Rhamnose-coated superparamagnetic iron ... - Wiley Online Library

Research article Received: 14 October 2015,

Revised: 5 November 2015,

Accepted: 6 November 2015

Published online in Wiley Online Library: 28 December 2015

(wileyonlinelibrary.com) DOI 10.1002/jat.3273

Rhamnose-coated superparamagnetic iron-oxide nanoparticles: an evaluation of their in vitro cytotoxicity, genotoxicity and carcinogenicity Alessandro Paolinia,b*, Constança Porredon Guarchc, David Ramos-Lópezc, Joaquín de Lapuentec, Alessandro Lascialfarid, Yannick Guarie, Joulia Larionovae, Jerome Longe and Rosanna Nanob ABSTRACT: Tumor recurrence after the incomplete removal of a tumor mass inside brain tissue is the main reason that scientists are working to identify new strategies in brain oncologic therapy. In particular, in the treatment of the most malignant astrocytic tumor glioblastoma, the use of magnetic nanoparticles seems to be one of the most promising keys in overcoming this problem, namely by means of magnetic fluid hyperthermia (MFH) treatment. However, the major unknown issue related to the use of nanoparticles is their toxicological behavior when they are in contact with biological tissues. In the present study, we investigated the interaction of glioblastoma and other tumor cell lines with superparamagnetic iron-oxide nanoparticles covalently coated with a rhamnose derivative, using proper cytotoxic assays. In the present study, we focused our attention on different strategies of toxicity evaluation comparing different cytotoxicological approaches in order to identify the biological damages induced by the nanoparticles. The data show an intensive internalization process of rhamnose-coated iron oxide nanoparticles by the cells, suggesting that rhamnose moiety is a promising biocompatible coating in favoring cells’ uptake. With regards to cytotoxicity, a 35% cell death at a maximum concentration, mainly as a result of mitochondrial damages, was found. This cytotoxic behavior, along with the high uptake ability, could facilitate the use of these rhamnose-coated iron-oxide nanoparticles for future MFH therapeutic treatments. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: iron-oxide nanoparticles; cytotoxicity; genotoxicity; carcinogenicity; hyperthermia treatment; iron content

Introduction

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Among brain neoplasms, astrocytic tumors are a class of malignant cells for which treatments appear to be one of the biggest challenges in the field of oncology. Glioblastoma multiforme (GBM) tumors represent 40% of all primary brain tumors, 60% of all astrocytic tumors and 78% of malignant tumors of the central nervous system (CNS) (Miller and Perry, 2007). Glioblastoma is predominately found in the male population, is diagnosed mainly in 60 and 70 year olds, although patients of all ages can be affected by this tumor (Kleihues and Cavenee, 2000), and congenital cases are very rare. The World Health Organization (WHO) considers glioblastoma one of the most aggressive tumors classifying it as a grade IV tumor. Actually, conventional therapies such as surgical resection, radiotherapy and chemotherapy, although approved treatments for malignant astrocytomas, so far have not produced significant results in improving the survival of patients, therefore, researchers are exploring new and alternative horizons (Kilic et al., 2000). The main reason of the failure of the conventional therapies is the impossibility to eradicate completely the tumor mass. Glioblastoma, in particular, not only shows resistance to the post-surgical treatments but is also characterized by a high infiltrative ability, which leads to the generation of relapses (Sipos et al., 2002).

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In this context, in recent years, a great deal of attention has been given to possible new therapeutic strategies that, in combination or in place of conventional therapies, may offer patients a better quality of life and an increased survival (Kilic et al., 2000). Moreover, additional difficulties encountered in the removal of this type of

* Correspondence to: Alessandro Paolini, Bambino Gesù Children’s Hospital-IRCCS, Gene Expression – Microarrays Laboratory, Viale di San Paolo 15, 00146 Rome, Italy and University of Pavia, Department of Biology and Biotechnology, ’Lazzaro Spallanzani‘, Laboratory of Experimental Neuro-Radio Biology, Via Ferrata 9, 27100 Pavia, Italy. E-mail: [email protected] Bambino Gesù Children’s Hospital-IRCCS, Gene Expression – Microarrays Laboratory, Rome, Italy

a

Department of Biology and Biotechnology ’Lazzaro Spallanzani‘, University of Pavia, Pavia, Italy

b

c

Unit of Experimental Toxicology and Ecotoxicology (UTOX-CERETOX), Barcelona Science Park, Barcelona, Spain

d

Dipartimento di Fisica and INSTM Unit, Università degli Studi di Milano, Milano, Italy

e

ICGM - UMR5253- Equipe IMNO, Université de Montpellier, Montpellier CEDEX 5, France

Copyright © 2015 John Wiley & Sons, Ltd.

Cytotoxicity of rhamnose-coated iron oxide nanoparticles

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The idea to apply MFH to a glioblastoma tumor has induced the necessity to synthesize new magnetic nanoparticles in order to increase the heat efficiency. At the same time, MFH being an new biomedical application, this strategy imposes the toxicological investigations on the nanomaterials, which are necessary to provide the insight in the possibility of in vivo employment of these nanoobjects and their integration into future medical applications. Moreover, they may offer rational approaches for the nanoprobes optimization in order to limit the nanoparticles toxicity. Generally, nanotoxicity denotes to the nano-objects aptitude to disturb the normal physiological processes and/or to interrupt the normal structure of organs and tissues. Several review reports on a current toxicology of frequently used inorganic nanoparticles (iron oxides, gold, quantum dots nanoparticles) state that the nanoparticles toxicity mainly depends on numerous intrinsic parameters, such as the nanoparticles composition, stability, size and shape, surface charge and the nature of the surface coating as well as on administered dose and host immunological integrity (Maurer-Jones et al., 2009; Soenen et al., 2011a, b; Yildirimer et al., 2011). In particular, IONPs classified, by Scientific Commitee on Emerging and Newly Identified Health Risks (SCENIHR, 2009: http://ec. europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o _022.pdf), as new chemical. In addition, the lack of appropriate toxicology testing methods (Kimbrell, 2009), induce the characterization of nanoparticles the most important step in identifying common features that may pose a risk to human health under certain conditions linked to exposure (Oberdörster et al., 2005; Thomas et al., 2006). In the present study, we evaluated the cytotoxic effects of rhamnose-coated iron oxide nanoparticles in two glioblastoma cell lines (T98G and U251), in the human urinary bladder carcinoma cell line (ECV304) and in a fibroblast cell line (BALB/3T3). The choice to use colorimetric approaches allowed us to assess the nanoparticles effects on cell uptake, cytotoxicity, viability and induction of reactive oxygen species (ROS). The use of the different cell types allowed us to obtain an overview on the risk assessment of these IONPs in brain tumors, in other kinds of tumors and in healthy fibroblast.

Materials and methods Nanoparticles synthesis and characterization The rhamnose-coated iron oxide nanoparticles (IONPs) (Fe3O4) of 18 nm were synthesized by using two steps procedure. First, the starting Fe3O4 nanoparticles stabilized by oleic acid were synthesized according to the published procedure (Yu et al., 2004) by thermal decomposition of iron oxide hydroxide in the presence of the oleic acid and the docosane. Second, the water-dispersible rhamnosederivative coated nanoparticles were obtained by exchange of the stabilizing agents according to a previously published procedure (Lartigue et al., 2009; Lartigue et al., 2011) (Fig. 1A). The physicochemical features were characterized by transmission electron microscopy (TEM) and magnetism (Zero Field-Cooled, ZFC, and Field Cooled, FC, magnetization curves). The magnetic measurements were performed using an SQUID-MPMS magnetometer working in the temperature range 1.8 to 350 K up to 7T. In the ZFC experiment, the sample was cooled in the absence of a static magnetic field and the magnetization was then recorded as a function of temperature under a 100 Oe. The FC curve was obtained by recording the magnetization upon cooling under the same magnetic field. Finally, the


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cancer are due to the specific genetic background of each patient, and to the difficulty of some compounds to cross the blood–brain barrier and deliver the drug in the target tissue. Future studies, therefore, will have the aim of developing new therapeutic approaches characterized by the lowest possible toxicity, a high specificity for tumor cells and the ability to significantly decrease the growth and/or recurrence of the neoplastic mass. Many therapeutic approaches are actually being studied and scientific attention has focused not only on the generation of new methods that allow a surgical resection as more complete as possible but also on new technologies using stem cells, immunotherapy, new targeting molecules, new natural anticancer agents and nanoparticles (NPs) (Galanis et al., 2011). In particular, some recent studies have turned the attention on nano-oncology and especially on the use of magnetic nanoparticles for Magnetic Fluid Hyperthermia (MFH) treatment associated with a Magnetic Resonance Imaging (MRI) diagnosis (Gautier et al., 2013). An important advancement in the past two decades has been realized in the design of magnetic nanoprobes for imagery. Indeed, numerous iron oxides nanoparticles, such as dextran-coated superparamagnetic iron oxides (SPIO) and related nanoparticles for instance EndoremTM (Guerbet, BP50400, Cedex, France), also called Feridex, Feraheme® (AMAG Pharmaceuticals, Waltham, MA, USA), Primovist™ and Eovist® (Bayer Schering Pharma AG, Leverkusen, Germany) are under clinical evaluation or already used as contrast agents (CAs) for MRI in current clinical practice, whereas other products as SupravistTM - SHU555C (Bayer Schering Pharma AG), VSOP-C184 (Ferropharm GmbH, Brandenburg, Germany), Ferumoxtran - AMI-227 and Ferumoxide - AMI-25 (AMAG Pharmaceuticals) will enter the market soon (Reimer et al., 1995; Jung and Jacobs, 1995; Josephson et al., 1999; Corot et al., 2006; Geraldes and Laurent, 2009). Such interest in iron oxide nanoparticles lies with the fact that they have historically been considered biocompatible and non-toxic to humans owing to their demonstrated relatively large LD50 and apparent lack of acute toxicity in vivo. However, multiple studies, particularly in vitro, have called these claims into question specifying that the toxicity should be evaluated for each nanoscale system (Leslie-Pelecky and Rieke, 1996; Diehl et al., 2001; Klabunde, 2001; Hyeon, 2003;). In parallel to the design of magnetic nanoparticles for imagery, SPIO nanoparticles could be used in MFH treatment. The simultaneous application of diagnostic tests and therapeutics treatments take the name of theranosis (Choi et al., 2012) that have the main purpose in the development of individually designed therapies against various diseases to accomplish personalized medicine (Thakare et al., 2010). MFH, thanks to the use of Iron Oxide NanoParticles (IONPs) that can act as hyperthermia mediators and MRI contrast agents, allow the tumor mass to be identified (by MRI) and, at the same time, to heat the tumor tissue inducing apoptosis to neoplastic cells. The increase in temperature is due to IONPs which, previously inserted in the cells, release heat as a result of successive applications of an alternating magnetic field (AMF) ( Jordan et al., 2001). The main idea is to heat up the tumor cells, which have been demonstrated to be weakened by heat more efficiently if compared with healthy cells (Cavaliere et al., 1967). An increase in intracellular temperature results in: a reduction of permeability, modification of the function of many enzymatic proteins, structural changes in the membrane potential, alteration of nucleic acid synthesis, inhibition of repair enzymes and conformational change of the DNA with consequent activation of apoptotic mechanisms (Coss and Linnemans, 1996; Lepock, 2003; Wong et al., 2003).

A. Paolini et al.

Figure 1. (A) Sketch representing schematically rhamnose-coated Fe3O4 nanoparticles; (B) transmission electron microscopy (TEM) image of the nanoparticles and the corresponding histograms of their size distribution (inset); (C) ZFC/FC magnetization curves obtained with an applied field of 100 Oe.

IONPs for the toxicity investigations were provided at a stock concentration of 5.2 mg l–1. Cell culture. Human glioblastoma cell lines (T98G and U251MG) and the human urinary bladder carcinoma cell line (ECV304) were obtained from the European Collection of Cell Cultures. All the cell lines were cultured in Eagle’s minimum essential medium (EMEM; Euroclone SpA, Italy) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 units ml–1 penicillin/ streptomycin (Euroclone SpA), 2 mM L-glutamine (Euroclone SpA) and 0.01% sodium pyruvate (Sigma-Aldrich) at 37 °C in an atmosphere of 5% CO2 and 90% humidity. Stock cultures were maintained in exponential growth as monolayers in 75-cm2 plastic tissue-culture flasks (Euroclone SpA). Fibroblast, BALB/3T3 cell line from ATCC was used for the cytotoxicity and genotoxicity tests. Mouse fibroblast contact inhibited the cell line, the Balb/c-3T3 clone A31-1-1 from JCRB Cell Bank was used for the Cell Transformation Assay (CTA). Both cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; SigmaAldrich) supplemented with 10% fetal bovine serum (HyClone, GE Healthcare Life Sciences), 4 mM L-glutamine (Sigma-Aldrich), 0.5% penicillin/streptomycin (Sigma-Aldrich). Cells were maintained at 37 °C in an incubator (ThermoForma) with 5% CO2 and a humid atmosphere in 100 × 15 mm plates (Nunc) for the BALB/3T3 cell line and in 60 × 15 mm plates for the Balb/c-3T3 clone A31-1-1.

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Cell exposure to rhamnose-coated iron nanoparticles. For cell exposure to rhamnose-coated iron-oxide nanoparticles, tumoral cell types were seeded, at different concentrations, and allowed to settle overnight in a humid atmosphere at 37 °C and 5% CO2.


Prior to cell labeling, the nanoparticles were diluted in complete culture medium at the following concentrations: 0, 1, 2, 5, 10, 25, 50 and 100 μgFe ml–1. Different dilutions in culture medium were used depending on the use of 25-cm2 flasks or 96-well plates. As a control, cells were exposed to complete culture media containing no particles. In all experiments, the cells were incubated with nanoparticles for 24 h at 37 °C and 5% CO2. A different rhamnose-coated iron-oxide nanoparticles exposition was performed for fibroblast cell lines which were exposed for 48 h to seven concentrations of IONPs ranging from 1000 to 15.63 μgFe ml–1 with a factor of 2. Intracellular iron content measurement. The colorimetric method was used to study the iron concentration of rhamnose Fe3O4 nanoparticles thanks to the ability of iron to give highly colored complexes when reacted with the thiocyanate iron. For the intracellular iron content quantification, 3 × 105 cells were seeded in 25-cm2 flasks for 24 h and then incubated with nanoparticles at different concentrations. After nanoparticles’ incubation, the cells were washed energetically with 1× phosphate-buffered saline (PBS) (Sigma-Aldrich), harvested by trypsinization and the pellet was incubated with 100 μl 12% HCl solution at 60 °C for 4 h. After incubation, the suspension obtained was centrifuged at 12000 g for 10 min and a volume of 50 μl of supernatant sample was put in a 96-well plate. Each well was incubated with 50 μl of 1% ammonium persulfate (Sigma-Aldrich) and 100 μl of 0.1 M potassium thiocyanate (Sigma-Aldrich) for 5 min to obtain a red color iron-thiocyanate solution. The absorption of the iron-thiocyanate solution was measured at the wavelength of 490 nm by an ELISA plate reader (iMarkTM; Bio-Rad Laboratories, USA).


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Cytotoxicity of rhamnose-coated iron oxide nanoparticles Trypan Blue assay. The estimation of cell viability was performed using Trypan Blue staining in 3 × 105 cells seeded, for 24 h, in 25-cm2 flasks. After 24 h of incubation with nanoparticles, the cells were gently washed with 1× PBS, trypsinized and centrifuged at 460 g for 6 min. The pellet obtained was suspended in 1 ml of medium and a volume of 100 μl was diluted 1:1 with a solution of Trypan Blue 0.5% (Euroclone SpA). The count of cells was performed using a hemocytometer chamber. MTT assessment of cell viability. To measure the cell viability, cells were seeded onto a 96-well plate at a density of 1.2 × 104 in 200 μl medium per well. After 24 h of incubation, the culture medium was removed and complete medium containing 0, 1, 2, 5, 10, 25, 50 and 100 μgFe ml–1 nanoparticles was added. Cells with different NPs concentration and the control (untreated) cells were tested in triplicate. After 24 h of treatments, 50 μl of MTT solution (Sigma-Aldrich) was added to each well and incubated for an additional 1.5 h. The medium was then aspirated, and the formazan crystals obtained were solubilized by adding 100 μl of DMSO. The samples, after treatment with nanoparticles, show a dark halo at the bottom of the plate owing to the black nanoparticles solution. For this reason, thanks to a magnet under each well, the formazan crystal solution was taken and transferred in a new 96-plate well to eliminate the dark interference for spectrophotometric reading. The absorbance of formazan crystals was measured at the wavelength of 595 nm by an ELISA plate reader (iMarkTM; Bio-Rad Laboratories, USA). The solvent control was considered as 100% cell viability or proliferation and the cell viability or proliferation in treatment groups was calculated as the percentage of solvent control. LDH assay evaluation of cytotoxicity. The release of LDH in culture supernatants and, consequently cell membrane damage by nanoparticles incubation, was measured using the Pierce LDH Cytotoxicity Assay kit (Thermo Scientific, USA). For this experiment, 1 × 104 cells in each well of 96-well plate were seeded. After 24 h, 10 μl of sterile ultrapure water, in spontaneous LDH activity control conditions and, contemporarily, different concentrations of nanoparticles in LDH nanoparticles-mediated activity conditions were added. After incubation, 10 μl of Lysis Buffer (10×) in wells containing the treated cells were added and incubated for 45 min. Successively, 50 μl of each sample medium was transferred, thanks to a potent magnet under each well, in a new 96-well plate and incubated with 50 μl of Reaction Mixture for 30 min. Finally, after the addition of 50 μl of Stop Solution, the absorbance of each sample was measured at 490 nm and 655 nm. NBT assay evaluation of radical oxygen species. To evaluate the presence of ROS in cells treated with rhamnose-coated magnetite nanoparticles, the nitroblue tetrazolium salt (NBT) colorimetric assay was performed (Sigma-Aldrich). A total of 2.5 × 104 cells were seeded into the wells of a 96-well plate, adding 200 μl of culture medium. After 24 h, the cells were incubated with different concentrations of nanoparticles, and at the end of the treatment the medium was substituted with 150 μl of NBT solution (1 mg ml–1) (Sigma-Aldrich). After 5 h, the NBT solution was removed and 95 μl of lysis buffer (1% Triton X-100 and 0.04 M HCl in isopropanol) was added and shaken at room temperature for 30 min. Finally, 105 μl KOH (10 M) was added and shaken for another 30 min. The absorbance of the reaction obtained was read by a plate reader at a wavelength of 620 nm.

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Carcinogenicity assay. The mouse fibroblast cell line Balb/c-3T3, clone A31-1-1 was used for this test. A colony forming efficiency (CFE) assay was developed prior to the final CTA assay, as a preliminary cytotoxicity test to determine which concentrations will be tested in the final assay. Exponentially grown Balb/c-3T3 cells were plated in 60-mm plates at a density of 50 cells per plate in 4 ml of culture media. After 24 h maintenance in cell culture medium cells were exposed for 72 h at five different concentrations. The tested concentrations in the CFE assay were 300, 150, 75, 37.5 and 18.75 μg ml–1. Balb/c-3T3 cells treated with 3-Methylcholanthrene (MCA) at 5 μg ml–1 served as \ positive control, and 5% water was used as a negative control and to dissolve the test product. Three replicates per condition were used. After exposure time, the cells were washed twice with PBS and left in full media for 1 week. Then cells were fixed with methanol and stained with 20% Giemsa for observation. Finally, colonies were counted and the CFE was estimated for each concentration. Due to the results obtained in the CFE assay, the concentrations selected for the final CTA were adjusted. As the results did not show any cytotoxic effects for rhamnose-coated Fe3O4 nanoparticles, it was decided to test higher concentrations with a narrow dilution factor. Therefore, five concentrations ranging from 300 to 37 μg ml–1 with a factor of 3 with 10 replicates per condition were used. Five concentrations with an interval factor of 2 between doses and 5 replicates per condition were used. Carcinogenicity was carried out in a single phase Cell Transformation Assay (CTA). For the single-phase CTA, exponentially growing Balb/c-3T3 cells were plated at a density of 104 cells per plate of a plate of 60 mm in 4 ml of culture media. After 24 h maintenance in cell culture medium, cells were exposed for 72 h to the selected concentrations of NPs. After 7 weeks of incubation without products, the plates were fixed with methanol and stained with 20% Giemsa for its observation. Finally, colonies were counted in the optical microscope Nikon TS100 according to the type of Foci (Table 1) and carcinogenicity was assessed. The cell survival was expressed as the CFE of the treatment in accordance with the solvent control. For the final CTA assay, every focus per plate was counted in accordance of its foci type and the mean and standard deviation



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Genotoxicity assay. BALB/3T3 cells were seeded at a high density on 96-well plates and they were exposed for 48 h to the same

concentrations than the ones used in the main cytotoxicity test stated above (from 1000 to 15.63 μgFe ml–1 with a factor of two between doses). Sterile water was used as a solvent and in the negative control with a 16.7% rate of the total culture volume for assays. Next, 400 mM MMS was used as a positive control in all cases. After the exposure time, cells were trypsinized from the plate and collected into microtubes. Collected cells were then mixed with a suspension of 0.9% low melting point agarose and the mixture was placed on slides previously covered with the first layer of 1% agarose. Once the agarose had solidified, the slides were submerged in lysis liquid for 1 h to release DNA. Later they remained in electrophoresis buffer for 40 min at 4°C to allow DNA unwinding. Then, electrophoresis at 25 V and 300 mA was carried out for 30 min. Once the electrophoresis ended, three washes were performed in Tris buffer and the slides were dried and kept protected from the light until their analysis. For sample analysis, the slides were stained with DAPI at a concentration of 5 μg ml–1 and the percentage of DNA in the tail in regard to cell intensity for 60 cells per condition were analyzed according to Comet Assay IV software. However, only concentrations below its IC50 (doses with a non-altered cell viability) were analyzed for its genotoxicity evaluation.

A. Paolini et al. Table 1. Definition, classification and properties of foci types used as parameters in the carcinogenicity assay

Type III foci increases significantly in regard to the solvent control and the total foci.

Foci classification

Statistical analysis. Data are presented with the mean and standard deviation. Statistical significance was analyzed using the t-test to compare two means using the GraphPad QuickCalcs. P -values smaller than 0.05 (P < 0.05) were considered significant. Statistical analysis of cytotoxicity, genotoxicity and CTA data was performed using PASW Statistics 18 software. IC50 was estimated by Probit analysis from cell viability data and concentration tested. For the genotoxicity test, the Mann–Whitney U-test was used to compare samples (each concentration group was compared to the solvent control group), setting the confidence interval at 95%. In the Balb/c-3T3 cell transformation assay, results are given as the mean with standard deviation. To perform the statistic analysis, the number of foci type III, total and type III/total were used. Normality of the samples was checked previously with the Shapiro– Wilk test and homogeneity of variances by Levene statistic. When this condition was accomplished, one-way ANOVA analysis with the DMS Test was carried out to analyze solvent control versus all other concentrations. Nevertheless, when data showed no normal distribution, the Mann–Whitney U-test was carried out to compare the solvent control versus all other concentrations.

Type I

Type II

Type III

Foci properties Foci with cells highly packaged but not stacked Not invasive Slightly basophilic cytoplasm Stacked cells Markedly basophilic cells Invasive Defined and regular edges with some occasional criss-cross area or starred shape not excessively pronounced Very dense foci Highly basophilic cells Spindle shaped Invasive (invasion of surrounding monolayer) With several layers of cells and starred edges

of every type of foci was calculated per each treatment group. To determine a positive result, the values for the number of type III foci, total foci and type III foci in relation to the total of foci were compared with regard to the negative control. It is considered that the substance tested causes carcinogenesis when the number of

Results Nanoparticles synthesis and characterization The rhamnose-derivative coated Fe3O4 nanoparticles were obtained using a two steps procedure including first the synthesis of the

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Figure 2. (A) Examples of Prussian blue staining in ECV304 cells treated with different concentrations of rhamnose-coated Fe3O4 nanoparticles (arrows), 400× magnification. (B) Standard curve for rhamnose-coated Fe3O4 nanoparticles using an known amount of nanoparticle per ml. (C) Intracellular concen–1 tration of iron (μgFe ml ) after 24 h of incubation with a different concentration of rhamnose-coated Fe3O4 nanoparticles. (D) Increase in intracellular iron in a single cell compared with the control.



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Cytotoxicity of rhamnose-coated iron oxide nanoparticles Fe3O4 nanoparticles stabilized by oleic acid and then the exchange reaction between the oleic acid and the rhamnose derivative at the nanoparticles surface (Fig. 1A). Figure 1B shows the TEM analysis of IONPs indicating the presence of a spherical shape, uniform and not aggregated nanoparticles with an average size of 19.37 ± 2.15 nm. The temperature dependence of the magnetization performed according to Zero Field-Cooled (ZFC)-Field Cooled (FC) procedure for the nanoparticles is shown on Fig. 1C. The ZFC curve exhibits a peak with a maximum at 245 K, corresponding to the blocking temperature of the mean size NPs, whereas the FC curve presents a maximum a 222 K as the temperature decreases and remains constant down to 1.8 K, that confirms the presence of superparamagnetic behavior with possible dipolar inter-particles interactions (Lartigue et al., 2011) The absence of a large or multiple additional maximum in the ZFC curves confirms the relative monodispersity of the sample previously observed by TEM. Intracellular iron content measurement analysis. Through the use of a standard curve for the iron ion, obtained using known concentrations of rhamnose-coated iron-oxide nanoparticles (Fig. 2B), it is

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Trypan Blue and MTT assessment of cell viability analysis. The analysis of the cytotoxic activity of rhamnose-coated iron-oxide nanoparticles began with the comparison between two classic assays, the MTT test and the Trypan Blue exclusion test, widely used in cytotoxicity experiments. The cells, incubated with different concentrations of nanoparticles, show a concentration-dependent decrease in viability (Fig. 3); the results of each line were plotted in a single graph in order to evidence the comparison between the MTT and Trypan Blue exclusion test (Fig. 3A–C). As can be seen, the results obtained for MTT and Trypan Blue are qualitatively similar in U251 and ECV304, whereas for T98G cells the viability obtained by Trypan Blue is higher than the one given by MTT. Thus, these results partly confirm the overestimation of the cell viability by the MTT test described in the study of Hoskins et al. (2012a). From a quantitative point of view, in T98G and ECV304 cell lines, the highest toxic effects were observed at the concentration of 100 μgFe ml–1 with a decrease in surviving cells of 30% for T98G and 35% for ECV304. It should be further noticed that in these two cases the difference between the MTT and Trypan Blue tests is not statistically significant. In contrast, in the U251 cell line, it is possible to see a marked discrepancy between the MTT and Trypan Blue results. In particular, this discrepancy is statistically significant (P < 0.05) up to the concentration of 50 μgFe ml–1. The two tests, in addition, show different trends of cytotoxicity, the highest toxic effects being observed in different conditions. In fact, in the MTT curve, the highest value was measured for c = 100 μgFe ml–1 with a decrease in surviving cells of 30%. On the contrary, in the Trypan Blue curve, the highest value was measured for c = 25 μgFe ml–1 with a decrease in surviving cells of 42%. These last considerations put in evidence the difficulty to compare the MTT and Trypan Blue experimental approaches but, at the same time, underlines the necessity in nanoparticles cytotoxicity experiments to always juxtapose a quantitative analysis (e.g. MTT, MTS and flow cytometry) to a qualitative one (e.g. Trypan blue and Clonogenic assay).



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Figure 3. Histogram for the percentage of living cells after 24 h by treatment with rhamnose-coated Fe3O4 nanoparticles. (A) T98G, (B) U251 and (C) ECV304. Data obtained using MTT and Trypan Blue (Tryp) tests compared with the control (*P < 0.01).

possible to evaluate the exact concentration of intracellular ironoxide nanoparticles at different test conditions. Figure 2C shows the total quantity of iron in each cell line at different concentrations of nanoparticles and underlines the increase in intracellular nanoparticles at higher experimental concentrations. In particular, at concentrations of 25, 50 and 100 μgFe ml–1, the T98G cell line expresses more intracellular iron than the other two cell lines (i.e. U251 and ECV304). Moreover, having a known number of cells in each flask and considering the cytotoxic effect of each single concentration (Fig. 3), the increase, compared with the control, of the cytoplasmic iron concentration in each cell line was calculated (Fig. 2D). These results show an uptake ability of U251 line higher than T98G line and, at a concentration of 100 μgFe ml–1, it was possible to measure an iron concentration CFe in U251 cells ~170 times higher than the control. In contrast in T98G cells, CFe is only 120 times higher than the control. These results appear to be in contrast to those in Fig. 2C, and a possible explanation could be given by considering the number of cells in the flask at the end of the entire protocol. The cells were seeded in the same number (3 × 105), but after 48 h (experimental time) in the flask it is probable to have more T98G cells than U251 and ECV304. This phenomenon may justify the higher value of iron in T98G with respect to the other two cell lines.

A. Paolini et al. Figure 4B shows the quantitative expression of ROS by T98G, U251 and ECV304 cells compared to the control where all the ROS values were calculated according to the MTT values in Fig. 3A–C.

Figure 4. (A) Concentration-dependent membrane damage as determined by lactate dehydrogenase leakage in T98G, U251 and ECV304 cell lines incubated with different concentrations of rhamnose-coated Fe3O4 nanoparticles for 24 h. (B) Relative levels of ROS in T98G, U251 and ECV304 cells incubated with various concentrations of rhamnose-coated Fe3O4 nanoparticles for 24 h. (*P < 0.01, **P < 0.05).

For BALB/3T3, no cytotoxicity was observed and no IC50 could be estimated in this case (IC50 > 1000 μgFe ml–1) (Fig. 5A).

Figure 5. (A) Graph expressing cell viability of rhamnose-coated Fe3O4 nanoparticles in BALB/3T3 cells, (B) Graph of the mean and standard deviation of the percentage of DNA in tail obtained for the rhamnose-coated Fe3O4 nanoparticles. Experimental controls: water was the negative control (solvent), and 400 mM methyl mehanesulfonate (MMS) was the positive control. (*P < 0.05).

LDH assay evaluation of cytotoxicity analysis. In the second part of our cytotoxic study, the cell membrane integrity was determined via quantification of the LDH leakage from cells incubated with rhamnose-coated iron-oxide nanoparticles compared with the control cells (Fig. 4A). The data obtained reflect a very high release of lactate dehydrogenase that in tumor cells reach values also 13 times higher than the control. In particular, the T98G cell line appears to be the less damaged line. In fact, both at low and high concentrations the release of LDH is similar suggesting a membrane damage not concentrationdependent. The ECV304 cell line shows a similar behavior with the same value of LDH released at the concentration of 100 μgFe ml–1; here, however, the concentration-dependent release of LDH confirms the increase in membrane damage with the increase in nanoparticles incubated (Fig. 4A). Finally, the U251 seems to be the weakest cell line showing massive cell membrane damage at the highest concentration (100 μgFe ml–1). This phenomenon may be related to the highest presence of intracellular iron in U251 cells described in Fig. 2D and induces a correlation between high values of intracellular iron, owing to a more marked internalization activity, and a higher probability of damage in the cell membrane.

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NBT assay evaluation of radical oxygen species analysis. To evaluate the oxidative damage of rhamnose-coated iron-oxide nanoparticles, the level of ROS produced was measured by means of an NBT assay.


Figure 6. Percentage of the number type III foci in relation to the total foci ones obtained after 7 weeks of exposure to rhamnose-coated Fe3O4 nanoparticles by the cell transformation assay. 3-Methylcholanthrene (MCA) (positive control); solvent was water (negative control). (*P < 0.05).


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Cytotoxicity of rhamnose-coated iron oxide nanoparticles Table 2. Results in the colony forming efficiency assay. Colony forming efficiency (CFE) reflects the percentage of colonies formed in relation to the control group whereas Effect reflects the percentage of cytotoxicity in relation to the solvent group NP

18 nm NP

Concentration (μg ml–1) 5% H2O 18.75 37.5 75 150 300 3% DMSO

Number of Colonies

Total

Replicate 1

Replicate 2

Replicate 2

75 75 63 93 86 95 27

82 89 66 75 90 89 13

89 76 70 81 99 98 23

246 240 199 249 275 282 63

CFE (%)

100.00 97.56 80.89 101.22 111.79 114.63 25.61

Table 3. The mean and standard deviation of the percentage of type III foci in relation to the total foci from Cell Transformation assay after 7 weeks of exposure to rhamnose-coated Fe3O4 nanoparticles. Solvent water was the negative control (solvent), and 3-Methylcholanthrene (MCA) was the positive control Treatment Concentration (μg ml–1) Solvent 5% 18 nm 3.70 18 nm 11.11 18 nm 33.33 18 nm 100 18 nm 300 MCA 0.005

Foci I Mean ± SD

Foci II Mean ± SD

Foci III Mean ± SD

Foci Total Mean ± SD

10.13 ± 2.90 12.20 ± 4.26 12.33 ± 5.24 13.33 ± 4.47 11.70 ± 4.35 11.10 ± 3.35 1.50 ± 2.42

16.88 ± 3.60 15.30 ± 8.11 24.56 ± 4.77 22.89 ± 6.58 23.10 ± 4.33 20.60 ± 7.46 12.20 ± 4.80

9.50 ± 6.59 8.90 ± 4.95 13.22 ± 3.27 8.67 ± 6.25 11.70 ± 3.65 7.60 ± 1.65 41.30 ± 8.33

36.50 ± 7.23 36.40 ± 12.03 50.11 ± 5.49 40.89 ± 9.53 46.50 ± 3.63 39.30 ± 6.27 55.00 ± 7.35

The ROS expression is strongly correlated with the numbers of living cells, obtained in MTT experiments, able to produce the NBT salt. Taking into account this observation, all data were elaborated considering the percentage of cell death in each condition. The values follow a concentration-dependent increase in ROS in relation to nanoparticles incubated, and this trend was found in all cell lines tested. In addition, one can observe the following. By comparing the two glioblastoma cell lines, the levels of ROS in U251 are higher than the ones in T98G and this difference appears, at the high concentrations, to be statically significant (P < 0.05). At medium and low concentrations, both glioblastoma cell lines show a similar trend reaching values 1.5/2 times higher than the control. Finally, regarding the comparison between glioblastoma lines (T98G and U251) and human urinary bladder carcinoma (ECV403), Fig. 4B shows, in general, a statistically high significant difference (P < 0.01) and this remarks a low production of ROS in ECV304 line that reach a (low) maximum value of 0.3 times higher than the control.

J. Appl. Toxicol. 2016; 36: 510–520

24.67 ± 12.63 22.59 ± 10.43 26.79 ± 7.75 18.43 ± 11.29 25.13 ± 7.64 19.81 ± 5.53 75.17 ± 11.74

was different from the previously tested MTT assay in the BALB/3T3 cell line. The CFE assay supported that IONPS were not cytotoxic (Fig. 6 and Table 2) Results from CTA of IONPs reflect there was not a biologically significant increase in type III foci in relation to the total number of focus for any of the doses and sizes tested (Fig. 6 and Table 3).

Discussion and conclusions Starting with the idea that, actually, no specific procedure exists to investigate the unique properties and the toxicity of a material with a nanometric (