of EpCAM aptamer functionalized PLGA-lecithinPEG based hybrid nanoformulation of curcumin in colon adenocarcinoma cells and advocate toward the advantage of active targeted nanotechnology-based drug delivery approach for cancer therapy. Further, our group has recently developed an EpCAM specific aptamer using cell SELEX (systematic evolution of ligands by exponential enrichment) based approach which is very stable in nature as it contain 2-fluoropyrimidines and a 3′-inverted deoxythymidine cap, leading to a 3′–3′ linkage that inhibits degradation by 3′ exonucleases. We also evaluated its binding specificity with EpCAM in various EpCAM expressing and nonexpressing cell lines and results suggest significantly higher specificity and binding of EpCAM aptamer (Apt) toward EpCAM expressing cancer cells than that of EpCAM non-expressing cells. 32,33
Semiconductor nanocrystals known as quantum dots (QDs) have been increasingly utilized as biological imaging and labeling probes because of their unique optical properties. 34,35 Nanoteranostic approaches for achieving superior cancer treatment options are now utilizing quantum dots in combination with anticancer drugs to amalgamate cancer therapy and imaging. 36,37 Thus in the present study, we aimed to develop a theranostic nanocarrier system capable of targeted cancer therapy and imaging. The approach taken in this work is to prepare and characterize biodegradable PLGA nanoparticulate systems loaded with nutlin-3a and surface functionalized with a targeting ligand i.e. Apt and an imaging agent i.e. quantum dots. Further, to validate our hypothesis that Apt targeted theranostic nanoparticles may exhibit an augmented therapeutic and imaging capability via EpCAM in diverse cancer, therapeutic and imaging potentiality of our theranostic carrier system was evaluated in several different types of solid cancer cell lines that are non-expressing or expressing high or moderate level of EpCAM. The in vitro study includes uptake, cytotoxicity, apoptosis analysis and imaging in 2D monolayer and 3D spheroid model. The molecular mechanism underlining apoptosis was investigated by studying the induction or inhibition of key proteins involved in the regulation of apoptosis. The results suggest that our theranostic nanocarriers may act as a multimodal vehicle capable of eliciting enhanced cancer therapeutics and functioning in molecule imaging in diverse cancer type.
Methods Materials Nutlin-3a was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Poly(D, L-lactide-co-glycolide) PLGA with a copolymer ratio of 50:50 and inherent viscosity (IV = 0.41) was obtained from Birmingham Polymers, Inc. (Birmingham, AL). All chemicals were obtained from Sigma–Aldrich Co. (St. Louis, MO) unless mentioned. All primary antibodies used were either obtained from Cell Signaling Technology, Inc. (Danvers, MA) or from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies (FITC conjugated or horseradish peroxidase-conjugated) were procured from Santa Cruz Biotechnology (Santa Cruz, CA). Qdot® 605 ITK™ amino (PEG) quantum dots (QD605) was purchased from Invitrogen Corp. (Carlsbad, CA). Cell culture MCF-7 (human breast cancer), SKOV3 (human ovarian cancer), and ZR751 (human breast cancer) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). HEK-293 (human embryonic kidney) cell line was obtained from National Centre for Cell Science, Pune, India. MCF-7, SKOV3, and ZR751 cells express EpCAM and HEK-293 is negative for EpCAM expression. All cells were grown in DMEM (Invitrogen, CA) media supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, 1%
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(PAN-Biotech GmbH, Germany) at 37 °C in an incubator with 5% CO2 supply (Hera Cell, Thermo Scientific, Waltham, MA).
studied over a period of time in phosphate buffer at 37 °C (Supplementary Materials).
Formulation of nutlin-3a nanoparticles PLGA NPs loaded with the drug nutlin-3a were prepared by oil-in-water single emulsion-solvent evaporation method as per our previously reported protocol (Supplementary Materials). 19
Immunostaining for EpCAM expression by laser-scanning confocal imaging. To investigate the expression of EpCAM in various cancer cell lines, immunostaining assay was performed according to the protocol followed by Maaser and Borlak 40 (Supplementary Materials).
Conjugation of Apt and quantum dots to PLGA nanoparticles EpCAM specific RNA aptamer was synthesized as per our previously reported protocol. 32 The Apt has sequence 5′-aminoC12-.GCGACUGGUUACCCGGUCG-idT-3′, containing 2′ fluoropyrimidines and a 3′-inverted deoxythymidine cap, leading to a 3′–3′ linkage that inhibits degradation by 3′ exonucleases. Conjugation of QD and Apt onto the surface of PLGA NPs was performed according to the protocol followed by Savla et al. 26 and Farokhzad et al. 27 (Supplementary Materials). Analysis of surface conjugation Agarose gel electrophoresis. The conjugation of Apt onto PLGA NPs surface was investigated by agarose gel electrophoresis. In brief, 2% agarose gel with 0.5 μg/ml of EtBr was prepared in Tris boric acid EDTA (TBE) buffer, Apt conjugated NPs (with EDC and without EDC), before washing and after washing and free Apt was loaded into well and the gel electrophoresis was performed at 60 V in TBE buffer. 38 Imaging was performed by Chemi Doc™ XRS+ Imaging System (Bio-Rad, CA, USA). FTIR analysis. The Fourier transform infrared (FTIR) spectra for void-PLGA-NPs, Apt-PLGA-NPs were obtained from FTIR spectrophotometer SPECTRUM RX I (Spectrum 1, PerkinElmer, San Jose, CA) for studying the surface modification of PLGA-NPs after Apt conjugation to PLGA NPs surface. Briefly, the samples were pressed with KBr to make a pellet by applying a pressure of 300 kg/cm 2 before obtaining their IR absorption spectra. The spectra were detected in KBr disks over a range of 4500–300 cm − 1. Fluorescence spectrophotometer analysis. Briefly, QDPLGA-NPs, void-PLGA-NPs suspension (0.1 mg/ml) in 0.1 M PBS was placed into a quartz cuvette and emission spectrum was recorded with spectrofluorophotometer (ParkinElmer, Massachusetts, USA) at excitation wavelength of 405 nm and emission wavelength of 605 nm. 36 Physiochemical characterization of nutlin-3a loaded PLGA NPs. Particle size, size distribution (polydispersity index) and surface charge of the nanoformulations were measured by dynamic light scattering (DLS), using Zetasizer. Size was further confirmed by TEM analysis. Surface morphology of NPs was studied by AFM analysis. Entrapment efficiency of nutlin-3a in NPs was estimated by high performance liquid chromatography (RP-HPLC) method following established protocol. 19 In vitro release kinetics of nutlin-3a from Apt-Nut-NPs was performed in serum containing media as per previously published protocol. 4,39 The stability of Apt and QD functionalized NPs was
Cellular uptake study of aptamer conjugated NPs. Qualitative and quantitative cellular uptake of native 6-coumarin, 6coumarin loaded NPs (conjugated or unconjugated) was investigated in different cell lines by confocal microscopy and fluorescence spectrophotometer respectively (Supplementary Materials). 19 In vitro cellular cytotoxicity of Apt functionalized nutlin-3a loaded NPs. The cytotoxic effect of Apt functionalized nutlin3a loaded NPs over unconjugated NPs and native nutlin-3a was investigated by MTT assay (Supplementary Materials). 5 Cell cycle arrest and apoptosis study. Cell cycle arrest and induction of apoptotic cell death following treatment with nutlin3a in native or nanoformulations having drug concentration of 1 μg/ml were performed in ZR751 cell line by flow cytometer (Supplementary Materials). 19,5 Immunoblotting. For Western blot analysis, ZR751 cells at cell density 1 × 10 5/ml were treated with 2 μg/ml concentrations of native nutlin-3a or equivalent concentration of drug loaded in nanoformulations (Nut-NPs and Apt-Nut-NPs) for 48 hours. Following various treatments, cell protein was extracted with lysis buffer and subjected to Western blot analysis with specific primary antibody against various proteins studied (Supplementary Materials). Imaging of cancer cells with Apt-QD-Nut-NPs by confocal laser scanning microscope. To image the cancer cells grown in monolayer or three dimensional (3D) spheroid tumor model, Apt-QD-Nut-NPs and QD-Nut-NPs were used as an imaging probe and imaging was performed by confocal microscope (Supplementary Materials). 41,42 Statistical analysis. Data were represented as means ± standard deviation (SD). Comparison of data in quantum dots conjugation assay was performed using a two tailed Paired Student's test. Uptake analysis was done by one-way analysis of variance (ANOVA) with Tukey post-test and cyototoxicity assay was performed by two-way analysis of variance (ANOVA) with Bonferroni multiple comparisons. P values less than 0.05 were considered statistically significant. Originpro 8 was used to fit dose–response and exponential equations to calculate IC50 values. GraphPad Prism Software was used for all statistical analyses. Results Physiochemical characterization of drug loaded PLGA nanoparticles Nutlin-3a loaded PLGA nanoparticles were formulated by emulsion-solvent evaporation method and result indicates that
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Figure 1. Physiochemical characterization of nanoformulations (A, B, C), Analysis of conjugation of Apt (D) and quantum dots (E) with PLGA nanoparticles and in vitro release kinetics of Apt-Nut-NPs (F). Size of Nut-NPs (A) was measured by DLS. (B) TEM analysis of nutlin-3a loaded NPs. Scale bar is 100 nm. (C) AFM image of Nut-NPs. (D) Conjugation of Apt to PLGA NPs (following EDC–NHS conjugation chemistry) was analyzed by 2% agaroge gel electrophoresis. Lanes 1, 2, 3, 4, and 5 correspond to native Apt, Apt-NPs with EDC and NHS before wash, Apt-NPs without EDC and NHS before wash, Apt-NPs with EDC and NHS after wash, Apt-NPs without EDC and NHS after wash respectively. (E) Conjugation of quantum dots to NPs was confirmed by fluorescence spectrophotometer by measuring the fluorescence intensity of void-PLGA-NPs and QD-PLGA-NPs. Error bar shows mean ± SD, n = 3, **p b 0.001 for void-PLGA-NPs versus QD-PLGA-NPs. (F) Sustained release of nutlin-3a exhibited by Apt-Nut-NPs in 10% FBS containing media. Error bar shows mean ± SD, n = 3.
the formulated NPs are of nanometer size range with negative zeta potential (Figure 1, A, B and Table 1) and having smooth surface morphology (Figure 1, C). The EDC/NHS activation technique enables the conjugation of amine functional group of the Apt/QD with the carboxyl group of PLGA through the formation of an amide bond (Figure 1, D, E; Figures S1 and
S2). 27,43 Further, the nanoformulation depicts a sustain release profile of nutlin-3a from Apt-Nut-NPs, over 72 hours as depicted in Figure 1, F. The in vitro stability study of Apt and QD functionalized Nut-NPs indicates the presence of QD or Apt on nanoparticles surface till 48 hours (Figure S3, A and B). Details are shown in supplementary information.
M. Das et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 379–389 Table 1 Physico-chemical characterization of Nutlin-3a loaded nanoparticle. Zeta potential (mV) b
Formulation Size (nm) a Void-NPs Nut-NPs Apt-Nut-NPs QD-Nut-NPs a b c d
237 253 292 257
± ± ± ±
13 − 10.99 ± 0.5 13 − 16.58 ± 0.95 10 − 20.3 ± 0.66 27 − 8.91 ± 3.1
Polydispersity Entrapment index c efficiency (%) d 0.24 0.18 0.36 0.20
± ± ± ±
0.13 0.03 0.02 0.02
– 65.98 ± 2.98 51.24 ± 6.7 –
Size in nm was measured by Zetasizer. Zeta potential in mV was measured by Zetasizer. Polydispersity index was measured by Zetasizer. Entrapment efficiency of nutlin-3a was measured by HPLC.
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treatment (native or nanoformulation) for 24 hours (Figure 4, A). Figure 4, A, shows that 39.54% of cells were arrested at G2/M phase of cell cycle treated with Apt-Nut-NPs compared to 27.45% in Nut-NPs and 16.42% native nutlin-3a in solution. Execution of enhanced apoptosis mediated by Apt-Nut-NPs Nutlin-3a has been advocated for its effectiveness in inducing apoptosis. In the present study, we have tested the efficacy of Apt functionalized Nut-NPs over native drug and unconjugated NutNPs in induction of apoptosis. Apt-Nut-NPs induced 21.08% early apoptotic cell death in comparison to 8.8% and 14.5% early apoptotic cell death exhibited by free nutlin-3a and unconjugated Nut-NPs (Figure 4, B). Thus, Apt-Nut-NPs elicited more potent activation of apoptosis in ZR751 cells than Nut-NPs or free drug.
Comparative expression study of EpCAM The immunofluoroscence result for EpCAM expression indicates that ZR751, MCF-7 and SKOV3 are positive for EpCAM expression with varying degrees of expression level whereas HEK-293 cell line do not express any detectable level of EpCAM (Figure S4). Cellular uptake analysis The cellular uptake analysis by confocal microscope depicts an augmented uptake of Apt-6-coumarin-NPs in all EpCAM expressing cell lines compared to its unconjugated counterpart (Figure 2, A), whereas no such significant difference in uptake efficiency between Apt-6-coumarin-NPs and 6-coumarin-NPs was evident in HEK-293 cell line (Figure 2, A). The uptake of Apt-6-coumarin-NPs in comparison to unconjugated NPs and native 6-coumarin was further validated by fluorescence spectrophotometer in ZR751 and HEK-293 cell line (Figure 2, B). The result indicates a higher uptake of Apt-6-coumarin-NPs compared to unconjugated counterpart in EpCAM expressing ZR751 cell line (Figure 2, B). However, no significant difference in uptake efficiency was observed between Apt-6-coumarin-NPs and 6-coumarin-NPs in HEK-293 cell line. Cellular cytotoxicity study The cytotoxicity results shows an augmented cytotoxic activity of Apt-Nut-NPs over unconjugated counterpart in EpCAM expressing cell lines (ZR751, MCF-7, SKOV3), however there is no substantial difference in cytotoxic effect in EpCAM non-expressing HEK-293 cell line (Figure 3). As shown in Table 2, the IC50 (inhibitory concentration required to induce fifty percent cytotoxic effect) for Apt-Nut-NP is 4-fold in ZR751, 2-fold in SKOV3 and 3-fold in MCF-7 cells lower than that for Nut-NPs, thus substantiating the above findings and indicating that Apt-Nut-NPs is more efficient in causing cell death in a targeted manner compared to unconjugated NPs. Void NPs (without any drug) equivalent to the doses of drug concentration are non-toxic in nature. Cell cycle analysis Nutlin-3a induces cell cycle arrest in G1 and/or G2 phase of cell cycle. To explore the cell cycle regulatory effect of nutlin-3a in ZR751 cells, cell cycle analysis was performed following drug
Modulation of proteins involved in the regulation of apoptosis Effect of Apt-Nut-NPs on p53 activation was studied by Western blot analysis. The result indicates enhanced expression of p53 proteins following treatment with Apt-Nut-NPs compared to native nutlin-3a and unconjugated counterpart (Figure 5). Activation of p53 modulates many signaling molecules involved in apoptotic events. Proapoptotic proteins like BAX and antiapoptotic proteins like bcl2 are the downstream targets of p53. Further, cleavage of caspase 3 also acts as an event during apoptosis process. Thus, we investigated the expression of key proteins like BAX, bcl2, and caspase 3 by Western blotting. The result reveals over-expression of BAX, cleavage of caspase 3 and down-regulation of bcl2 following drug treatment (Figure 5) and compared to Nut-NPs and native nutlin-3a, targeted Apt-NutNPs exhibited enhanced modulation of proteins involved in apoptosis pathway. In vitro bio-imaging To investigate the potentiality of QD conjugated Apt-Nut-NPs as targeted imaging modality; we carried out an in vitro imaging study in a 2D monolayer culture and 3D tumor spheroid model. Confocal microscope image of ZR751 and HEK-293 cell lines grown in monolayer indicates that, the fluorescence intensity of Apt-QD-Nut-NPs exposed to EpCAM over-expressing ZR751 cell line was higher compared to non-targeted counterpart (QD-NutNPs) (Figure 6, A). However, no significant difference in fluorescence intensity was evident in EpCAM negative HEK293 cell line treated with QD-Nut-NPs and Apt-QD-Nut-NPs depicting the potentiality of targeted imaging in vitro (Figure 6, A). To further validate the concept of targeted in vivo imaging by QD functionalized Apt-Nut-NPs, we tested its penetrating ability (a prerequisite for in vivo imaging) in ZR751 cells grown as a tumor spheroid model. Spheroids provide an in vitro avascular 3dimensional model that mimics the in vivo tumor and its interaction with tumor microenvironment. The Z-stack laser scanning confocal images at stack 8 (at the center of the tumor spheroid) exhibited higher fluorescence intensity than that of stack 0 or stack 16 located at the top and bottom, respectively for ZR751 tumor spheroid treated with Apt-QD-Nut-NPs in comparison to QD-NutNPs (Figure 6, B), indicating the ability of the targeted system to penetrate deep into a solid tumor.
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Figure 2. Analysis of cellular uptake of nanoformulations by confocal microscopy and fluorescence spectrophotometer. MCF-7, ZR751, and SKOV3 cell lines positive for EpCAM expression and HEK-293 cell line negative for EpCAM expression were treated with 90 ng/ml of native 6-coumarin, equivalent concentration of 6-coumarin loaded nanoparticles (6-coumarin-NPs) and Apt conjugated 6-coumarin NPs (Apt-6-coumarin-NPs) for 2 hours and uptake of 6coumarin was observed by confocal microscope (A). Quantitative uptake of above formulations was further investigated in ZR751 and HEK-293 cells by fluorescence spectrophotometer (B). Values are shown as mean ± SD, n = 5, significance level was set at p b 0.05.
Discussion In spite of the ability of the contemporary chemotherapeutic treatment regimens to achieve relatively high rates of remission induction and survival, still there are challenges for successful cancer therapy. In this setting to overcome the challenges associated with cancer chemotherapy there has been progressively heightened interest in the development of a combinational approach for targeted drug delivery and controlled release technology which may pave the road to more effective yet safer chemotherapeutic options for cancer therapy. Apart from developing a targeted sustained drug delivery technology, monitoring the effect of chemotherapeutic agents on tumor progression is very much essential. In this setting, molecularly targeted theranostic approach i.e. fusion of therapeutic and diagnostic for simultaneous cancer detection, inhibition and therapeutic response assessment has emerged as a promising therapeutic strategy against cancer. 44 Noteworthy, advances in
nanobiotechnology have enabled the development of such targeted theranostic nanoplatforms. 7,26,37,45 To this end, with an aim to achieve a therapeutic strategy enabling delivery of high therapeutic payload at target site in a controlled fashion and facilitating imaging, in the present investigation, we have surface functionalized nutlin-3a loaded nanocarrier system with a targeting ligand (aptamer specific for targeting EpCAM over-expressed in most of cancer) and an imaging agent (quantum dots) and tested its therapeutic and imaging prospective. In the present work, we have developed nanoparticles of nanometer size range with negative zeta potential (Figure 1, A, B, C and Table 1). Developing a multifunctional nanoformulation capable of both targeted drug delivery and imaging is a challenging task. By implementing the EDC-NHS chemistry, we performed simultaneous conjugation of both targeting (Apt) and imaging modalities (quantum dot) onto nutlin-3a loaded PLGA nanoparticle surface in an efficient way (Figure 1, D and E) to formulate a
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Figure 3. Cytotoxicity assay. Briefly, 3000 cells were seeded per well in 96 well plates for overnight and then incubated with 0.005–6 μg/ml drug concentration (of native nutlin-3a, Nut-NPs, Apt-Nut-NPs) and corresponding amount of void NPs. Cells left untreated serve as control. The extent of growth inhibition was measured at the 5th day using MTT assay and inhibition was calculated with respect to untreated control cells. Data are expressed as mean ± SD., n = 4, *p b 0.05, **p b 0.01, ***p b 0.001, for native nutlin-3a vs Nut-NPs or Nut-NPs vs Apt-Nut-NPs.
Table 2 Cytotoxicity study by MTT assay. Cell line
Treatments
IC50 (μg/ml) a
MCF-7
Native Nutlin-3a Nut-NPs Apt-Nut-NPs Native Nutlin-3a Nut-NPs Apt-Nut-NPs Native Nutlin-3a Nut-NPs Apt-Nut-NPs Native Nutlin-3a Nut-NPs Apt-Nut-NPs
2.46 0.99 0.36 1.02 0.33 0.08 5.30 1.60 0.68 4.08 1.08 1.15
ZR 751
SKOV3
HEK-293
± ± ± ± ± ± ± ± ± ± ± ±
0.40 0.13*** 0.06* 0.15 0.13*** 0.02* 1.01 0.39*** 0.15* 0.61 0.09*** 0.38
a
Inhibitory concentration causing 50% cell death. Data as mean ± SD, n = 4. *p b 0.05, **p b 0.01, ***p b 0.001 for Native Nutlin-3a vs Nut-NPs or NutNPs vs Apt-Nut-NPs.
nanotheranostic system. Further our Apt functionalized NPs exhibited a sustained drug release profile which may facilitate a prolonged cytotoxic effect at tumor site for better cancer remission.
Various site-specific targeting strategies have been adopted in recent years to augment the cellular interaction and internalization of nanoformulations. 2 Thus, to authenticate our concept that, Apt functionalization to drug loaded NPs will result in enhanced cellular internalization of the therapeutic payload, cellular uptake efficiency of the Apt targeted carrier system was assessed by confocal microscopy. Results demonstrate that, uptake of Apt-6-coumarin-NPs was significantly higher than that of the unconjugated nanoparticle in EpCAM expressing cell lines but no significant difference in cellular uptake pattern was evident in EpCAM non-expressing HEK293 cell line (Figure 2, A). In a recent study, Hussain et al. 21 have documented a higher cellular binding and uptake of EpCAM-targeted immunoliposomes compared to non-targeted control liposome in EpCAM expressing MCF-7 and SW2 cell lines, however no remarkable difference was evident in EpCAM negative RL cells. In accordance with above findings, in our cellular uptake study the greater uptake of Apt-6coumarin-NPs in EpCAM positive cells indicates that, EpCAM expression may be playing a crucial role in receptor mediated binding and uptake of targeted NPs. EpCAM mediated internalization of Apt targeted NPs was further validated by
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Figure 4. Cell cycle arrest analysis and apoptosis assay by flow cytometry. In brief, 2 × 10 5 ZR751 cells were seeded overnight in 6-well plates and then treated with 1 μg/ml of native nutlin-3a, Nut-NPs, Apt-Nut-NPs for 24 hours and 36 hours for cell cycle analysis and apoptosis assay respectively. Cell cycle distribution was analyzed by PI staining (A). Content of DNA is represented on the x-axis; number of cells counted is represented on the y-axis. The regions marked M1, M2, M3 and M4 represent G1, S, G2 and G0 phases respectively of the cell cycle. Experiment was performed in duplicates and representative image has been given. Apoptosis percentage was analyzed using standard annexin V assay by FACS (B). The experiment has been performed three times and representative image has been provided. Data represented as mean ± SD.
fluorescence spectrophotometer analysis in ZR751 and HEK293 cell lines (Figure 2, B). To validate the therapeutic potentiality of targeted nanoformulations upon internalization for sustained killing of cancer
cells, MTT based cytotoxicity assay was performed. The enhanced cytotoxic effect of Apt-Nut-NPs than unconjugated NPs in EpCAM over-expressing cell lines and a similar pattern of cytotoxicity in EpCAM non-expressing cell line, stress the key
Figure 5. Study of signaling proteins by Western blot analysis. ZR751 cells were incubated with native nutlin-3a, Nut-NPs, and Apt-Nut-NPs (2 μg/ml) for 48 hours, and various apoptosis associated proteins were analyzed by Western blotting. β-Actin serves as internal control. The experiment has been performed two times and representative image of an experiment has been provided.
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Figure 6. Confocal bio-imaging. (A) HEK-293 and ZR751 cells were grown in 2D monolayer and exposed to 5 μg/ml of Apt-QD-Nut-NPs or QD-Nut-NPs for 4 hours and images were captured by confocal microscope. (B) ZR751 cells were grown as 3D spheroid and treated with 20 μg/ml of Apt-QD-Nut-NPs or QDNut-NPs for 4 hours and Z-stack laser scanning fluorescence images (Z-0 to Z-17) were captured by confocal microscope. Z-0, Z-8, and Z16 images for all samples were given for presentation. Experiment has been performed twice and representative image of an independent experiment has been provided.
role of receptor mediated NPs binding and enhanced internalization in enhancement of cytotoxic activity (Figure 3, Table 2). In a recent study Subramanian et al. 46 have documented the enhanced cytotoxic effect of chimeric EpCAM aptamer doxorubicine conjugate in EpCAM expressing retinoblastoma cells, while no significant cytotoxicity was observed in EpCAM negative non-cancerous Muller-glial cells following higher cellular uptake by EpCAM receptor mediated endocytosis. The above study clearly suggests the key role of EpCAM in mediating targeted cytotoxic effect, thus substantiating our finding. Most of the chemotherapeutic agents exert their effect either by arresting the cell cycle progression or through induction of apoptosis. In the present context, treatment of ZR751 cells with Apt-Nut-NPs resulted in a greater proportion of cells in G2 phase (Figure 4, A) and higher apoptotic cell death (Figure 4, B) compared to unconjugated counterpart and native nutlin-3a. Enhanced efficiency of Apt-Nut-NPs compared to Nut-NPs in arresting more number of cells and inducing enhanced apoptotic cell death may have resulted due to higher intracellular drug levels at the site of action (due to enhanced cellular uptake
following receptor mediated endocytosis) for a longer period of time (following sustain release). 47,48 As loss of proapoptotic signals and gain of anti-apoptotic mechanisms lead to resistance of cancer cells toward apoptosis, most of the current chemotherapeutic agents exert their antitumor effect by modulating these signal transduction pathways. 49,50 We have studied the expression of few signaling molecules playing a role in apoptosis by Western blot analysis. In the present work, we observed enhanced expression of p53, BAX, cleavage of caspase 3, and down-regulation in expression of antiapoptotic protein bcl2 in cells treated with native drug or nanoformulation (Figure 5). Notably, in all cases Apt functionalized nanopaticles showed greater efficacy in modulating the expression of above mentioned proteins compared to native nutlin-3a and Nut-NPs following site specific drug delivery. In relation to cancer, bio-imaging of tumor presents a unique challenge because of the urgent necessity for highly specific and sensitive imaging agents. Among various imaging modalities, semiconductor QDs have received much attention as a new generation imaging agent for molecular, cellular and in vitro/in vivo
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imaging. However, lack of site specific imaging ability may hamper the use of QD in clinical setting. Thus in this study, we have surface functionalized Apt and QD605 quantum dots on to PLGA NPs surface for site specific imaging application and the in vitro results of imaging in tumor spheroid and 2D monolayer suggest that, the Apt targeted theranostic nanosystem is capable of target specific imaging compared to unconjugated counterpart (Figure 6). Recently Gao et al. 45 have shown the higher cancer cell labeling potentiality of prostate specific membrane antigen (PSMA) antibody targeted QD compared to non-targeted QD for PSMA-positive C4-2 cells, however no remarkable difference was observed compared with that in PSMA-negative PC-3 cells. In corroboration with above finding, in our imaging study the greater internalization of Apt-QD-Nut-NPs in EpCAM-positive cells resulting in enhance cellular labeling indicates toward the crucial role of Apt in receptor mediated binding and internalization of conjugated NPs. Further, our Apt-QD-Nut-NPs have also demonstrated deeper penetrating capability compared to unconjugated counterpart as shown from different z-scan confocal images (Figure 6, B). In accordance to above observation Savla et al. 26 have also demonstrated, higher internalization and penetration potentiality of MUC1 aptamer conjugated QD in comparison to unconjugated QD in MUC1 expressing A2780/AD ovarian carcinoma cells grown as 3D tumor spheroids. Thus results suggest that, the above developed multifunctional theranostic nanosystem may be applied for achieving better imaging and therapeutic response in cancer. Extensive investigation by Savla et al. 26 has validated the application of mucin 1 aptamer targeted nanothranostics for their preferential accumulation in the ovarian tumor and tumor imaging in vivo. In relation to this, the preliminary but significantly important findings observed in the present investigation are currently in the process of in vivo validation and may be implemented in clinical settings in the future. In conclusion, current diagnosis and treatment options for cancer must be improved, despite significant advances over the past decades to combat this disease. Extensive preclinical research in cancer nanotechnology holds much potential to generate improved options for diagnosing and treating the disease. In this context, in the present state of art, we have developed a multifunctional nanosystem capable of both cancer therapeutics and imaging. Our results indicate that, Apt targeted nutlin-3a loaded NPs resulted in enhanced cellular accumulation of drug, and greater cytotoxicity in EpCAM overexpressing cancer cells. Further, Apt functionalized nanoparticle induces higher apoptosis by triggering signaling molecules. Moreover, our carrier system has shown its potency as an imaging modality in in vitro tumor spheroid model. Overall, we anticipate that this multifunctional nanosystem may act as a treatment option for cancer, however, for implementing the use of the theranostic nanosystem in clinical settings a detailed investigation on in vivo biodistribution and antitumor effect of the targeted nanoformulations is warranted in the near future. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2014.09.002.
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