Toxicology and Applied Pharmacology 275 (2014) 232–243

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Ligand-conjugated mesoporous silica nanorattles based on enzyme targeted prodrug delivery system for effective lung cancer therapy Shenbagamoorthy Sundarraj a,⁎, Ramar Thangam a,b, Mohanan V. Sujitha a, Karuppaiya Vimala a, Soundarapandian Kannan a,c,⁎ a b c

Proteomics and Molecular Cell Physiology Laboratory, Department of Zoology, Bharathiar University, Coimbatore 641 046, TN, India Department of Virology, King Institute of Preventive Medicine and Research, Guindy, Chennai 600 032, TN, India Department of Zoology, Periyar University, Salem 636 011, TN, India

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Article history: Received 17 August 2013 Revised 3 January 2014 Accepted 14 January 2014 Available online 25 January 2014 Keywords: Silica nanorattles Epidermal growth factor receptor (EGFR) Pyrrolidine-2 cPLA2α activity Drug delivery system (DDS) Lung cancer therapy

a b s t r a c t Epidermal growth factor receptor antibody (EGFRAb) conjugated silica nanorattles (SNs) were synthesized and used to develop receptor mediated endocytosis for targeted drug delivery strategies for cancer therapy. The present study determined that the rate of internalization of silica nanorattles was found to be high in lung cancer cells when compared with the normal lung cells. EGFRAb can specifically bind to EGFR, a receptor that is highly expressed in lung cancer cells, but is expressed at low levels in other normal cells. Furthermore, in vitro studies clearly substantiated that the cPLA2α activity, arachidonic acid release and cell proliferation were considerably reduced by pyrrolidine-2 loaded EGFRAb-SN in H460 cells. The cytotoxicity, cell cycle arrest and apoptosis were significantly induced by the treatment of pyrrolidine-2 loaded EGFRAb-SN when compared with free pyrrolidine-2 and pyrrolidine-2 loaded SNs in human non-small cell lung cancer cells. An in vivo toxicity assessment showed that silica nanorattles and EGFRAb-SN-pyrrolidine-2 exhibited low systemic toxicity in healthy Balb/c mice. The EGFRAb-SN-pyrrolidine-2 showed a much better antitumor activity (38%) with enhanced tumor inhibition rate than the pyrrolidine-2 on the non-small cell lung carcinoma subcutaneous model. Thus, the present findings validated the low toxicity and high therapeutic potentials of EGFRAb-SN-pyrrolidine-2, which may provide a convincing evidence of the silica nanorattles as new potential carriers for targeted drug delivery systems. © 2014 Elsevier Inc. All rights reserved.

Introduction Cancer is a leading cause of death in economically developed countries and the second leading cause of death in developing countries (WHO, 2004). Advances in our knowledge of molecular biology of cancer and pathways involved in malignant transformation are revolutionizing the approach to cancer treatment with a focus on targeted cancer therapy. In the present study we have mainly focused on cytosolic phospholipase A2α (cPLA2α) because it has fascinated attention to target the controlling arachidonic acid and eicosanoid related expressions in inflammation and cancer. Cytosolic phospholipase A2α has been proposed to play an important role in cell cycle regulation. The unique functions of cPLA2α are emphasized by the earlier findings endorsed that most of the tumor cells are found to produce elevated levels of eicosanoids, resulting in increased tumor growth, invasiveness and upgradation of metastatic activity of the tumor cells (Lagorce-Pages et al., 2004; Laye and Gill, 2003; Reich and Martin, 1996). Accordingly, cPLA2α is remarkably found to be over-expressed in a range of human ⁎ Corresponding authors. Fax: +91 4272345124. E-mail addresses: [email protected] (S. Sundarraj), [email protected] (S. Kannan). 0041-008X/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2014.01.012

tumors, including non-small cell lung cancer (Heasley et al., 1997; Sundarraj and Kannan, 2010). Furthermore, PLA2 inhibitors are able to suppress proliferation of tumor cells by inducing apoptosis (Korystov et al., 1998). In addition to the above factors, the 15Shydroxyeicosatetraenoic acid has been shown to decrease the percentage of cells in S phase. This observation is concomitant with an increase in the numbers of cells in G0/G1 phase in prostate carcinoma cells (Shappell et al., 2001). These data may be acquainted with the pivotal role of cPLA2α inhibitors as therapeutics for cancer treatment. The silica nanoparticles are believed to be non-toxic and are currently used in several industrial and biomedical applications including food, cosmetics and drug delivery carrier systems. The drug delivery system will not only be an important therapeutic and pharmacological application, but also be of great interest in medical imaging and diagnosis. The EGFR and its ligands are important in normal and neoplastic epithelial cell growths. EGFR has been recognized as a potential cancer biomarker since the activation of EGFR is associated with the tumorigenic mechanisms such as autonomous cell growth, invasion, angiogenesis, and metastasis (Grünwald and Hidalgo, 2002; Ritter and Arteaga, 2003). The epidermal growth factor receptor is used as the target ligand (Rusch et al., 1996), and it can be employed for more specific recognition and interaction with cancer cells because EGFR is overexpressed in human

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tumors, especially on non-small cell lung cancer (Fontanini et al., 1998; Fujino et al., 1996; Rusch et al., 1993, 1997; Salomon et al., 1995; Volm et al., 1998). Xu and Amiji (2012) recently reported the therapeutic gene delivery through EGFRAb conjugated gelatin nanoparticle to target human pancreatic cancer. Slowing et al. (2006) have confirmed that surface-functionalized 100 nm size silica particles are effectively internalized by HeLa cells regardless of the surface composition of the particles. Li et al. (2010, 2011) have proved that the SN-PEG–Dtxl has low systemic toxicity and high therapeutic efficacy, which provides convincing evidence for the silica nanorattles as a promising candidate for a drug delivery system. The recent study witnessed that the silica nanoparticles have internalized in Balb/3T3 mouse fibroblasts as it did not trigger any cytotoxic or genotoxic effect and did not induce a morphological transformation (Uboldi et al., 2012). To our knowledge, no reports are available with emphasis on the use of EGFRAb conjugated silica nanorattles to delivery drug of interest. Recently a class of pyrrolidine containing compounds has been reported to act as potent inhibitors of cPLA2α in vitro and to block arachidonate release in calcium ionophore-stimulated human acute monocytic leukemia THP-1 cells (Seno et al., 2000) and CHO cells (Ghomashchi et al., 2001). They block the production of prostaglandins E2 and leukotriene (Ghosh et al., 2007; Seno et al., 2000). In this study, we aimed to develop as a novel targeted drug delivery system for the cPLA2α inhibitor pyrrolidine-2 against non-small cell lung carcinoma cells. We further investigated the Silica nanorattles encapsulated pyrrolidine-2 reduced systemic toxicity and also enhanced therapeutic efficacy in tumor bearing Balb/c mice. Materials and methods Synthesis and characterization of EGFRAb-SN-pyrrolidine-2 Silica nanorattles were fabricated according to a procedure described by Chen et al. (2010) with a slight modification. In a typical synthesis, 0.5 g of tetraethyl orthosilicate (TEOS) (Sigma-Aldrich, India) was mixed under inert atmosphere and added to an ethanol solution containing the structure directing agent sodium do-decyl sulfate (SDS, 99%). The resulting synthesis mixture had a molar ratio of 0.4 TEOS:0.5 SDS:1439 EtOH:2560 H2O. The solution was stirred 24 h at room temperature, and thereafter aged for 6 h at static conditions. The precipitate was filtered off, washed with ethanol, and dried at 60 °C in vacuo for 48 h. The SDS was subsequently removed by ultrasonication in ethanol three times (Moller et al., 2007). Morphology and structure of the resulting SNs were observed with a Technai G2 Transmission Electron Microscope (TEM); the size of nanorattles was determined by Dynamic Light Scattering (DLS) (Malvern ZetaSizer Nano); Fourier transform infrared (FTIR) analysis was carried out using KBr disks in the region of 4000–400 cm−1 (Shimadzu, IR Affinity-1, Japan). Polyethylenimine (PEI) was grown onto the silica nanorattles by hyperactive surface polymerization according to a modified reaction described by Rosenholm et al. (2009). Thus, before polyethylenimine alteration, the surfactant extracted nanorattles were carefully vacuumdried and subsequently subject to argon atmosphere. The nanorattles (0.125 g) were immersed in toluene under inert atmosphere. Catalytic amounts of acetic acid and 45 μL of aziridine were added, and the reaction mixture was stirred overnight at 75 °C. After the reaction, the nanorattles were filtered off, washed with copious amounts of toluene, and vacuum-dried for at least 24 h. The nanorattles plain and PEI coated, were labeled with FITC (fluorescein isothiocyanate, 98%) by suspending 25 mg of nanorattles in carbonate buffer (pH 9.0), to which 250 μL of an ethanolic FITC solution (1 mg/mL) was added and stirred for 30 min. After this, the PEI-functionalized nanorattles were collected by centrifugation, washed with de-ionized water repeatedly, and subsequently suspended in MES buffer (pH 5.0). Anti EGFR was purchased from Santa Cruz Biotechnology (sc-1724, Santa Cruz, CA). 50 μg of EGFRAb was sonicated

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in MES, to which 20 μL of a 1 μL/mL EDC solution (1-ethyl-3-(3dimethylaminopropyl) carbodiimide) was added to activate the carboxylic acid groups of EGFRAb. This solution was rapidly added to the nanorattle suspension, after which 25 μL of (1 mg/mL in MES) NHS (N-hydroxysuccinimide) solutions was mixed with the suspension. The suspension was disturbed for over-night and washed with copious amounts of de-ionized water and ethanol, dried in vacuo and stored at 4 °C. The ζ-potential was measured as a function of pH by titrating with 0.1 or 0.5 M HCl and NaOH at 25 °C. The detailed schematic synthesis of mesoporous silica nanorattles with a functional core, hollow, and mesoporous structure was explained in Scheme 1. Pyrrolidine-2 was synthesized as described previously by Seno et al. (1998). To load pyrrolidine-2 into the pores of the nanorattles, SN and EGFRAb-SN were dispersed in a solution of pyrrolidine-2 (10 mg/mL in ethanol) and stirred for 24 h, followed by centrifugation with extensive washing of PBS to obtain the pyrrolidine-2 loaded SN and EGFRAb-SN spheres, which were used for subsequent in vitro and in vivo studies. Cell culture and culture conditions The human lung epithelial cell line L-132 and human large cell lung cancer H460 cells were purchased from the National Centre for Cell Sciences (Pune, India). L-132 cells were maintained in DMEM with 10% fetal bovine serum and H460 cells were cultured in RPMI 1640 (HiMedia, Inc.) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. Both the cells were maintained at 37 °C with 5% CO2 in a humidified incubator. Quantification of pyrrolidine-2-loaded EGFRAb-SN uptake by L-132 and H460 cells To compare the uptake rate of pyrrolidine-2-loaded EGFRAb-SN, L-132 and H460 cells were plated 24 h prior to starting the experiment in chamber slides at a density of 5 × 103 cells/cm2. After incubation with 10 μg/mL FITC or RITC labeled sphere shaped EGFRAb-SN-pyrrolidine-2 for 4 h, the L-132 and H460 cells were washed twice with PBS and incubated with 0.1% Triton X-100 plus 1% BSA in PBS at room temperature for 15 min. The slides were washed twice with PBS and then examined with a LEICA-Sp5 confocal microscope. L-132 and H460 cells were seeded 5 × 105 cells/well in six-well plates and allowed to adhere for 24 h. To determine the quantity of fluorescence silica nanorattles (FSNs) taken up by L-132 and H460 cells, the cells were incubated with FSN in the specific medium for 4 h and the cells were washed thrice with PBS, and finally harvested through trypsinization. After the cell pellet was centrifuged, it was re-washed once again and re-suspended with PBS containing 0.1% FBS. The cellular uptake of FSN was quantitatively determined by Fluorescence Activated Cell Sorting (FACS) (Beckman Coulter Inc., CA). Cell viability assay The cytotoxicity of SN-PEI, pyrrolidine-2, pyrrolidine-2-loaded SN, and pyrrolidine-2-loaded EGFRAb-SN was evaluated by MTT cell viability assay (Mosman, 1983; Sundarraj et al., 2012a). Pyrrolidine-2 was dissolved using DMSO, and the final concentration of DMSO in culture media was less than 0.5%. For 24 h detection, the cells were seeded at a density of 8000 cells/well on 96-well plates (Nunc, USA), and for 72 h detection, the cell density was 2000 cells/well. After incubating the cells with SN, pyrrolidine-2, SN-pyrrolidine-2, and EGFRAb-SNpyrrolidine-2 for 24 h, MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide) (Hi-Media, India) (final concentration of 5 mg/mL) was added to each well. After 4 h of incubation at 37 °C, colorimetric measurements were performed at 570 nm on a microtiter plate reader (Thermo Electron Corporation, USA). Data were

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Scheme 1. Schematic representation of synthesis of pyrrolidine-2 loaded epidermal growth factor receptor antibody (EGFRAb) conjugated mesoporous silica nanorattles.

expressed as mean ± standard deviation (SD) of at least six independent experiments.

counting. Finally the AA release was expressed as the percentage of total cellular radioactivities.

Measurement of cPLA2α activity

Proliferation assay

Arachidonoyl thio-PC was used as the substrate to measure cPLA2 activity in vitro which was measured by cPLA2 assay kit from Cayman Chemicals according to the manufacturer's protocols. After a 24 h treatment of cells with pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SNpyrrolidine-2, the cells were homogenized. Twenty micro-liters of cell lysate was finally subjected to the assay, and the absorbance values were measured at 414 nm and normalized to protein concentration.

L-132 and H460 cell proliferation rates were assessed using a 5bromo-2′-deoxyuridine (BrdUrd) incorporation based ELISA (Roche Diagnostics). Cells were seeded at 1 × 104 cells per well (0.55 × 104 cells/cm2) in 96-well plates, grown for 24 h, incubated for 24 h with pyrrolidine-2 (10 μM), SN-pyrrolidine-2 (10 μM), and EGFRAbSN-pyrrolidine-2 (10 μM) and BrdUrd, and then processed according to manufacturer's instructions.

Arachidonic acid release assay

Cell cycle analysis

Arachidonic acid release assay was performed as described previously by Shimizu et al. (2008). Briefly, L-132 and H460 cells were cultured to the required density in six well culture dishes and incubated for 24 h with 0.5 μCi/ml [3H] AA in growth media. Sub-confluent, confluent, or wounded cells were washed with PBS and incubated with pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2, an inhibitor of cPLA2α for 24 h. For measurement of passive AA release, media were collected following the 24 h incubation, cleared by centrifugation, and assayed for radioactivity by using liquid scintillation

For measurement of cellular DNA content, flow cytometric analysis was used (Thangam et al., 2012). Briefly, the cells (1 × 106) were seeded in 100 mm culture dishes, and incubated for 24 h. the cultured cells were treated with SN (100 μg/mL), pyrrolidine-2 (10 μM), SNpyrrolidine-2 (10 μM), and EGFRAb-SN-pyrrolidine-2 (10 μM) for 24 h. The cells were then harvested by trypsinization. Pellet out the trypsinized cells at 2500 rpm/5 min/RT and re-suspend cells in 300 μL of PBS-EDTA. Add drop wise 700 μL of chilled 70% ethanol with slow vortex. Tap mix lightly to ensure complete mixing of ethanol and store at

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0 °C overnight. Re-suspend the cells in PBS EDTA with 1% FBS. Add 1:100 volumes of 20 mg/mL RNase and incubate at 37 °C for 1 h. Add propidium iodide to a final concentration of 50 μg/mL and incubate the cells for fixation about 10–20 min at room temperature. The PI fixed and stained cells were analyzed for cell cycle phase distribution by flow cytometry (Becton Dickinson, FACS CALIBUR, USA). Data from 10,000 cells per sample were collected and analyzed with the software (BD, Inc., CA). Apoptosis assay L-132 and H460 cells were seeded at 5 × 105 cells in 6-well plates and incubated with SN (100 μg/mL), pyrrolidine-2 (10 μM), SNpyrrolidine-2 (10 μM), and EGFRAb-SN-pyrrolidine-2 (10 μM) for 24 h. The cells were washed with PBS, resuspended in 0.2 mL of staining buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 2.5 mM CaCl), and incubated for 10 min at room temperature with 20 μL of propidium iodide and then analyzed immediately using the flow cytometry (Becton Dickinson, FACS CALIBUR).

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pyrrolidine-2 (1 mg/kg, 200 μL, dispersed in saline), (c) EGFRAb-SNpyrrolidine-2 (1 mg/kg, 200 μL, dispersed in saline), and (d) control group (200 μL saline) every 4 days (day 1, day 5, day 9, day 13 and day 17) through tail veins until a total of three administrations were completed. Body weights were observed and recorded carefully throughout the experimental period. On the 21st day, the coefficients of liver, kidneys, and spleen to the body weight were calculated as the ratio of tissues (wet weight in mg) to body weight (g). Tissues recovered from the necropsy were fixed with formaldehyde 10% and embedded in paraffin for sectioning, and stained with hematoxylin and eosin (H&E) for histological examination using standard techniques. After hematoxylin/eosin staining, the slides were observed and photos were taken using an optical microscope.

In vivo antitumor effect

Male Balb/c mice (aged 6 weeks, 18 ± 2 g) were purchased from King Institute of Preventive Medicine, Chennai. All experimental protocols in this study were approved by the Institutional Animal Ethical Committee of the Sankaralingam Bhuvaneshwai College of Pharmacy (622/PO/c/02/CPCSEA/2014) in accordance with the policies established in the Guide to the Care and Use of Experimental Animals prepared by the Committee for the Purpose of Control and Supervision on Experiments on Animals. A total of 24 healthy male Balb/c mice were allocated to four groups and intravenously administrated with (a) pyrrolidine-2 (1 mg/kg, 200 μL, diluted with saline), (b) SN-

The male mice were injected subcutaneously into hind limbs with 1 mL of cell suspension containing H460 cells in the number of 1 × 106. After tumor size had reached about 200 mm3, the mice were divided into four groups (n = 5), minimizing weight and tumor size differences. The mice were intravenously administered with (a) pyrrolidine-2 (1 mg/kg, 200 μL, diluted with saline), (b) SNpyrrolidine-2 (1 mg/kg pyrrolidine-2, 200 μL, dispersed in saline), (c) EGFRAb-SN-pyrrolidine-2 (1 mg/kg pyrrolidine-2, 200 μL, dispersed in saline), and (d) saline (200 μL) (control group) every 4 days through tail veins (day 1st, 5th, 9th, 13th and 17th). On the 21st day, after five times of therapy, all the animals were killed and the tumor sections were dissected by weight. Tumor growth inhibition rates were calculated using the formula inhibition (%) = 100 × (C − T) / C, where C is the average tumor weight of the control group and T the average tumor weight of each treated group.

Fig. 1. (A, B & C) TEM image of silica nanorattles size was 84.31 nm; (D) dynamic light scattering (DLS) measurements of silica nanorattles with its average size.

Fig. 2. (A) Zeta potential measurements of SN, PEI-SN and EGFRAb-SN. The plain SN shows a negative potential. After the PEI functionalization as well as after FITC-and EGFRAb conjugation shows an increased positive zeta potential from +15.5 to +23.4 mV respectively, (B) FT-IR spectra of SN, PEI functionalized SN, EGFRAb conjugated SN and EGFRAb conjugated pyrrolidine-2 loaded silica nanorattles.

In vivo systemic toxicity

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Statistical analysis All in vitro experiments were accomplished in triplicate, and the experiments were repeated at least thrice. The statistical software SPSS version 17.0 was used for analysis. p values were determined using the t test; p b 0.001 was considered significant. Results and discussion Characterization of EGFRAb-SN-pyrrolidine-2 Silica nanoparticles represent a modern class of silica nanorattles with a functional core, hollow and mesoporous structure. It attributes to the special structure and perfection with biocompatibility of the silica nanorattles. It is successfully employed as an efficient anticancer drug docetaxel delivery system for cancer therapy (Li et al., 2010; Liu et al., 2011a,b, 2012). Silica nanorattles showed superior performance for increasing of the therapeutic efficacy and decreasing of the systemic toxicity of the cytotoxic drug. The TEM images of silica nanorattles were shown in Fig. 1. Our findings revealed that the partially amino functionalized silica nanorattles with a mean diameter of about 84.31 nm have been acquired in a first time using tetraethyl orthosilicate (TEOS) as a precursor and sodium do-decyl sulfate (SDS) as the structuredirecting agent (Figs. 1A–C). The hydrodynamic diameter of the silica nanorattles was determined by dynamic light scattering (DLS), after hyperactive polymerization of polyethylenimine and 3-aminopropyl triethoxy silane (APTES), which corroborated that the morphology

and diameter of the nanorattles had no obvious changes. The size distribution of silica nanorattles was measured by dynamic light scattering which indicated that there was no aggregation of nanorattles during the modification (Fig. 1D). As anticipated, these silica nanorattles were considerably mono-dispersed which attributed to the excellent biocompatibility in accordance with previous reports of Li et al. (2010). The surface modification was determined by ζ-potential measurement, which was mainly sensitive to the outer surface of the nanorattles. In contrast to the Zeta potential of − 14.3 mV for plain nanorattles, the values for PEI functionalized-nanorattles and EGFRAbnanorattles in PBS buffer at pH 7.4 are found to increase from + 15.5 mV to + 23.4, respectively, as shown in Fig. 2A. These results confirmed that the functional end groups were primary amines in PEI modified nanorattles and the high positive charge density was provided by polyethylenimine. Thus, the changes in the ζ-potential of the nanorattles declared that the surface conjugation of PEI, FITC, and EGFRAb to the silica nanorattles was successful. The ζ-potential value at physiological pH of 7.4 was measured to be +23.4 mV, which made the nanorattles to be fully dispersible in PBS buffer. Interestingly, the presented data further showed that the ζ-potential value and the dispersion were more affirmative to remain unchanged at physiological pH in HEPES buffer solution, which was typically used for buffering cell medium. Thus, the charge based analysis of the synthesized silica nanorattles correlates roughly with its ability to disperse in cellular environments. Apparently, our results were in agreement with a similar report postulated by Gao et al. (2012) and Hu et al. (2011).

Fig. 3. (A) Quantification of cellular uptake of FSN and EGFRAb-SN-pyrrolidine-2 by fluorescence-activated cell sorting (FACS), and (B) confocal microscopy images of L-132 and H460 cells after a 4 h incubation at 37 °C with EGFRAb-SN-pyrrolidine-2 conjugated with FITC or RITC.

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Fig. 4. Cytotoxicity of pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 on L-132 and H460 cells by MTT assay. (A) Cell viability with concentrations of PEI-SN from 0 to 200 μg/mL for 24 h. Inhibition rate of pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 (concentration of pyrrolidine-2 from 2 to 12 μM) on (B) L-132 and (C) H460 cells; (D) corresponding IC50 value on L-132 and H460 cells.

Fourier Transform Infrared (FTIR) spectra of the different reaction mixtures were recorded using a Biorad FTS 6000 spectrometer at room temperature using KBr pellets. The synthesis of hybrid PEIsilica nanorattles results in hyperactive PEI covalently linked to the surface of silica by growing PEI via surface-polymerization as similar to the observations made by Rosenholm et al. (2010a,b). Then, EGFRAb grafted with PEI that has been chemically immobilized on the surface of silica. Fig. 2B designated the FTIR spectra of SN, PEISN, EGFRAb-SN and EGFRAb-SN-pyrrolidine-2. It showed that there was a well pronounced band appearing at 1072 cm − 1 , together with two less pronounced bands at 788 cm− 1 and 452 cm− 1 in the spectrum of SN, which were corresponding to the vibration absorption of Si\O\Si groups. PEI-modification of SN was confirmed by the appearance of new bands at around 1631 cm− 1 and 2925 cm− 1 which was revealed by a typical absorption peak of NH2 group presented on the surface of SN. The EGFRAb-SN spectrum was characterized by a broad band at 3391 cm− 1 that also corresponding to two narrow bands at 2853 cm − 1 and 2923 cm− 1 that were attributed to CH2 stretching vibrations and NH stretching vibration, respectively. The registered bands between 1750 cm−1 and 1500 cm−1 were corresponding to the vibration peaks of amide group and a narrow peak at 1488 cm−1 indicated the presence of COO− the carboxylate groups. The peak intensity was observed to be high at 1622 cm−1 and 1383 cm−1 representing a typical absorption peak of NH bend (primary amines) and C\F vibration, respectively. The pyrrolidine-2 contained NH and C\F as functional groups; we confirmed the presence of pyrrolidine-2 in the nanorattles. The appearances of these new peaks in the IR spectra further endorsed the successful loading of the drug into the synthesized nanorattles. The triply degenerated stretching and bending vibration modes of the (SiO4) tetrahedron at 1091 cm−1 and 470 cm−1, the Si\OH vibration at 981 cm−1 and the Si\O\Si bending vibration mode at 789 cm−1 were depicted in Fig. 2B. In order to enable active targeting of lung cancer cells, epidermal growth factor receptor antibody was covalently conjugated with the

outer PEI layer of silica nanorattles. As the EGFR was over-expressed on the surface of NSCLC cells, the hyperactive PEI polymer chains on the surface of the pyrrolidine-2 loaded silica nanorattles covalently conjugated with EGFR antibodies bind with the receptor due to the affinity of EGFRAb-SN-pyrrolidine-2 with EGFR of NSCLC cells. Our findings were in concordance with the results of Zhang and Chang (2008) and Mamaeva et al. (2011). They reported that the hyperactive PEI polymer chain attached to the nanorattles covalently conjugated with the ligands binds specifically to the target receptor and hence the non-specific binding of other proteins or substances is significantly blocked.

Fig. 5. (A) Inhibition of cPLA2α activity by functionalized drug loaded silica nanorattles. Cell lysates of L-132 and H460 cells treated with DMSO (control) or pyrrolidine-2, SNpyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 were measured for cPLA2α activity. (B) Passive [3H] AA release from proliferating pre-labeled L-132 and H460 cells were assessed by liquid scintillation counting following 24 h of growth in the presence of the indicated inhibitors. All treatments significantly reduce AA release; mean activity relative to control was expressed (±SD), *p b 0.05 when compared with control.

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Fig. 6. Inhibition of L-132 and H460 proliferation by pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2. Quantitation of L-132 and H460 growth in the presence of nanorattles for 24 h (n = 3, ±SEM). Proliferation was determined using a colorimetric ELISA based on BrdU incorporation. *p b 0.05; **p b 0.001 versus control.

Specific internalization of EGFRAb-SN-pyrrolidine-2 by L-132 and H460 cells The ability of EGFRAb-SN-pyrrolidine-2 to target H460 cancer cells was identified. It was found that the human lung epithelial cells (L-132) express a much lower level of the epidermal growth factor receptor than the lung cancer cells (H460), as shown in Fig. 3. This result

is in part consistent with the previous reports of Paciotti et al. (2006), Mossman et al. (2007), and Melancon et al. (2008). Gelatin nanoparticles (GPs) are modified with NeutrAvidinFITC-biotinylated epidermal growth factor (EGF) to form an EGF receptor (EGFR) seeking nanoparticles (GP-Av-bEGF) which resulted in higher transformation efficiency in adenocarcinoma cells (A549) than in the normal lung cells (HFL1) (Tseng et al., 2007, 2008). Flow cytometry analyses were performed to quantify the number of H460 and L-132 cells targeted by EGFRAb-SNpyrrolidine-2. The internalization of silica nanorattles was almost twofold higher in greater number of H460 cells as compared with L-132 cells (Fig. 3A). Further we demonstrated the cellular uptake ability of silica nanorattles, wherein the percentage of internalized EGFRAb-SNpyrrolidine-2 in L-132 and H460 cells were quantified as 29.28 and 44.57%, respectively. The fluorescent intensity of internalized EGFRAbSN-pyrrolidine-2 was less in L-132 than that of H460 cells. Similar study is undertaken to make use of quantum dots (QDs), which depicted a successful conjugation of monoclonal anti-EGFR antibody (cetuximab) to QDs. It is achieved using PEG-conjugated polymercoated QDs and two long-chain hetero-bi-functional linkers, sulfo-LCSPDP and sulfo-SMCC. The dissociation constant of the QD–cetuximab conjugates to EGFR overexpressing A549 lung cancer cells authenticates that there is no significant nonspecific binding toward EGFR-negative CT26.WT cells. Cetuximab (or Erbitux) is the first monoclonal antibody (mAb) drug that targets the epidermal growth factor receptor (EGFR) overexpressed in most cancer cells. As cetuximab has been known to induce internalization of EGFR, live cell imaging is performed to examine

Fig. 7. Cell cycle analysis of L-132 and H460 cells treated with pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 by flow cytometry. Cells (1 × 106 cells/mL) were incubated with MSN (100 μg/mL), pyrrolidine-2 (10 μM), SN-pyrrolidine-2 (10 μM), and EGFRAb-SN-pyrrolidine-2 (10 μM) for 24 h. The percentage of each phase distribution was determined and expressed as a percentage of total cell number (*p b 0.05 versus control. **p b 0.001 vs respective control).

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whether the QD–cetuximab conjugates are internally internalized by endocytosis (Lee et al., 2010). Cellular toxicity The cytotoxicity of the EGFRAb-SN-pyrrolidine-2 was evaluated and compared with free pyrrolidine-2 and SN-pyrrolidine-2 via MTT assay. To assess cellular toxicity of the synthesized silica nanorattles, we determined the number of dead cells in a culture medium treated with high and low concentrations of PEI coated nanorattles for up to 24 h as compared with untreated cells. As the cellular uptake of silica nanoparticles is fairly rapid and has been observed even after 2–3 h, toxicity has generally been observed within 24 h (Lison et al., 2008; Yu et al., 2009). The results showed no difference in cell death overtime in the treated versus untreated cells. To exclude the possible influence of SN-PEI on cell viability, various concentrations of SN-PEI were incubated for 24 h with L-132 and H460 and then cell viability was evaluated, within that tested concentration range even as high as 200 μg/mL, the SN-PEI had no ostensible adverse effect on cell viability (Fig. 4A). It demonstrated the SN-PEI itself had no substantial cytotoxicity. Then, L-132 and H460 cells were incubated with a series of equivalent concentrations of free pyrrolidine-2, SN-pyrrolidine-2, or EGFRAb-SN-pyrrolidine-2 for 24 h. Free pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 executed obvious cell inhibition after a 24 h incubation (Figs. 4B & C) with pyrrolidine-2 concentration varying from 1 to 20 μM. EGFRAbSN-pyrrolidine-2 exhibited obvious advantage over pyrrolidine-2 and SN-pyrrolidine-2 in cytotoxicity at all concentrations in H460 cells when compared with L-132 cells. The free pyrrolidine-2 showed a similar cytotoxicity to the equivalent concentration of SN-pyrrolidine-2

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in all concentrations. The half-maximum inhibiting concentration (IC50 value) of free pyrrolidine-2 was 12.2 μM for L-132 and 16.4 μM for H460 cells compared with that of EGFRAb-SN-pyrrolidine-2 which was 15.8 μM and 7.1 μM for L-132 and H460 cells, respectively (Fig. 4D). Functionalized nanorattles inhibits cPLA2α and arachidonic acid release Pyrrolidine-2 inhibited cell growth by inhibiting cPLA2α activity. We determined the effect of pyrrolidine-2, SN-pyrrolidine-2 and EGFRAbSN-pyrrolidine-2 on inhibition of cPLA2α activity in vitro. Cytosolic phospholipase A2α activity was quantified by measuring conversion of a PLA2 substrate, arachidonoyl thio-PC, to free thiol by cPLA2α. Fig. 5 confirms that at 10 μM EGFRAb-SN-pyrrolidine-2 (100 μg/mL) results in a significant reduction of both cPLA2 activity and AA release in H460 cells compared with L-132 cells. Pyolidine-2 and SN-pyrrolidine2 revealed that there was no significant inhibition of both cPLA2 activity and AA release in H460 and L-132 cells. Ghomashchi et al. (2001) demonstrated the role of pyrrolidine to inhibit cPLA2α dependent release of arachidonate in a variety of mammalian cells. Pyrrolidine-2 is a very potent and specific inhibitor of cPLA2α and reduced the level of cell proliferation in human lung cancer cells (Sundarraj et al., 2012b). Inhibition of cell proliferation The inhibitory effect of free pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 on L-132 and H460 cells was further confirmed using BrdU incorporation into the untreated and treated cells. EGFRAb-SN-pyrrolidine-2 convincingly suppressed H460 cell

Fig. 8. Pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 induce apoptosis of L-132 and H460 cells. The cells were disrupted and the DNA labeled with propidium iodide. The samples were analyzed by flow cytometry and the fraction of sub-G0/G1 events was detected as a measure of apoptotic cell death. The relative fluorescence intensity of propidium iodide per cell was measured at the FL3-H channel. Results are mean ± SEM (n = 3) *p b 0.001 versus control.

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proliferation compared with L-132 cells at a concentration of 10 μM (100 μg/mL) (Fig. 6). Our recent findings proved that the pyrrolidine2 inhibited non-small cell lung cancer proliferation by determining BrdU incorporation (Sundarraj et al., 2012b). Cell cycle regulation The effect of functionalized silica nanorattles in the regulation of cell cycle progression was assessed by analyzing the cell cycle distribution of L-132 and H460 cells using flow cytometry. The proliferation of L-132 and H460 cells was treated with free pyrrolidine-2 (10 μM), SN-

pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 for 24 h. The PI stained cells were subjected for cellular DNA content and cell cycle phase analyses. As determined by FACS, the number of cells in S and G2/M phases were markedly reduced (Fig. 7). As a result, more cells resided in the G0/G1 phases of the cell cycle for a longer period, which implied that a modified silica nanorattles modulated the G1 to S phase progression. Pyrrolidine treated endothelial cells displayed both reduced numbers of cells in the S phase and delayed in attaining the S phase compared with control cells. A similar study has been explained to justify the impact of pyrrolidine against several cancer cells (Herbert et al., 2009).

Fig. 9. Histological sections of liver, spleen, kidney, lungs, heart and testis samples collected after 17 days post-injection of the free pyrrolidine-2 (left) and pyrrolidine-2 loaded SN-EGFRAb (right). Sections were stained with H&E and observed under a light microscope at 10× and 40× magnifications.

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Targeted SNs induce apoptosis in cancer cells Apoptosis is experienced in which pyrrolidine curb the cells via diminished tubule formation, induced cell cycle arrest and cyclin A/ cdk2 dependent apoptosis in human umbilical vein endothelial cells (Herbert et al., 2005). The cells treated with a high concentration of SN-PEI (100 μg/mL) did not show any abnormality compared with the control cells, which further confirmed the biocompatibility of SN-PEI. The apoptosis of L-132 and H460 cells was induced by pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 as it was proven using flow cytometry. EGFRAb-SN-pyrrolidine-2 treatment induced apoptosis in a population of H460 lung cancer cells compared with the normal lung epithelial cells (Fig. 8). The significant level of apoptosis in cancer cells may occur due to the proper delivery of the selected anticancer drug due not only to the support of silica nanorattles, but also to cell specific recognition of EGFR and its antibody. Systemic toxicity and antitumor efficacy of functionalized SNs Subsequently, we evaluated the systemic toxicity of SN-PEI, pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 formulation in vivo. SN-PEI suspended in saline was injected through the tail vein into Balb/c mice with a single dose of 1 mg/kg. The gross anatomy and patho-morphology examinations showed that all of the organs including mononuclear phagocyte system (MPS) as well as the spleen had no evidence of change in morphology including the lymphoid follicles or the area of the white pulp. We had carried out the histological analysis of the major organs (liver, spleen, kidney, lung, heart and testis) at 408 h post-injection of the pyrrolidine-2 and pyrrolidine-2 conjugated EGFRAb-SN nanorattles. Tissues were harvested and fixed at 10% neutral buffered formalin, processed routinely into paraffin, and 4 μm sections were cut and stained with hematoxylin and eosin (H&E) and which were then examined using light microscopy. As shown in Fig. 9, no apparent tissue/cellular damages were observed in the mice injected with the pyrrolidine-2 loaded EGFRAb-SN, when compared with that obtained from mice injected with free pyrrolidine-2. The histological results also indicated that the liver had no obvious histopathological abnormalities in the pyrrolidine-2 loaded EGFRAb-SN group relative to the control (Fig. 9), but in the pyrrolidine-2 group, 5 of 6 mice had a remarkable microgranuloma formation in the liver. It was estimated that the necrosis of liver cells was induced by pyrrolidine-2 which recruited the monocytes and Kupffers to the necrosis location. Other tissues including spleen, kidney, lung, heart and testis had no apparent histopathological abnormalities or lesions in all groups. These results showed that the drug carrier of SN-PEI was biocompatible. By encapsulating pyrrolidine-2 into silica nanorattles conjugated EGFRAb, the formulation can be readily dispersed in the physiological media without any disturbance to normal cells. To determine the function of SN-PEI for reducing the toxicity of pyrrolidine-2, the systemic toxicities of pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2 were evaluated in healthy mice. Yang et al. (2009) reported that single-chain Fv anti-EGFR antibody (ScFvEGFR) has been successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR-expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells are demonstrated after systemic delivery of ScFvEGFR-QDs or ScFvEGFR-IO magnetic nanoparticles into an orthotopic pancreatic cancer model. This report significantly supports our results that the EGFRAb enhanced the internalization of drug loaded silica nanorattles into the lung cancer cells exclusively. Twenty-four healthy male Balb/c mice with a body weight of about 20 g were randomly divided into four groups, pyrrolidine-2 group (1 mg/kg), SN-pyrrolidine-2 group (1 mg/kg), EGFRAb-SNpyrrolidine-2 group and control group (physiological saline). A total of five intravenous administrations (day 1, day 5, day 9, day 13 and day

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17) in a span of 17 days were given. The mice were then kept free for another 4 days. After the administration for 17 days, a lesser amount of body weight gain in pyrrolidine-2 and SN-pyrrolidine-2 groups was found at 4.8% and 6.2%, respectively. These were indicated severe toxicity. A significant amount of body weight gain in the control group was observed about 21.4% due to the development of tumors, where appreciable gains in body weight (15.9%) in EGFRAb-SN-pyrrolidine-2 group was noticed due to ameliorate in feeding habits of the mice (Fig. 10A). Human H460 tumor cells were injected subcutaneous into the right hind limbs of 6-week-old male Balb/c mice. Tumor-bearing mice were received daily intra-peritoneal injections of vehicle or 1 mg/kg pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SN-pyrrolidine-2. Treatment was repeated for four successive days, and tumor volume was determined by peripheral caliper measurements. Several studies suggest that the PLA-PLL-EGFRmAb NPs can efficiently target to hepatocellular carcinoma (HCC) and are specifically internalized in EGFR over-expressing tumor cells. The PLA-PLL-EGFRmAb NPs also has been shown better targeting for tumor as compared with the plain nanoparticles (Liu et al., 2010). Fig. 10B showed that in the H460 model, the average inhibition rate of tumor volume was calculated about 22% by pyrrolidine-2, whereas by SN-pyrrolidine-2 group, it was intended about 47% and the highest inhibition rate of tumor volume was estimated by the EGFRAb-SN-pyrrolidine-2 group (64%) (Table S1). Although lung cancer is resistant to most anticancer drugs, our results here

Fig. 10. In vivo antitumor activities of pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SNpyrrolidine-2 on H460 lung cancer subcutaneous model (1 mg/kg of pyrrolidine-2, five doses, i.v.). (A) Change of body weight, and (B) mean value of H460 tumor volumes in each of the treatment groups (control, pyrrolidine-2, SN-pyrrolidine-2 and EGFRAb-SNpyrrolidine-2).

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proved that the pyrrolidine-2 has a high suppressive effect on H460 lung cancer cells. Conclusion Beyond the existing reports on synthesis and uses of silica nanoparticles, the present study was summarized that the silica nanorattles were particularly well suited for targeting lung cancer cells with the assistance of its conjugates namely EGFRAb (supports internalization) and the pyrrolidine-2 (possesses anticancer properties). The EGFRAb-SNpyrrolidine-2 was also shown to have better targeting of tumor on par with the SN-pyrrolidine-2. The better targeting of EGFRAb-SNpyrrolidine-2 was attributed to more cellular uptake by lung cancer cells, which were mediated by the ligand–receptor recognition, making the nanorattles highly promising candidates for targeted drug delivery for cancer treatment, or imaging agents for early tumor diagnosis. These silica nanorattles can be advantageous for in vivo enhancement of cancer therapy as well as for reducing the systemic toxicity of the antitumor pro-drugs. These results suggested that the EGFRAb-SNpyrrolidine-2 may assist in an efficient targeted drug delivery for cancer therapy. We believe that this preliminary works would promote further understanding of other nanomaterials and pave the way for future clinical translation. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the Department of Science and Technology-Nanomission, Government of India (SR/NM/NS-60/2010) and DST-FIST. TEM measurements were carried out in the Sophisticated Analytical Instrumentation Facility, AIIMS, New Delhi. Flow cytometry and fluorescence microscopy studies were analyzed in the Central Research Instrumentation Facility, Shankara Nethralaya, Chennai and DLS was assisted by the Department of Nanoscience and Technology, Karunya University, Coimbatore. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.taap.2014.01.012. References Chen, Y., Chen, H., Guo, L., He, Q., Chen, F., Zhou, J., Feng, J., Shi, J., 2010. Hollow/rattle-type mesoporous nanostructures by a structural difference-based selective etching strategy. ACS Nano 4, 529–539. Fontanini, G., De Laurentiis, M., Vignati, S., Chine, S., Lucchi, M., Silvestri, V., Mussi, A., De Placido, S., Tortora, G., Bianco, A.R., Gullick, W., Angeletti, C.A., Bevilacqua, G., Ciardiello, F., 1998. Evaluation of epidermal growth factor-related growth factors and receptors and of neoangiogenesis in completely resected stage I–IIIA non-small cell lung cancer: amphiregulin and microvessel count are independent prognostic indicators of survival. Clin. Cancer Res. 4, 241–249. Fujino, S., Enokibori, T., Tezuka, N., Asada, Y., Inoue, S., Kato, H., 1996. A comparison of epidermal growth factor receptor levels and other prognostic parameters in non-small cell lung cancer. Eur. J. Cancer 32, 2070–2074. Gao, F., Li, L., Liu, T., Hao, N., Liu, H., Tan, L., Li, H., Huang, X., Peng, B., Yan, C., Yang, L., Wu, X., Chen, D., Tang, F., 2012. Doxorubicin loaded silica nanorattles actively seek tumors with improved anti-tumor effects. Nanoscale 74, 3365–3372. Ghomashchi, F., Stewart, A., Hefner, Y., Ramanadham, S., Turk, J., Leslie, C.C., Gelb, M.H., 2001. A pyrrolidine-based specific inhibitor of cytosolic phospholipase A(2)alpha blocks arachidonic acid release in a variety of mammalian cells. Biochim. Biophys. Acta 1513, 160–166. Ghosh, M., Loper, R., Ghomashchi, F., Tucker, D.E., Bonventre, J.V., Gelb, M.H., Leslie, C.C., 2007. Function, activity and membrane targeting of cytosolic phospholipase A2zeta in mouse lung fibroblasts. J. Biol. Chem. 282, 11676–11686. Grünwald, V., Hidalgo, M., 2002. The epidermal growth factor receptor: a new target for anticancer therapy. Curr. Probl. Cancer 26, 109–164.

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