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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2017) 41: 322-332© TÜBİTAKdoi:10.3906/biy-1608-61

Biocompatible polymeric coatings do not inherently reducethe cytotoxicity of iron oxide nanoparticles

Aylin ŞENDEMİR ÜRKMEZ1,2,3,*, Ece BAYIR2,3, Eyüp BİLGİ1, Mehmet Özgün ÖZEN4

1Department of Bioengineering, Faculty of Engineering, Ege University, İzmir, Turkey2Department of Biomedical Technologies, Graduate School of Natural and Applied Sciences, Ege University, İzmir, Turkey

3Application and Research Center for Testing and Analysis (EGE-MATAL), Ege University, İzmir, Turkey4Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Department of Radiology, Canary Center at Stanford for Cancer Early

Detection, Stanford University, Palo Alto, CA, USA

* Correspondence: aylin.sendemir@ege.edu.tr

1. IntroductionMagnetic nanoparticles (MNPs) are nanoscale materials (1–100 nm) and they commonly consist of magnetic elements such as iron, nickel, or cobalt and their chemical compounds. Their unique physical, chemical, electrical, thermal, and magnetic properties offer many potential uses in various fields, such as physics, medicine, biology, and materials science (Li et al., 2008; Bao et al., 2016). Because of the toxicity of nickel and cobalt elements, iron-based nanoparticles are more commonly used for biomedical applications (Mahmoudi et al., 2011b). In the last few decades, MNPs have been increasingly used in biomedicine, especially for drug delivery, magnetic resonance imaging, hyperthermia therapy, biosensors, theranostic devices, cell sorting, cell labeling, tissue repair, and magnetofection studies (Gupta and Gupta, 2005; Kaushik et al., 2008; Mahdavi et al., 2013; Bayir et al., 2014; Gudovan et al., 2015).

Iron oxide nanoparticles (IONPs) often agglomerate due to their physicochemical properties (Li et al., 2008;

Wahajuddin, 2012). IONPs can be coated by various suitable molecules in order to prevent the formation of iron oxide clusters. Although IONPs are relatively biocompatible, many coating materials such as poly(ethylene glycol), dextran, heparin, silica, and phospholipids are used in order to prevent the generation of reactive oxygen species (ROS) and improve their biocompatibility for in vivo and in vitro applications (Mikhaylova et al., 2004; Fang and Zhang, 2009; Ling and Hyeon, 2013).

Tumor cell lines, due to their easy handling in vitro and highly proliferative capacity, have been utilized frequently for the investigation of various tumor cell biology characteristics, as well as mechanisms of carcinogenesis (Susa et al., 2009). In this study, a human osteosarcoma-derived cell line, SaOs-2, was used as a model cell line to test polymer-coated IONPs’ cytotoxicity. Osteosarcoma is the most common bone cancer with an overall survival rate of <20%, predominantly seen in children and adolescents (Mohseny et al., 2011; Lauvrak et al., 2013).

Abstract: Nanotechnology in biomedical research is an emerging and promising tool for different purposes, like high-resolution medical imaging, diagnostics, and targeted drug delivery. Although some experimental efforts focused on determination of the cytotoxicity of nanoparticles (NPs) are present, there are many controversial results. In this study, chitosan (CS)- and poly(acrylic acid) (PAA)-coated iron oxide nanoparticles (IONPs) were used to investigate the possible cytotoxicity of coated IONPs on model mammalian cell line SaOs-2 by evaluating cellular viability and membrane integrity. Increasing concentrations of IONPs increased the cytotoxic effect on SaOs-2 cells with both CS- and PAA-coated IONPs. Cell viability on day 3 was as low as 40% and 48% at 1000 µM concentration for PAA- and CS-coated IONPs, respectively, in 1% FBS-supplemented media. Cytotoxicity determined by the released lactate dehydrogenase (LDH) was as high as 163% with 1000 µM concentration of CS-IONPs, while there was no significant change in LDH release with PAA-coated IONPs at any concentration. This study reveals that IONPs coated with a biocompatible polymer, which are usually assumed to be nontoxic, show cytotoxicity with increasing concentration and incubation time. The cytotoxicity of nanoparticles intended for biomedical purposes must be evaluated using more than one approach.

Key words: Iron oxide nanoparticles, poly(acrylic acid), chitosan, human osteoblast-like cell line, cytotoxicity

Received: 22.08.2016 Accepted/Published Online: 07.12.2016 Final Version: 20.04.2017

Research Article

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Synthetic and natural polymers are widely used in the biomedical field for different purposes. In this study, IONPs were coated by both a synthetic polymer (poly(acrylic acid): PAA) and a natural polymer (chitosan: CS) to investigate the effects of different concentrations of coated IONPs on in vitro cultures of human osteoblast-like cells (SaOs-2). CS is an attractive biopolymer for biomedical applications because chitin, which is the natural source of CS, is the second-most abundant polysaccharide on earth after cellulose and it is highly biocompatible and biodegradable by human enzymes (Bhattarai et al., 2010). Fibrous CS scaffolds, in a biomineralized form, have high biocompatibility with SaOs-2 cells, allow cell attachment and extracellular matrix formation, and cause very low (<10%) apoptotic or necrotic effect on these cells (Ucar et al., 2016). CS is particularly attractive for drug conjugates and targeted delivery applications (particularly for anionic drugs), since its chemistry allows it to be easily modified and functionalized in order to incorporate specificity and selectivity. CS-conjugated NPs were recently shown to target osteosarcoma (SaOs-2) cells in vitro effectively through cancer-specific integrin receptors (Mansur et al., 2016). CS-based NPs, as carriers for ursolic acid (Zhang et al., 2015) and retinoic acid (Qin et al., 2015), were shown to inhibit proliferation of osteosarcoma (SaOs-2, U2OS, and MG-63) cells due to a decrease in membrane potential and release of cytochrome C as well as cell cycle arrest. CS was also used for encapsulation and slow release of c-jun (an oncogene) DNAzymes to induce apoptotic cell death in SaOs-2 cells (Dass et al., 2008).

These properties make CS a good candidate for coating IONPs. Even though CS is a biocompatible polymer, an additional modification of CS is needed to make water-soluble composites (Li et al., 2012). Thus, another biocompatible biopolymer, PAA (Tripathi et al., 2016), was chosen to make the IONPs both biocompatible and well dispersed in culture medium without additional chemical treatment. PAA is a well-characterized weak polyanion. Besides its water solubility, PAA is FDA-approved as an inactive ingredient (as a stabilizer and thickener to tune rheological properties) in drug formulations, and it is used clinically for oral, mucosal, and even ocular delivery of drugs due to its safety in pharmaceutical applications (Kadajji and Betageri, 2011; Morton et al., 2014). PAA, in a side-chain functionalized form with alendronate (a clinically used bisphosphonate), binds and is rapidly internalized by human osteosarcoma (143B) cells (Morton et al., 2014).

Currently, no universally accepted procedure to investigate the cytotoxicity of NPs is available. Cytotoxicity evaluation of NPs by a single analysis method can lead to false negative or false positive results due to the fact that NPs can affect animal cells via different pathways, e.g., the

destruction of the membrane structure or the changing of the mechanism of the intracellular pathway. Cell–nanoparticle interaction is a dynamic and complicated process, which includes several known and unknown parameters, such as interactions of nanoparticles with the cell membrane, culture media components, cytoplasmic fluids, or DNA as well as interactions between the culture media components and coating materials. It has been shown that naked IONPs could lead to cytotoxicity in animal cells above a certain concentration (Hussain et al., 2005; Ankamwar et al., 2010; Soenen et al., 2011). Therefore, it is a common practice to coat IONPs with biocompatible polymers to reduce their cytotoxicity (Mura et al., 2011; Bahring et al., 2013; Grabowski et al., 2015).

In this study, it is hypothesized that IONPs can be cytotoxic even after being coated by biopolymers that are assumed to be biocompatible. Therefore, cytotoxicity of IONPs must be examined via different analysis methods if they are intended for use in medical procedures. The hypothesis was tested by investigation of the biocompatibility of CS- and PAA-coated IONPs (CS-IONPs and PAA-IONPs, respectively) in vitro and by comparing the results of two different cytotoxicity assays, lactate dehydrogenase (LDH) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT).

2. Materials and methodsIn order to determine the cytotoxic effects of CS- and PAA-IONPs on the SaOs-2 cell line, the cells were subjected to various concentrations of IONPs (10, 100, and 1000 µM) for 24 and 72 h. Two different analysis methods, MTT and LDH, were used for determining cytotoxicity. Since serum proteins affect the results, the assay medium must include 1% serum for LDH analysis. MTT was also examined with 1% serum-containing media in addition to the standard serum concentration (10%) to compare the results properly. The morphology of the cells and the dispersion of the IONPs were observed with inverted microscopy, scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX) techniques.

Independent variables in the experimental design were the concentration of the IONPs, the coating materials, and IONP exposure time. Dependent variables were the cell viability and membrane integrity.2.1. Cell cultureThe SaOs-2 cell line was obtained from HUKUK (Culture Collection of Animal Cells, Foot and Mouth Disease Institute, Ankara, Turkey). The cells were used between passages 5 and 10 and were cultured at 37 °C in an atmosphere of 5% CO2 in a humidified incubator. The culture medium was Dulbecco’s modified Eagle’s medium with Ham’s F-12 (1:1) (HyClone DMEM/F-12; GE Healthcare Lifesciences, Marlborough, MA, USA)

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supplemented with 10% (v/v) fetal bovine serum (FBS; Biochrom, Berlin, Germany) and 100 U/mL penicillin and 100 µg/mL streptomycin (Biochrom). The culture medium was replaced with fresh medium every 2–3 days; cells were passaged before reaching 85% confluence, and split into 1/3 in tissue culture dishes by lifting with trypsin-EDTA solution (0.05% trypsin, 0.02% EDTA; Sigma-Aldrich, St. Louis, MO, USA). 2.2. Treatment of SaOs-2 cells with IONPsCS-IONPs and PAA-IONPs were kindly provided by Dr Serhat Küçükdermenci from the Ege University Department of Electrical and Electronics Engineering. Küçükdermenci et al. described the production method and characterization results in their study (2013). Briefly, in their polyol method, NaOH, diethylene glycol, biopolymers, and FeCl3 were vigorously mixed at a high temperature under nitrogen, and IONPs were obtained after washing, sedimentation, and drying steps. The average size of the IONPs was approximately 10 nm.

SaOs-2 cells were plated at a density of 1 × 104 cells/well in 96-well plates (Greiner Bio-One; Frickenhausen, Germany) for MTT (Sigma-Aldrich) and LDH cytotoxicity (Roche; Stockholm, Sweden) assays. To determine cellular uptake and morphological changes (if any) with SEM, the cells were plated at a density of 2 × 105 cells/well in 12-well plates (Greiner Bio-One) on glass coverslips. After 24 h, cells were washed with PBS and the media were replaced with fresh media containing PAA- and CS-IONPs in different concentrations (10, 100, and 1000 µM). Media were supplemented with 1% and 10% FBS for MTT assay and 1% FBS for LDH assay. MTT and LDH assays were performed after periods of 24 and 72 h. 2.3. MTT assayViability of SaOs-2 cells after exposure to IONPs was assessed by MTT assay. After 24 and 72 h of treatment, the culture medium was replaced with fresh, serum-free medium containing 10% MTT solution, and the cells were incubated for 3 h at 37 °C in 5% CO2 atmosphere until the yellow MTT tetrazolium salt was reduced to purple formazan crystals by mitochondrial succinate dehydrogenase. Formazan crystals were solubilized with dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany), and the absorbance of each well was read on a microplate reader (Spectramax; Molecular Devices, Sunnyvale, CA, USA) at 570–690 nm. 2.4. LDH assayLDH is a cytoplasmic enzyme that leaks into the culture medium when plasma membrane integrity is disrupted. The amount of LDH released into the culture medium is related to cytotoxicity due to cell membrane disruption. After 24 and 72 h of IONP exposure, the 96-well plate was centrifuged to settle the nanoparticles and the cells in the medium. Supernatant (50 µL) was transferred to a fresh

well in a 96-well plate and 50 µL of serum-free medium was added for dilution. The reaction mixture (Cytotoxicty Detection Kit Plus (LDH), Roche) was added (100 µL) to the supernatant and incubated for 30 min at 25 °C. After incubation, 50 µL of stop solution was added and the absorbance of each well was read on a microplate reader at 490–690 nm.2.5. Sample preparation for SEM and EDXThe samples were prepared for SEM and EDX analysis after 3 days of IONP exposure to determine morphological changes of cells (if any), perform elemental analysis on cell membranes, and observe cellular IONP uptake. Briefly, the samples were washed with PBS and then kept in 0.1 M sodium cacodylate (Sigma-Aldrich) and 5% (v/v) glutaraldehyde (Merck) for 30 min on ice. After glutaraldehyde fixation, the samples were kept in 7% (w/v) sucrose (Sigma-Aldrich) in 1 M sodium cacodylate and in 2% (w/v) osmium tetroxide (Sigma-Aldrich) in 1 M sodium cacodylate for periods of 30 min on ice. The cells were then washed two times with distilled water, serially dehydrated in ethanol at room temperature (25 °C), and kept in hexamethyldisilazane (HMDS, Sigma-Aldrich) solution for 5 min. After removal of the HMDS, the samples were air-dried at room temperature. The samples were stored in a desiccator after the completion of drying. Before the SEM examination, samples were sputter-coated with gold-palladium.2.6. Statistical analysisAll values presented in this study are represented as means ± standard deviation of experiments made with five replicates. Statistical significance was determined by one-way analysis of variance (ANOVA). Multiple comparisons were analyzed using the Tukey post hoc method. Statistical significance was described at P < 0.05.

3. Results3.1. Cell cultureSaOs-2 cells show epithelial-like morphology. The morphology of the cells and agglomeration of the CS-IONPs after treatment with IONPs are shown in Figure 1. Control cells were grown in the same medium with experimental groups but were not treated with IONPs. PAA-IONPs cannot be seen in the images, since they are well dispersed in the medium. This is expected, because PAA has been used as a coating agent to control dispersibility before (Jeong et al., 2010; Huang et al., 2014), and the high dispersibility of PAA-IONPs in water makes them good candidates for dispersion of the nonsoluble nanoparticles in water-based media. Shen et al. reported that coating Ag NPs with PAA preserves and enhances the stability of NPs, and they called this phenomenon a “cage effect” (Shen et al., 2007). However, CS-IONP clusters can be seen in Figure 1, since CS-IONPs were not dispersed

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Figure 1. Inverted microscope images of control cells and PAA- and CS-IONP-treated SaOs-2 cells. Arrows show clustered NPs. Scale bars correspond to 100 µm.

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well in cell culture media. CS has a cationic structure due to its amino groups (Fang and Zhang, 2009; Coelho et al., 2010; Kim, 2013), and a hydrogen bond occurs between the hydrogen in the amino group and oxygen in Fe3O4 (Hong et al., 2010). The –OH groups that remain unbound and give CS its typical cationic nature are expected to act as a means for IONPs to repel each other, and prevent agglomeration. However, in this case, this positive charge was possibly hindered by the ionic microenvironment of the culture medium and could not prevent agglomeration (Hussain et al., 2009). 3.2. Cell viability Formazan crystals formed by MTT were dissolved in DMSO and quantified by measuring the absorbance of the solution at 570–690 nm. The absorbance values are proportional to the number of viable cells. Results show that cytotoxicity of the IONPs is more significant in the culture medium supplemented with lower serum concentration (1% FBS) (Figure 2). SaOs-2 cell viability was lower than that of the control at all concentrations of PAA-IONPs and further decreased with increasing amount of PAA-IONPs in both serum concentrations, except at 10 µM

on day 3 (Figures 2 and 3). CS-IONPs showed the same effect as PAA-IONPs; however, especially in the 10% FBS-supplemented medium, the effect did not dramatically increase with increasing CS-IONP concentration.

PAA-IONPs had a reduced cytotoxic effect compared to CS-IONPs at a concentration of 10 µM, but higher concentrations enhanced the decrease in cell viability. 3.3. Cell membrane integrityDisruption of cell membrane integrity is proportional to the amount of LDH that leaks from the cytoplasm. Figure 4 shows the effect of nanoparticles on cell membrane integrity after 24 and 72 h of exposure. As opposed to the MTT results, PAA-IONPs did not show any significant LDH leakage at any concentration over 72 h of incubation. On the contrary, there was an increase in LDH release levels of CS-IONP groups at both 10 and 100 µM concentrations at both time points, and this increase was more substantial at the 1000 µM concentration. 3.4. SEM and EDX analysisCell morphology and dispersion of IONPs were analyzed by SEM (Quanta 250 FEG, FEI, Hillsboro, OR, USA) after 72 h of exposure (Figure 5). Elemental analyses of the

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Figure 2. MTT graphics of SaOs-2 cells in the culture medium supplemented by 1% FBS and different concentrations of IONPs (10, 100, and 1000 µM): A) day 1, B) day 3. Asterisk indicates statistically significant differences between the data set and the control: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. MTT images of SaOs-2 cells in culture medium supplemented by 10% FBS and different concentrations of IONPs (10, 100, and 1000 µM): A) day 1, B) day 3. Asterisk indicates statistically significant differences between the data set and the control: *P < 0.05, **P < 0.01, ***P < 0.001.

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cell surface and IONPs for both experimental groups are shown in Figure 6. According to EDX results, bright parts of the cells indicate IONPs. Spectra 1 and 5, which were taken from dark regions, have significantly lower amounts of Fe (0.04% and 0.27% wt., respectively) than spectra 2 and 6 (7.88% and 6.21% wt., respectively), which were taken from bright regions (Table).

SEM images show that the morphology of the SaOs-2 cells treated with IONPs is not different from that of the control group. However, CS-IONP clusters and rings are observed clearly on the cell membranes, especially around the nucleus.

4. DiscussionThe results indicate that, on the IONPs tested, the CS coating induces a greater cytotoxic effect on SaOs-2 cells than the PAA coating. As seen in Figures 2 and 3, both CS and PAA have similar effects on the SaOs-2 cell line: both coatings decrease cell viability, particularly at lower serum concentrations, and the increasing concentration of IONPs decreases cell viability further. However, Figure 4 indicates that coating material is very important for the preservation of membrane integrity. High amounts of LDH

were released from cells treated with CS-IONPs, while cells treated with PAA-IONPs were not significantly affected compared to the control. The effects seem to be through the inhibition of cell proliferation, rather than decreased cell viability or disruption of membrane integrity for PAA-IONPS, while the CS coating definitely causes disrupted cell membrane integrity in IONPs. Toxicity levels increased as the NP concentration increased for both coating types. It was shown before that increasing IONP concentration in culture medium results in a decreased cell proliferation rate (doubling time). Particularly, high levels of IONP localization around a nucleus diminish the efficiency of protein expression and hinder actin maturation (Soenen et al., 2010). Similar localization of IONPs is visible in this study for 1000 µM CS-IONPs, which also showed the highest level of toxicity in both MTT and LDH tests. Although the effect mechanisms of nanoparticles have not been determined clearly yet, some studies indicate that NP–cell interaction can be affected via NP–culture media interaction and properties of NPs can be altered by culture media composition (Mahmoudi et al., 2010; Mura et al., 2011).

FBS is used to supplement culture media for in vitro tests to support cell proliferation and maintenance. Serum is animal-derived and has variable chemical composition, in which several proteins, amino acids, growth factors, trace elements, vitamins, etc. are found (van der Valk et al., 2010). It has been reported that CS-coated NPs form a bridge between NPs and cells because plasma proteins are adsorbed onto NPs (Wang et al., 2011). Several authors have shown that positively charged serum proteins are adsorbed on positively charged NPs, which often causes a shielding or protective effect on cell viability (Nafee et al., 2009; Schulze et al., 2009). These high-affinity proteins constitute the first layer of the biocorona, usually called the “hard corona” (Frohlich et al., 2012; Tenzer et al., 2013; Au et al., 2016), and this corona basically determines the binding/internalization, as well as the following functionality of the NP. The significant cytotoxic effect of CS-IONPs can be explained by the high protein-absorbing capacity of CS-coated surfaces, which might alter the conformation of absorbed proteins and lead to the disruption of physiological processes (Kim et al., 2013; Xu et al., 2016).

Although CS is a commonly used biocompatible polymer, the vast majority of papers testing CS cytotoxicity are based on cases where CS is not internalized by the cells. However, as shown in the study by Petri-Fink et al. (2005), due to the interaction of the amino groups on the surface of CS with the negatively charged domains on cell membranes, CS-IONPs pass through the cell membrane. Cationic NPs were shown to have enhanced cellular uptake compared to anionic or neutral NPs and to induce

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Figure 4. LDH images of SaOs-2 cells in the culture medium supplemented by 1% FBS and different concentrations of IONPs (10, 100, and 1000 mM): A) day 1, B) day 3. Asterisk indicates statistically significant differences between the data set and the control: * P < 0.05, ***P < 0.001.

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more ROS and mitochondrial and lysosomal damage (Mahmoudi et al., 2011a; Liu et al., 2013; Calatayud et al., 2014). They were also shown to cause more disruption

of plasma membrane integrity and more hole formation, particularly in nonphagocytic cells (Nafee et al., 2009; Frohlich, 2012; Carvalho et al., 2016).

Figure 5. SEM images of the SaOs-2 cells.

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CS-IONPs also showed significantly higher LDH leakage, particularly at 1000 µM, than the PAA-IONPs and controls. The membrane integrity of the cells treated with anionic PAA-IONPs was not affected. It has been well documented in the literature that NPs can cause structural damage to cells, and the extent of leakage is determined by the surface chemistry of the NPs (Li and Gu, 2010; Lin et al., 2010). It has been shown that cationic NPs show higher cytotoxicity compared to their neutral and anionic analogues, since they interact electrostatically with the negatively charged lipid bilayer and depolarize the cell membrane (Goodman et al., 2004; Calatayud et al., 2014). Cationic NPs also stimulate the Ca2+ influx through the membrane (Hwang et al., 2015). This change in membrane potential and Ca2+ influx enhances the internalization of NPs with the cost of increased toxicity.

The cytotoxic effect of CS-IONPs in both MTT and LDH assays can be explained by these phenomena: 1) cell proliferation could be inhibited or the proliferation rate could be decreased since the proteins carried inside the cytoplasm by the aid of the CS coating have altered conformations that might have detrimental effects on various physiological processes, and 2) cell membrane integrity could be disrupted because of the free positive charge of the CS coating.

The most explicit limitation of working with IONPs is that there is still no universally accepted method to investigate the possible cytotoxicity of these materials because cellular responses can be changed via several different pathways. Size, chemical structure, and physical and surface properties, as well as their interactions with culture media, could easily affect the results. As this paper clearly

Figure 6. EDX results of cell surface and IONPs: A) PAA- and B) CS-coated.

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indicates, PAA-IONPs do not show statistically significant cytotoxicity according to LDH results in Figure 2, but it has also been shown that they could decrease cell proliferation, even when they are used in lower concentrations (Figure 1). Using just one method could lead to false negative or false positive results. Existing methods focused on revealing only one cellular response remain insufficient for cytotoxicity evaluation. To overcome this issue, comprehensive studies have to be carried out with different cell lines and NPs with a wide range of concentration and sizes. Developing a standard and universally accepted method may not be achievable immediately, but consideration of these issues while working with IONPs could help prevent fluctuation of results among different groups.

These results emphasize the need for standardized test methods for preclinical testing of NPs. Biomaterials that are generally considered safe for clinical use, like CS, can induce significant cytotoxic effects when used as NP-coating layers. This fact curtails their potential in drug delivery systems for regenerative and/or therapeutic purposes, while it can be exploited as an advantage when targeting cancerous tissues.

Acknowledgment We thank Assist Prof Dr Serhat Küçükdermenci for supplying the PAA- and CS-IONPs.

Table. Quantitative results of EDX analysis.

Element Spectrum 1 Spectrum 2 Spectrum 5 Spectrum 6

Wt.% Atomic% Wt.% Atomic% Wt.% Atomic% Wt.% Atomic%

C 50.28 61.42 42.28 55.29 42.09 54.16 61.07 47.77

O 33.44 30.67 35.71 35.06 35.81 34.59 29.15 30.38

Na 1.79 1.14 1.44 0.98 2.35 1.58 1.30 1.95

Al 0.77 0.42 0.69 0.40 1.04 0.59 0.43 0.76

Si 10.00 5.22 8.96 5.01 13.51 7.43 9.55 5.22

P 0.00 0.00 0.12 0.06 0.00 0.00 0.15 0.07

K 1.74 0.65 1.51 0.61 2.44 0.97 1.67 0.65

Ca 0.18 0.07 0.17 0.07 0.07 0.03 0.10 0.04

Fe 0.04 0.01 7.88 2.22 0.27 0.07 6.21 1.71

Zn 1.77 0.40 1.24 0.30 2.41 0.57 1.47 0.34

Total 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

References

Ankamwar B, Lai T, Huang J, Liu R, Hsiao M, Chen CH, Hwu Y (2010). Biocompatibility of Fe3O4 nanoparticles evaluated by in vitro cytotoxicity assays using normal, glia and breast cancer cells. Nanotechnology 21: 075102.

Au JL, Yeung BZ, Wientjes MG, Lu Z, Wientjes MG (2016). Delivery of cancer therapeutics to extracellular and intracellular targets: determinants, barriers, challenges and opportunities. Adv Drug Deliver Rev 97: 280-301.

Bahring F, Schlenk F, Wotschadlo J, Buske N, Liebert T, Bergemann C, Heinze T, Hochhaus A, Fischer D, Clement JH (2013). Suitability of viability assays for testing biological effects of coated superparamagnetic nanoparticles. IEEE T Magn 49: 383-388.

Bao Y, Wen T, Samia ACS, Khandhar A, Krishnan KM (2016). Magnetic nanoparticles: material engineering and emerging applications in lithography and biomedicine. J Mater Sci 51: 513-553.

Bayir E, Bilgi E, Urkmez AS (2014). Implementation of nanoparticles in cancer therapy. In: Bououdina M, editor. Handbook of Research on Nanoscience, Nanotechnology, and Advanced Materials. 1st ed. Hershey, PA, USA: IGI Global, p. 447-491.

Bhattarai N, Gunn J, Zhang M (2010). Chitosan-based hydrogels for controlled, localized drug delivery. Adv Drug Deliver Rev 62: 83-99.

ŞENDEMİR ÜRKMEZ et al. / Turk J Biol

331

Calatayud MP, Sanz B, Raffa V, Riggio C, Ibarra MR, Goya GF (2014). The effect of surface charge of functionalized Fe3O4 nanoparticles on protein adsorption and cell uptake. Biomaterials 35: 6389-6399.

Carvalho SM, Mansur HS, Ramanery FP, Mansur AAP, Lobato ZIP, Leite MF (2016). Cytotoxicity investigation of luminescent nanohybrids based on chitosan and carboxymethyl chitosan conjugated with Bi2S3 quantum dots for biomedical applications. Toxicol Res 5: 1017-1028.

Coelho JF, Ferreira PC, Alves P, Cordeiro R, Fonseca AC, Góis JR, Gil MH (2010). Drug delivery systems: Advanced technologies potentially applicable in personalized treatments. EPMA J 1: 164-209.

Dass CR, Friedhuber AM, Khachigian LM, Dunstan DE, Choong PF (2008). Biocompatible chitosan-DNAzyme nanoparticle exhibits enhanced biological activity. J Microencapsul 25: 421-425.

Fang C, Zhang M (2009). Multifunctional magnetic nanoparticles for medical imaging applications. J Mater Chem 19: 6258-6266.

Frohlich E (2012). The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int J Nanomed 7: 5577-5591.

Frohlich E, Meindl C, Roblegg E, Griesbacher A, Pieber TR (2012). Cytotoxicity of nanoparticles is influenced by size, proliferation and embryonic origin of the cells used for testing. Nanotoxicology 6: 424-439.

Goodman CM, McCusker CD, Yilmaz T, Rotello VM (2004). Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjugate Chem 15: 897-900.

Grabowski N, Hillaireau H, Vergnaud J, Tsapis N, Pallardy M, Kerdine-Römer S, Fattal E (2015). Surface coating mediates the toxicity of polymeric nanoparticles towards human-like macrophages. Int J Pharm 482: 75-83.

Gudovan D, Balaure PC, Eduard Mihaiescu D, Fudulu A, Radu M (2015). Functionalized magnetic nanoparticles for biomedical applications. Curr Pharm Design 21: 6038-6054.

Gupta AK, Gupta M (2005). Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 26: 3995-4021.

Hong S, Chang Y, Rhee I (2010). Chitosan-coated ferrite (Fe3O4) nanoparticles as a T2 contrast agent for magnetic resonance imaging. J Korean Phys Soc 56: 868-873.

Huang Q, Shen W, Xu Q, Tan R, Song W (2014). Properties of polyacrylic acid-coated silver nanoparticle ink for inkjet printing conductive tracks on paper with high conductivity. Mater Chem Phys 147: 550-556.

Hussain S, Hess K, Gearhart J, Geiss K, Schlager J (2005). In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In Vitro 19: 975-983.

Hussain SM, Braydich-Stolle LK, Schrand AM, Murdock RC, Yu KO, Mattie DM, Schlager JJ, Terrones M (2009). Toxicity evaluation for safe use of nanomaterials: Recent achievements and technical challenges. Adv Mater 21: 1549-1559.

Hwang TL, Aljuffali IA, Lin CF, Chang YT, Fang JY (2015). Cationic additives in nanosystems activate cytotoxicity and inflammatory response of human neutrophils: Lipid nanoparticles versus polymeric nanoparticles. Int J Nanomed 10: 371.

Jeong S, Song HC, Lee WW, Choi Y, Lee SS, Ryu BH (2010). Combined role of well-dispersed aqueous Ag ink and the molecular adhesive layer in inkjet printing the narrow and highly conductive Ag features on a glass substrate. J Phys Chem C 114: 22277-22283.

Kadajji VG, Betageri GV (2011). Water soluble polymers for pharmaceutical applications. Polymers 3: 1972-2009.

Kaushik A, Khan R, Solanki PR, Pandey P, Alam J, Ahmad S, Malhotra BD (2008). Iron oxide nanoparticles–chitosan composite based glucose biosensor. Biosens Bioelectron 24: 676-683.

Kim SK (2013) Chitin and Chitosan Derivatives: Advances in Drug Discovery and Developments. Boca Raton, FL, USA: CRC Press.

Kim ST, Saha K, Kim C, Rotello VM (2013). The role of surface functionality in determining nanoparticle cytotoxicity. Accounts Chem Res 46: 681-691.

Küçükdermenci S, Kutluay D, Avgın İ (2013). Synthesis of a Fe3O4/PAA-based magnetic fluid for faraday-rotation measurements. Mater Tehnol 47: 71-78.

Lauvrak SU, Munthe E, Kresse SH, Stratford EW, Namlos HM, Meza-Zepeda LA, Myklebost O (2013). Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes. Brit J Cancer 109: 2228-2236.

Li GY, Jiang YR, Huang KL, Ding P, Chen J (2008). Preparation and properties of magnetic Fe3O4–chitosan nanoparticles. J Alloy Compd 466: 451-456.

Li X, Kong X, Zhang Z, Nan K, Li L, Wang X, Chen H (2012). Cytotoxicity and biocompatibility evaluation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery application. Int J Biol Macromol 50: 1299-1305.

Li Y, Gu N (2010). Thermodynamics of charged nanoparticle adsorption on charge-neutral membranes: a simulation study. J Phys Chem B 114: 2749-2754.

Lin J, Zhang H, Chen Z, Zheng Y (2010). Penetration of lipid membranes by gold nanoparticles: Insights into cellular uptake, cytotoxicity, and their relationship. ACS Nano 4: 5421-5429.

Ling D, Hyeon T (2013). Chemical design of biocompatible iron oxide nanoparticles for medical applications. Small 9: 1450-1466.

Liu X, Huang N, Li H, Jin Q, Ji J (2013). Surface and size effects on cell interaction of gold nanoparticles with both phagocytic and nonphagocytic cells. Langmuir 29: 9138-9148.

Mahdavi M, Ahmad MB, Haron MJ, Namvar F, Nadi B, Rahman MZA, Amin J (2013). Synthesis, surface modification and characterisation of biocompatible magnetic iron oxide nanoparticles for biomedical applications. Molecules 18: 7533-7548.

ŞENDEMİR ÜRKMEZ et al. / Turk J Biol

332

Mahmoudi M, Laurent S, Shokrgozar MA, Hosseinkhani M (2011a). Toxicity evaluations of superparamagnetic iron oxide nanoparticles: cell “vision” versus physicochemical properties of nanoparticles. ACS Nano 5: 7263-7276.

Mahmoudi M, Sant S, Wang B, Laurent S, Sen T (2011b). Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy. Adv Drug Deliver Rev 63: 24-46.

Mahmoudi M, Simchi A, Imani M, Shokrgozar MA, Milani AS, Häfeli UO, Stroeve P (2010). A new approach for the in vitro identification of the cytotoxicity of superparamagnetic iron oxide nanoparticles. Colloid Surface B 75: 300-309.

Mansur AA, de Carvalho SM, Mansur HS (2016). Bioengineered quantum dot/chitosan-tripeptide nanoconjugates for targeting the receptors of cancer cells. Int J Biol Macromol 82: 780-789.

Mikhaylova M, Kim DK, Bobrysheva N, Osmolowsky M, Semenov V, Tsakalakos T, Muhammed M (2004). Superparamagnetism of magnetite nanoparticles: dependence on surface modification. Langmuir 20: 2472-2477.

Mohseny AB, Machado I, Cai Y, Schaefer KL, Serra M, Hogendoorn PC, Llombart-Bosch A, Cleton-Jansen AM (2011). Functional characterization of osteosarcoma cell lines provides representative models to study the human disease. Lab Invest 91: 1195-1205.

Morton SW, Shah NJ, Quadir MA, Deng ZJ, Poon Z, Hammond PT (2014). Osteotropic therapy via targeted layer-by-layer nanoparticles. Adv Healthc Mater 3: 867-875.

Mura S, Hillaireau H, Nicolas J, Le Droumaguet B, Gueutin C, Zanna S, Tsapis N, Fattal E (2011). Influence of surface charge on the potential toxicity of PLGA nanoparticles towards Calu-3 cells. Int J Nanomed 6: 2591-2605.

Nafee N, Schneider M, Schaefer UF, Lehr CM (2009). Relevance of the colloidal stability of chitosan/PLGA nanoparticles on their cytotoxicity profile. Int J Pharm 381: 130-139.

Petri-Fink A, Chastellain M, Juillerat-Jeanneret L, Ferrari A, Hofmann H (2005). Development of functionalized superparamagnetic iron oxide nanoparticles for interaction with human cancer cells. Biomaterials 26: 2685-2694.

Qin YG, Zhu LY, Wang CY, Zhang BY, Wang QY, Li RY, Liu Z (2015). Glycol chitosan incorporated retinoic acid chlorochalcone (RACC) nanoparticles in the treatment of osteosarcoma. Lipids Health Dis 14: 70.

Schulze C, Schulze C, Kroll A, Schulze C, Kroll A, Lehr CM, Schäfer UF, Becker K, Schnekenburger J, Schulze Isfort C et al. (2009). Not ready to use – overcoming pitfalls when dispersing nanoparticles in physiological media. Nanotoxicology 2: 51-61.

Shen Z, Duan H, Frey H (2007). Water-soluble fluorescent Ag nanoclusters obtained from multiarm star poly(acrylic acid) as “molecular hydrogel” templates. Adv Mater 19: 349-352.

Soenen SJ, Himmelreich U, Nuytten N, De Cuyper M (2011). Cytotoxic effects of iron oxide nanoparticles and implications for safety in cell labelling. Biomaterials 32: 195-205.

Soenen SJ, Nuytten N, De Meyer SF, De Smedt SC, De Cuyper M (2010). High intracellular iron oxide nanoparticle concentrations affect cellular cytoskeleton and focal adhesion kinase-mediated signaling. Small 6: 832-842.

Susa M, Iyer AK, Ryu K, Hornicek FJ, Mankin H, Amiji MM, Duan Z (2009). Doxorubicin loaded polymeric nanoparticulate delivery system to overcome drug resistance in osteosarcoma. BMC Cancer 9: 399.

Tenzer S, Docter D, Kuharev J, Musyanovych A, Fetz V, Hecht R, Schlenk F, Fischer D, Kiouptsi K, Reinhardt C et al. (2013). Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 8: 772-781.

Tripathi SK, Ahmadi Z, Gupta KC, Kumar P (2016). Polyethylenimine-polyacrylic acid nanocomposites: type of bonding does influence the gene transfer efficacy and cytotoxicity. Colloid Surface B 140: 117-120.

Ucar S, Ermis M, Hasirci N (2016). Modified chitosan scaffolds: proliferative, cytotoxic, apoptotic, and necrotic effects on SaOs-2 cells and antimicrobial effect on Escherichia coli. J Bioact Compat Pol 31: 304-319.

Van der Valk J, Brunner D, De Smet K, Fex Svenningsen Å, Honegger P, Knudsen LE, Lindl T, Noraberg J, Price A, Scarino ML et al. (2010). Optimization of chemically defined cell culture media – replacing fetal bovine serum in mammalian in vitro methods. Toxicol In Vitro 24: 1053-1063.

Wahajuddin SA (2012). Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers. Int J Nanomed 7: 3445.

Wang JJ, Zeng ZW, Xiao RZ, Xie T, Zhou GL, Zhan XR, Wang SL (2011). Recent advances of chitosan nanoparticles as drug carriers. Int J Nanomed 6: 765-774.

Xu Y, Sherwood JA, Lackey KH, Qin Y, Bao Y (2016). The responses of immune cells to iron oxide nanoparticles. J App Toxicol 36: 543-553.

Zhang X, Lu X, Geng W, Qu G, Zhou Z, Jiang L, Li Y, Chen X, Nie L (2015). Role of glycol chitosan-incorporated ursolic acid nanoparticles in the treatment of osteosarcoma. Trop J Pharm Res 14: 1581-1588.