Preparation, characterization and toxicological investigation of copper loaded chitosan nanoparticles in human embryonic kidney HEK-293 cells

Materials Science and Engineering C 61 (2016) 227–234

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Preparation, characterization and toxicological investigation of copper loaded chitosan nanoparticles in human embryonic kidney HEK-293 cells Divya Arora a,b,1, Vandna Dhanwal c,1, Debasis Nayak a,c, Ankit Saneja a,b, Hina Amin c, Reyaz ur Rasool a,c, Prem Narayan Gupta a,b, Anindya Goswami a,c,⁎ a b c

Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu, India Formulation and Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India

a r t i c l e

i n f o

Article history: Received 24 July 2015 Received in revised form 6 November 2015 Accepted 16 December 2015 Available online 18 December 2015 Keywords: Chitosan CuCSNPs Cytotoxicity ROS Caspase cleavage

a b s t r a c t Metallic nanoparticles often attribute severe adverse effects to the various organs or tissues at the molecular level despite of their applications in medical, laboratory and industrial sectors. The present study highlights the preparation of copper adsorbed chitosan nanoparticles (CuCSNPs), its characterization and validation of cytotoxicity in human embryonic kidney HEK-293 cells. Particle size of the CuCSNPs was determined by using Zetasizer and the copper loading was quantified with the help of ICP/MS. Further characterization of CuCSNPs was carried out by FT-IR analysis to determine the formation of nanoparticles and SEM was conducted for the morphological analysis of the CuCSNPs. The CuCSNPs exhibited pronounced cytotoxic effects towards HEK-293 cells as analyzed by MTT assay. Moreover, the CuCSNPs inhibited the colony formation and induced nuclear damage at the dose of 100 μg/mL, much more effectively than the in built control copper sulfate (CuSO4). At the molecular level, the CuCSNPs were found to be triggering reactive oxygen species (ROS), activating effector caspases and subsequent PARP cleavage to induce cell death in HEK-293 cells. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In recent years, metallic nanoparticles have engrossed the interests of researchers because of their ease in synthesis and modification into desired forms, which facilitate them to conjugate with antibodies, drugs and ligands to bind with specific targets [1]. However, due to their larger surface area and specific structural properties, toxicity issues (killing of normal cells) are major concern. These issues minimize their use in specific cases and a major challenge to overcome [2–4]. Further, the use of metallic nanoparticles has gained increasing interest in the field of catalysis [5–8]. These nanoparticles possess a strong inherent tendency to aggregate and agglomerate due to their immense surface energy restricting their applications in catalysis [9]. To prevent this aggregation and recovery of these metals, a wide array of techniques viz. fixation on surfaces/in a matrix or using polymers as a coating support has been introduced [9,10]. Among these polymers, chitosan has gained a considerable interest as a support for metals due to its high affinity for sorption of metal ions owing to the presence of amine (− NH2) and ⁎ Corresponding author at: Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu, India. E-mail address: [email protected] (A. Goswami). 1 These authors contributed equally to this article.

http://dx.doi.org/10.1016/j.msec.2015.12.035 0928-4931/© 2015 Elsevier B.V. All rights reserved.

hydroxyl (−OH) groups [11]. Chitosan, a polysaccharide, poly[(1,4)-Dglucose-2-amine)], having molecular formula (C6H11NO4)n is the deacetylated product of chitin obtained from the exoskeleton of crustaceans such as shrimp, lobster, insects, fungi and algae. Chitosan– halloysite nanotube (HNT) nanocomposite (NC) scaffolds have their potential applications in tissue engineering or as drug/gene carriers. These NC scaffolds exhibit significant enhancement in compressive strength, compressive modulus, thermal stability and cytocompatibility compared to the pure chitosan [12]. Copper, a ductile metal of laboratory and industrial importance has been studied since years. Handful of reports clearly reveal copper nanoparticles (CuNPs) are being extensively used in electronics, films, ceramics, polymers, metallics, inks, lubricant oil and coatings. In addition, CuNPs have shown great potential in osteoporosis-treatment drugs, additives in livestock, poultry feed, antibacterial materials, and intrauterine contraceptive additives [13]. Cu(II) ions can be effectively adsorbed onto the chitosan and cross-linked chitosan beads at an optimum pH of 6.0. The adsorption isothermal data of the above interaction indicates that uptake of Cu(II) ions on chitosan beads obeys Langmuir isothermal equation. The Cu(II) ions can also be removed from the chitosan and cross-linked chitosan beads by treatment with an aqueous EDTA solution [14,15]. Several studies have shown that CuNPs possess excellent antibacterial activity against both gram +ve and gram − ve

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bacteria as well as cytotoxicity towards human cancer cells [10,13,16, 17]. Originally developed as a potential drug carrier [18,19], emerging evidences demonstrate chitosan containing diets and chitosan nanoparticles (CSNPs) are helpful in reducing the growth of tumor cells as well as lower the risk of cancer progression [20–22]. Thiolated chitosan (TCS) based mucoadhesive polymeric nanoparticles developed by ionic cross-linking reaction with pentasodium tripolyphosphate (TPP) has been proved for their in vitro cytocompatibility and less toxicity towards normal cells and hence can be effectively used for drug and gene delivery applications [23]. CSNPs can enhance the targeted delivery of anticancer drugs and can overcome the multidrug resistance (MDR) induced by P-glycoprotein (P-gp). It has been seen that a novel copolymer, chitosan-graft-D-α-tocopheryl polyethylene glycol 1000 (TPGS) (CT) synthesized for doxorubicin delivery showed enhanced cytotoxicity towards doxorubicin resistant human breast adenocarcinoma (MCF7) cells and 5-FU resistant human hepatocarcinoma (HepG2 and BEL-7402) cells compared to Adriamycin. This enhanced activity can be attributed to the P-gp blocking and downregulation of the ATP levels by the CT NPs [24]. CSNPs also offers a stable drug delivery system for proteins and siRNAs into the biological system [25]. CSNP containing microspheres designed for pulmonary administration of therapeutic macromolecules have shown to possess suitable aerodynamic properties, capacity to encapsulate proteins and in vitro biocompatibility [26]. CSNPs can inhibit the growth of human hepatocellular carcinoma tumor xenografts by reducing the microvascular density and tumor angiogenesis through the inhibition of vascular endothelial receptor 2 (VEGFR2) in a dose dependent manner [22]. Though, a few studies report copper loading to CSNPs potentiates the cytotoxic effect of CSNPs, the exact mechanism underlying was not clearly understood till date. The higher cytotoxic activities of CuCSNPs might be attributed to the superior affinity of the cupric ions towards negatively charged cell membrane and higher surface charge density compared to the CSNPs as well as the controlled release of cupric ions dissociated in the CSNPs [27]. Chitosan nanoparticles loaded with metal ions (Ag+, Cu2+, Zn2+, Mn2 +, or Fe2 +) possesses high antibacterial activities against broad spectrum of bacterial strains like; Escherichia coli, Salmonella choleraesuis and Staphylococcus aureus in vitro [28,29]. However, reports on the other biological activities such as anticancer efficacy of these metallic chitosan nanoparticles are still very limited. The major disadvantages of these nanoparticles include less stability, low mechanical resistance and toxicity to the normal tissues/organs. These issues restrict their use in human biological systems and needs modeling for its effective and safe use. Since, our laboratory is involved with this recent approach of nanotechnology, we were keen to explore the underlying mechanism by which these metallic nanoparticles exhibit their role in cytotoxicity. Here, our combined approaches unveil CuCSNPs effectively induced apoptosis in HEK-293 cells by nuclear cleavage, membrane damage and generating ROS. Further molecular mechanisms revealed CuCSNPs promoted caspase activation and PARP cleavage at a concentration dependent manner to impart toxicity in HEK-293 cells.

2. Experimental details 2.1. Chemicals and antibodies Chitosan (Low Molecular Weight; M.W. ~ 50,000–190,000 Da and 75–85% deacetylated), was purchased from Sigma-Aldrich., USA. Sodium tripolyphosphate (TPP) was obtained from Alfa Aesar, USA. Copper (II) sulfate pentahydrate (CuSO4.5H2O) was obtained from Himedia labs, India. RPMI-1640 media and fetal bovine serum (FBS) were from Invitrogen. Penicillin G, streptomycin, Trypsin–EDTA, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), crystal violet, paraformaldehyde, Triton X-100, UltraCruz DAPI mounting media, H2O2, 2′,7′-dichlorofluorescein diacetate (DCFDA) and dimethylsulfoxide (DMSO) were obtained from Sigma Chemicals. Caspase-(Glo) 3/7 detection assay kit was purchased from Promega. Antibodies for caspase-3 was procured from Abcam (USA), PARP from Santa Cruz Biotechnology (Santa Cruz, CA) and Actin from Sigma (St. Louis, MO).

2.2. Preparation of nanoparticles Chitosan nanoparticles were prepared by ionotropic gelation of chitosan with TPP anions. In brief, chitosan (0.2%, w/v) was dissolved in acetic acid aqueous solution (2%, v/v). Chitosan nanoparticles were formed by the dropwise addition of 0.7 mL of aqueous TPP solution (0.4% w/v) to 5 mL of chitosan solution and the opalescent solution was formed, which was further kept under magnetic stirring (500 rpm, 2 h) at room temperature. The nanoparticles were separated by centrifugation at 10,000 g (Sigma 3–30 K, Germany) and 25 °C for 10 min, freeze dried and stored at 5 ± 3 °C. The weights of freeze-dried NP's were also measured. After this, CuCSNPs were prepared by adding one part of Cu ions (1 Part × 3.932 mg of CuSO4) into the five parts of chitosan nanoparticles and the suspension was left for 12 h stirring. The resultant suspension was centrifuged and a wet pellet was obtained (Fig. 1).

2.3. Particle characterization The particle size distribution and the zeta potential were determined using Zetasizer Nano-ZS90 (Malvern Instruments). The particle size analysis was performed at a scattering angle of 90° at 25 °C. For zeta potential measurements, samples were diluted with 0.1 mM KCl and measured under the automatic mode. FT-IR spectra of the samples were taken with potassium bromide pellets on a Perkin–Elmer spectrometer in the range from 4000 to 500 cm−1. The morphology of chitosan nanoparticles and copper loaded chitosan nanoparticles were investigated under scanning electron microscopy (SEM). Particles were spread over carbon-tape attached to aluminum holder and then coated with gold prior to inspection under scanning electron microscope (S-3400N, Hitachi, Japan).

Fig. 1. Scheme of preparation of chitosan and copper adsorbed chitosan nanoparticles.

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Fig. 2. Particle size of (a) chitosan nanoparticles and (b) copper-adsorbed chitosan nanoparticles.

2.4. Estimation of copper adsorption

2.6. Morphological analysis

The amount of copper present in the sample was determined by Inductively Coupled Plasma Mass Spectroscopy (ICP/MS, Agilent Technologies). Briefly, the solids that weighed between 21 and 301.7 mg were digested in quartz vessel. To each vessel, 8 mL of concentrated nitric acid was added. After equilibrating the pressure, samples were subsequently digested using a microwave digester (Anton Paar 3000). After digestion, samples were removed and cooled before making a final dilution to 50 mL using ultrapure deionized water. A mass of copper was used for quantification. The amount of copper adsorbed was calculated using the formula:

HEK-293 cells (0.5 × 106) were plated into 6 well plates and treated with CS, CSNP, CuSO4 and CuCSNPs (100 μg/mL each) for 24 h. After the termination of the treatment, media was removed from the plates and washed with sterile PBS twice. The cells were observed under an inverted microscope and images were captured at 20 × magnification with the help of a digital camera (NIKON D3100).

Amount of copper adsorbed ð%Þ Amount of copper present in the sample  100 ¼ Amount of copper used

2.5. Cell viability assay The cell viability was determined by standard MTT dye uptake method [30]. Briefly HEK-293 cells from moderately confluent flasks were trypsinized properly and plated into 96 well tissue culture plates at a density of 3.0 x 103 cells/well. Treatment to the cells was given with various concentrations CS, CSNPs, CuSO4 and CuCSNPs in triplicates so that the final concentration of DMSO solvent was 0.2%. After 44 h incubation, MTT solution (2.5 mg/mL) was added and further incubated at 37 °C and 5% CO2 for 4 h. The amount of colored formazan derivatives was determined by measuring optical density (OD) using TECAN microplate reader (Infinite M200 PRO, Switzerland AG) at 570 nm. The percentage of cell viability was determined and the IC50 values were calculated using GraphPad Prism software [31].

2.7. Apoptosis assay Cells after trypsinization were seeded into 8 well chamber slides (Nunc) at a density of 35 x 103 cells/per well and treated with 100 μg/mL of CS, CSNP, CuSO4 and CuCSNPs. After 24 h of incubation, cells were washed twice with chilled PBS and fixed with 4% paraformaldehyde for 20 min. Then cells were permeabilized with 1% Triton X-100 followed by incubation with DAPI containing mounting medium for 15 min/RT in the dark. Apoptosis was observed under Floid Cell Imaging Station (Life Technologies) at 20× magnification [32,33].

2.8. Clonogenic assay As previously described [34], HEK-293 cells were plated at a seeding density of (1 × 103 cells/well) in 6 well tissue culture grade plates. After 24 h, the culture medium was replenished with fresh RPMI medium and cells were exposed to 100 μg/mL of CS, CSNP, CuSO4 and CuCSNPs for 5 days in 37 °C incubator with 5% CO2. Later on, the obtained colonies were fixed with 4% paraformaldehyde and were stained with 0.5% crystal violet solution. The colonies from the three random fields were counted, averaged and photographed under inverted microscope as described above.

Fig. 3. Zeta potential of (a) chitosan nanoparticles and (b) copper-adsorbed chitosan nanoparticles.

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Fig. 4. FT-IR spectrum of (a) chitosan, (b) chitosan nanoparticles and (c) copper adsorbed chitosan nanoparticles.

Fig. 6. Graph showing the % cell viability against the logarithmic concentrations of CuCSNP at various time points (24, 48 and 72 h) as determined by MTT assay.

2.9. Reactive oxygen species (ROS) determination Adherent HEK-293 cells were plated into 12 well plates at a density of 0.3 × 106 cells/well and treated with 100 μg/mL of CS, CSNP, CuSO4 and CuCSNPs along with H2O2 as positive control for 24 h. DCFDA dye solution (10 μM) was added at the completion of 24 h in dark and cells were further incubated for 30 min. Cells were then washed thrice with PBS thoroughly and images were captured under Floid Cell Imaging Station (Life Technologies) at 20× magnification. The intracellular ROS generation was also quantified by a fluorescence spectrometer with excitation and emission spectra of 495 nm and 529 nm respectively and values were presented as relative fluorescence unit (RFU) [35,36]. 2.10. Caspase activity assay

60 mM/L, NP-40 0.3%, EDTA 1.0 mM/L, DTT 1.0 mM/L, Sodium orthovanadate 1.0 mM/L, PMSF 0.1 mM/L, cocktail protease inhibitor). The cell extractions were centrifuged at 12,000 rpm for 10 min at 4 °C. Protein concentration was determined by the standard Bradford method. Equal amount (20 μg) of protein from each sample was subjected to SDS–PAGE and proteins were transferred to PVDF membrane (PALL), blocked with 5.0% (w/v) non-fat milk in PBS containing 0.1% Tween-20 and probed with relevant antibodies for 3 h at room temperature or overnight at 4 °C. Subsequently blots were washed and probed with species specific secondary antibodies coupled to horseradish peroxidase. Immunoreactive proteins were detected by enhanced chemiluminescence plus (Thermo). 2.12. Statistical analysis

The intracellular caspase activity was measured through a commercial Caspase-Glo (3/7) assay kit (Promega) [37]. Briefly, cells (10 × 103 per well) in a 96 well plate were treated with 100 μg/mL of CS, CSNP, CuSO4 and CuCSNPs and incubated for 24 h. The plate was brought to room temperature and 100 μL of Caspase-Glo (3/7) reagent was added into the wells. After 30 min of further incubation, the reading was taken with the help of a luminometer microplate reader (TECAN, Infinite M200 PRO, Switzerland, AG). The values were analyzed and expressed as relative luminescence unit (RLU). 2.11. Western blot analysis The experimental protocol was described previously [32]. Briefly, HEK-293 cells (1 × 106 cell per well) were incubated overnight in a 6 well plate and exposed to varying concentrations of CuCSNP and vehicle. Cells were accordingly rinsed with PBS, trypsinized and collected after 24 h and then lysed with lysis buffer (HEPES 1.0 mM/L, KCl

All the data were expressed as mean ± SEM of at least three independent experiments performed. IC50 values were determined with the help of GraphPad Prism software Version 5.0 (GraphPad Software, Inc., La Jolla, U.S.A) by taking log of inhibitor vs response. Comparisons used Student's t test and P b 0.05 values were assigned significance. 3. Results and discussion The essence of the current study underscores the preparation and characterization of the copper loaded chitosan nanoparticles together with its evaluation for cytotoxic and apoptosis inducing effects on HEK-293 cells. The study reveals that amount of copper loaded on to the chitosan nanoparticles was 17.41%, as quantified by ICP/MS. Moreover, the size distribution profile as shown in Fig. 2a, represents a typical batch of nanoparticles with a mean diameter ranging from 173.5 ± 2.829 nm and a narrow size distribution (polydispersity index 0.214 ± 0.023). The size of copper loaded nanoparticles is

Fig. 5. SEM images of (a) chitosan nanoparticles and (b) copper-adsorbed chitosan nanoparticles.

D. Arora et al. / Materials Science and Engineering C 61 (2016) 227–234 Table 1 Cytotoxic effects of CS, CSNP, CuSO4 and CuCSNPs in human embryonic kidney HEK-293 cells. Test compounds & formulations

CS CSNP CuSO4 CuCSNP

IC50 values in human embryonic kidney (HEK-293) cells (μg/mL) 24 h

48 h

72 h

N1000 N1000 223 ± 0.156 135 ± 0.212

N1000 980 ± 0.113 187 ± 0.133 105 ± 0.068

862 ± 0.127 735 ± 0.212 151 ± 0.198 92 ± 0.096

288.9 ± 5.024 nm (polydispersity index 0.344 ± 0.019) as shown in Fig. 2b. Intriguingly, the zeta potential of the surfaces of chitosan nanoparticles have a positive charge about +22.5 ± 1.55 mV and it increases with the adsorption of copper ions to about +52.3 ± 1.86 mV implying the reason probably is due to the positive charge carried by copper ions, which are adsorbed onto chitosan nanoparticles (Fig. 3a, b). For chitosan nanoparticles, the +ve zeta potential is because of the presence of protonated amine groups. However, when − ve charged TPP reacts with + ve charged chitosan, the net zeta potential decelerates. Therefore, an increase in zeta potential represents lower amount of TPP present in the sample. CSNPs are considered to be microporous biopolymers; therefore, they are having large cavity to let copper ions through. Copper ions are first adsorbed onto the external surfaces of CSNPs and then diffuse into the cavity of nanoparticles. Finally, these ions can be chelated onto the internal surface of CSNPs [38].

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The FT-IR spectral analysis was used to determine the formation of CSNPs and CuCSNPs. FT-IR spectra of CS, CSNPs and CuCSNPs are shown in Fig. 4. CS showed a characteristic peak around 3450 cm− 1 which is assigned to the stretching vibration of –OH groups (Fig. 4a). The peak observed at 1420 cm−1 can be attributed to –NH deformation vibration in –NH2, while the peaks at 1319 and 1152 cm−1 are due to the –CN stretching vibration. The peaks at 1076 and 1021 cm−1 are corresponded to the –CO stretching vibration in –COH [39]. A peak at 1638 cm− 1 is attributed to the CONH2 group [40]. In chitosan nanoparticles (Fig. 4b), the peak of 3450 cm−1 was geting broader; indicating that hydrogen bonding is enhanced. The peak at 1529 cm−1 in the CSNPs is indicative of interaction between NH+ 3 groups of chitosan and phosphate groups of TPP [41]. The intensity of the peaks at 1420, 1319 and 1152 cm− 1 are reduced in chitosan nanoparticles indicating that the ionic gelation of chitosan and TPP was through the complexation between –NH+ 3 and –O–P [39]. In copper adsorbed chitosan nanoparticles (Fig. 4c), the intensity of the broad band of the –OH stretching vibration at the wave number 3450 cm− 1 decreased due to the copper ions adsorption. The γ (C–N) peak at 1414 cm− 1 disappeared, and the γ (CH3) peak of the group acetyl at 1381 cm−1 shifted to 1384 cm− 1 illustrating copper bonding could cause the substantial redistribution of vibration frequencies. Copper ions adsorption was found to affect the bonds related to nitrogen atoms, indicating that nitrogen atoms could be the main sites for its attachment. Apart from that, the intensity at the wave number 1093 cm− 1 was reduced, suggesting that the oxygen atom in the hydroxyl groups of nanoparticles was also involved in the copper

Fig. 7. CuCSNPs are highly cytotoxic to human embryonic kidney (HEK-293) cells. (a) Morphological images of the HEK-293 cells, 24 h after treatment with CS, CSNP, CuCSO4 and CuCSNPs. (b) HEK-293 cells (5 × 104) were treated with 100 μM of CS, CSNP, CuSO4 and CuCSNPs for 24 h. Cells after fixation were stained with DAPI nuclear stain and photographed under fluorescence microscope at 20× magnification. (c) Effect of CS, CSNP, CuSO4 and CuCSNPs on the colony forming ability of HEK-293 cells. Data were calculated, compared with each control and expressed as ±SEM of three independent experiments performed (*P b 0.05).

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Fig. 8. CuCSNPs induce oxidative stress through generation of reactive oxygen species. (a) HEK-293 cells were cultured with CS, CSNP, H2O2, CuSO4 and CuCSNPs for 24 h and with DCFDA for 30 min. Images were captured under a fluorescent microscope at 20× magnification. (b) The intracellular ROS generation was also measured by a fluorescence spectrometer with excitation and emission spectra of 495 nm and 529 nm respectively and values were presented as relative fluorescence unit. Data from three independent experiments were subjected to statistical analysis and P b 0.05 values were assigned significance.

adsorption [39,40]. These observations demonstrate that the adsorption of copper ions is probably due to the formation of complexes with the nitrogen atom of NH2 group (which are not involved during ionic-cross-linking process) and of the oxygen atom of hydroxyl group. The morphology of the chitosan nanoparticles was investigated under SEM. The chitosan nanoparticles exhibited spherical shape

without any aggregation (Fig. 5a). However, copper adsorbed chitosan nanoparticles showed somewhat blurry boundary surrounding a central sphere which may result from the adsorbtion of copper ions (Fig. 5b). The mean diameter of the nanoparticles also increased significantly after the loading of copper ions as illustrated by SEM image. This might be due to the + ve charge and high surface energy of the copper ions that tends to form agglomerates.

Fig. 9. CuCSNPs induces apoptosis through caspase activation and PARP cleavage. (a) HEK-293 cells after treatment with CS, CSNP, CuSO4 and CuCSNPs in 96 well plates were incubated for 24 h. The caspase activity was measured by a commercial Caspase-Glo 3/7 assay kit (Promega) with the help of a microplate reader luminometer. The data obtained were compared with each control and expressed as relative luminescence unit (RLU) (*P b 0.05). (b, c) The HEK-293 cells were treated with CuCSNPs at indicated concentrations for 24 h and whole cell lysates were employed for the western blot analysis for checking the expression of Caspase-3, cleaved-Caspase-3, PARP and cleaved PARP. Actin expression was considered as the loading control for each western blotting experiment performed.

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Metal based nanomaterials including, quantum dots, various metallic nanoparticles such as Au, Ag, Pt and FePt nanoparticles and metal oxides, such as TiO2, ZnO2, Fe3O4, Al2O3, CrO3 and SiO2 impart adverse effects mostly by causing oxidative stress, apoptosis, inflammation to the endothelial cells [2,42,43]. Studies also demonstrate that various inhalable metal oxide nanoparticles (TiO2, Ag, Al, Zn, Ni) are cytotoxic to the human alveolar epithelial cells (A549) in a dose dependent manner through DNA fragmentation and apoptotic damage [44]. To the best of our knowledge, the toxic effects of CuCSNPs in human normal cellular environments have not been studied in detail till date. Keeping this in mind, we were interested to evaluate the toxic effects of our prepared chitosan based nanoparticles. CSNPs and CuCSNPs were studied for their cytotoxicity/antiproliferative activity against HEK-293 cells through MTT assay. The toxicity profile of CSNPs for HEK-293 cells was very less even at the higher concentration (~1000 μg/mL); whereas, CuCSNPs substantially amplified the toxicity (almost 10 times to that of CSNPs). The 24, 48 and 72 h IC50 values for CuCSNPs were 135 ± 0.212, 105 ± 0.068 and 92 ± 0.096 μg/mL respectively (Fig. 6), whereas the IC50 values for CuSO4 was 223 ± 0.156, 187 ± 0.133 and 151 ± 0.198 μg/mL respectively (Table 1). The CS showed less or negligible toxicity (N1000 μg/mL) towards HEK-293 cells. Hence, CuCSNPs confer more pronounced cytotoxicity compared to the CSNPs. This increase in activity might be due to the greater zeta potential of CuCSNPs that leads to the stronger interaction with cell membrane, thus higher the cytotoxicity [27]. Further, we seek to determine the effect of CuCSNPs on apoptosis in HEK-293 cells. After 24 h of treatment with 100 μg/mL of CuCSNPs, the cells showed distorted morphology and features of early apoptotic signatures compared to the empty chitosan nanoparticles. Though, the effect of an equal dose of CuSO4 was remarkable, it was lesser than the toxicity induced by CuCSNPs, probably due to the increase in delivery of the metal into the cell (Fig. 7a). Further from DAPI staining, we observed majority of cells showing early features of apoptosis viz. membrane blebbing, chromatin condensation around nuclear periphery and nuclear fragmentation in the CuCSNPs (100 μg/mL) treated cells, compared to the CuSO4 alone and empty nanoparticles within the 24 h of treatment (Fig. 7b). Additionally, to examine the effect of CuCSNPs on cell proliferation, colony formation assay was performed. Interestingly, CuCSNPs (100 μg/mL) effectively abrogated the cell proliferation and colony formation capability (N70%) than the CSNPs treated cells (Fig. 7c). Together, these results demonstrate that CuCSNPs are substantially toxic, promote cell death and more noxious than the CuSO4 towards HEK-293 cells. CuCSNPs were further examined whether they could generate ROS, which is a prime hallmark of cellular damage and apoptosis. ROS levels increase dramatically during the exposure of cytotoxic substances/ drugs to the cells resulting in severe damages to the cell structures [45]. In order to check this, cells were first exposed to 100 μg/mL of CS, CSNP, CuSO4 and CuCSNPs for 24 h and then incubated with ROS specific dye DCFDA for 30 min. Our experimental results clearly revealed that CuCSNPs are able to generate sufficient mitochondrial ROS similar to positive control H2O2. In comparison, very few cells yield ROS when exposed to the similar concentration of CS and CSNPs (Fig. 8a). The RFU for the CuCSNP treated cells illustrate 6–7 times higher than that of the CSNPs, indicating a significant quantity of ROS production (Fig. 8b). Collectively, these results demonstrate that the toxic effects of copper increase significantly, when it adsorbs onto a polymeric (chitosan) nanoparticle. To further find out the mechanism of toxicity of CuCSNPs, we investigated its effect on caspase activity. Caspases are a family of cysteine proteases that play essential roles in mediating apoptosis. We have found a significant increase in caspase-3 activity (~ 2 fold) in HEK-293 cells treated with 100 μg/mL of CuCSNPs compared to CS and CSNP treated cells. The RLU for the CuCSNP treated cells was also ~2000 units higher than the cells treated with only CuSO4, indicating a sufficient increase in caspase-3 activity (Fig. 9a). To validate, we

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conducted western blot analysis to check the expression of caspase-3 in CuCSNP treated cells. Our results found cleavage of caspase-3 even at the lower dose (25 μg/mL) of CuCSNP and increased consistently upto the higher dose (100 μg/mL) (Fig. 9b). Poly (ADP-ribose) polymerase (PARP) is a nuclear protein that detects DNA single strand break. Hence, PARP activation is an early response to chemical or radiation induced DNA damage. Apoptotic caspases targets PARP-1 for proteolysis and facilitates the synthesis of poly (ADP-ribose), which is an indicator of the cells undergoing apoptosis [46]. Interestingly, we also observed a remarkable PARP cleavage and increase in cleaved-PARP expression in 50 μg/mL and 100 μg/mL of CuCSNPs (Fig. 9c). Therefore, the above results clearly demonstrate that the copper loaded chitosan nanoparticles induce apoptotic cell death via the mechanisms of caspase activation and PARP cleavage in HEK-293 cells. 4. Conclusion In conclusion, the present study uncovers the development, characterization and cytotoxic effects of CuCSNPs in HEK-293 cells. The biological mechanisms unveil CuCSNPs mediate reactive oxygen species generation, caspase activation and subsequent PARP cleavage to induce cell death. The study also demonstrates that the toxic effects of CuSO4 increases significantly, when it is adsorbed onto the surfaces of polymeric chitosan nanoparticles, supporting chitosan as an effective carrier for the delivery of chemicals/drugs into the biological system. Conflict of interest The authors have no such interests to disclose. Acknowledgment We thank our director, Dr. Ram Vishwakarma, for his encouragement and support behind the work. The authors would also like to thank the Council of Scientific and Industrial Research for providing the fellowship. References [1] V.V. Mody, R. Siwale, A. Singh, H.R. Mody, Introduction to metallic nanoparticles, J. Pharm. Bioall. Sci. 2 (4) (2010) 282. [2] A.M. Schrand, M.F. Rahman, S.M. Hussain, J.J. Schlager, D.A. Smith, A.F. Syed, Metal based nanoparticles and their toxicity assessment, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (5) (2010) 544–568. [3] H.A. Jeng, J. Swanson, Toxicity of metal oxide nanoparticles in mammalian cells, J. Environ. Sci. Health Part A 41 (12) (2006) 2699–2711. [4] S. Park, Y.K. Lee, M. Jung, K.H. Kim, N. Chung, E.-K. Ahn, Y. Lim, K.-H. Lee, Cellular toxicity of various inhalable metal nanoparticles on human alveolar epithelial cells, Inhal. Toxicol. 19 (S1) (2007) 59–65. [5] V. Calo, A. Nacci, A. Monopoli, F. Montingelli, Pd nanoparticles as efficient catalysts for Suzuki and Stille coupling reactions of aryl halides in ionic liquids, J. Org. Chem. 70 (15) (2005) 6040–6044. [6] B. Chen, F. Li, Z. Huang, F. Xue, T. Lu, Y. Yuan, G. Yuan, Highly stable, recyclable copper nanoparticles as catalysts for the formation of C–N bonds, ChemCatChem 4 (11) (2012) 1741–1745. [7] M. Kidwai, N.K. Mishra, V. Bansal, A. Kumar, S. Mozumdar, Novel one-pot Cunanoparticles-catalyzed mannich reaction, Tetrahedron Lett. 50 (12) (2009) 1355–1358. [8] L. Rout, S. Jammi, T. Punniyamurthy, Novel CuO nanoparticle catalyzed CN cross coupling of amines with iodobenzene, Org. Lett. 9 (17) (2007) 3397–3399. [9] S. Bokern, J. Getze, S. Agarwal, A. Greiner, Polymer grafted silver and copper nanoparticles with exceptional stability against aggregation by a high yield one-pot synthesis, Polymer 52 (4) (2011) 912–920. [10] M. Valodkar, P.S. Rathore, R.N. Jadeja, M. Thounaojam, R.V. Devkar, S. Thakore, Cytotoxicity evaluation and antimicrobial studies of starch capped water soluble copper nanoparticles, J. Hazard. Mater. 201 (2012) 244–249. [11] F. Zhao, B. Yu, Z. Yue, T. Wang, X. Wen, Z. Liu, C. Zhao, Preparation of porous chitosan gel beads for copper (II) ion adsorption, J. Hazard. Mater. 147 (1) (2007) 67–73. [12] M. Liu, C. Wu, Y. Jiao, S. Xiong, C. Zhou, Chitosan-halloysite nanotubes nanocomposite scaffolds for tissue engineering, J. Mater. Chem. B 1 (15) (2013) 2078–2089. [13] F. Rispoli, A. Angelov, D. Badia, A. Kumar, S. Seal, V. Shah, Understanding the toxicity of aggregated zero valent copper nanoparticles against Escherichia coli, J. Hazard. Mater. 180 (1) (2010) 212–216.

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