Acute Toxicity of Silver Free and Encapsulated in Nanosized Zeolite

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Acute toxicity of silver free and encapsulated in nanosized zeolite for eukaryotic cells Clément Anfray, Biao Dong, Sarah KOMATY, Svetlana Mintova, and Samuel Valable ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00265 • Publication Date (Web): 06 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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ACUTE TOXICITY OF SILVER FREE AND ENCAPSULATED IN NANOSIZED ZEOLITE FOR EUKARYOTIC CELLS Clément Anfray1, Biao Dong2, 3, Sarah Komaty2, Svetlana Mintova2* and Samuel Valable1* 1

ISTCT/CERVOxy group, Normandy University, UNICAEN, CEA, CNRS, 14000 Caen, France.

2

Laboratoire Catalyse et Spectrochimie (LCS), CNRS, ENSICAEN, Normandy University, 6

boulevard du Maréchal Juin, 14050 Caen, France. 3

State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and

Engineering, Jilin University, China. KEYWORDS Silver cations, silver nanoparticles, zeolite, tumor cell, brain, toxicity.

ABSTRACT

The potential toxicity of encapsulated silver in EMT-type nanosized zeolites on prokaryotic cells, human tumor cell lines from various origins and primary cultures of neurons and astrocytes, was investigated. Silver in cationic form (Ag+) was encapsulated in EMT-type nanosized zeolites via ion exchange process (Ag+-EMT), and compared with the reduced silver (Ag0) in the zeolite (Ag0-

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EMT). As reference samples for the toxicity measurements, pure EMT- type zeolite and silver perchlorate were used. Cells were exposed to silver containing zeolites (50, 100 and 400 µg/ml) for 24h and 48h. After exposure to Ag+-EMT (50 µg/ml) for 24h, a loss in cell viability independent on the cell type was observed, ranging from -34.37% ±23.90 for astrocytes to -99.5% ±0.24 for U87-MG cells. These results were comparable with the toxicity for silver perchlorate. The Ag0-EMT sample showed lower toxicity on human cell lines in comparison to the Ag+-EMT. A decrease in cell viability, i.e., -73.46% ±20.78 and -62.3%±17.96 for U87-MG and HEK 293 cells, respectively under exposure only to high concentration of Ag0-EMT (400 µg/ml) for 24h was measured. However, the Ag0–EMT was as toxic as the Ag+-EMT for astrocytes and neurons (-97.95% ±3.31 and -100% ±1.11, respectively after exposure to 50 µg/ml for 24h). No decrease in cell viability exposed to pure EMT zeolite was found. The results demonstrate the severe toxicity of silver cations, either free or encapsulated, in comparison to reduced silver encapsulated in zeolite nanocrystals. Therefore, silver cations, either free or encapsulated, have to be used with great caution regarding their toxicity on eukaryotic cells.

Introduction Nanoparticles (NP) defined as objects ranging from 1 to 100 nm are being used for various applications varying from medical treatments to energy storage and daily life products1. Thanks to their antimicrobial properties, silver nanoparticles (SNP) are one of the most widely used since they have proven excellent antimicrobial efficacy against bacteria, viruses and other eukaryotic micro-organisms2–4. In 2008, approximately 500 tons of silver in the form of silver ions, silver proteins and colloids worldwide were produced5. However, serious concerns have arisen since NP may be at the origin of adverse effects in the pulmonary, cardiovascular and central nervous systems6.

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Concerning the effects on the central nervous system, SNP have demonstrated a huge, sizedependent cytotoxicity elicited by a strong oxidative stress on primary cultures of neurons and astrocytes7. This vast toxicity has, interestingly, been proposed as an original cancer therapy for glioblastoma8. At the cellular level, SNP have been described to interact with various intracellular cytosolic proteins, to induce the expression of inflammatory mediators on cell lines of various origin, to elicit calcium or to induce oxidative stress9. Among NP, studies for biomedical applications have focused on zeolites nanocrystals, which possess exceptional sorption properties and the possibility to precisely control the synthesis process and thus to predetermine their properties10. Zeolites are crystalline inorganic microporous materials with unique framework structures consisting of interconnected channels and cages, with variable sizes and shapes. The interest in zeolites for environmental and biological application is constantly growing. Several studies report on the use of zeolites as a host for the encapsulation and delivery of anti-cancers drugs using for instance 5-fluorouracil11,12 but also as an hemostatic agent to prevent uncontrolled bleeding after injuries13. The development of these biomedical applications raises, however, the question of a potential toxicity of zeolites. Recently, pure ultra-small zeolites with EMT- and LTL-type structures, both with high alumina content and large pores (above 0.7 nm), showed no sign of toxicity on the cell14. In addition to the unique properties of zeolites, the introduction of different cations and further stabilization of metal (Fe, Cu, Pd, Pt, etc.) and semiconducting (CdS, ZnS, PbS, etc.) clusters within the nanosized zeolites change their properties. As an example, the preparation of the EMT- type nanosized zeolite from organic template free suspensions, followed by iron ion-exchange procedure directly in suspensions for brain applications was reported15. The iron incorporated in the zeolite

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nanoparticles did not cause any cell toxicity for both tumor cell line and primary cell culture of astrocytes. The high porosity, low Si/Al ratio, and high concentration of sodium of the EMT-type ultrasmall crystals also permitted the introduction of a high amount of silver16. The antibacterial activity of the silver cations incorporated in the zeolites was discussed. The induced toxicity has been proposed via mitochondrial dysfunction, reactive oxygen species release and oxidative damage8. Until now, the potential toxicity of silver containing zeolites has never been investigated for eukaryotic cells. The primary objective of this study was to assess the potential toxicity of encapsulated silver in nanosized zeolite crystals on tumor cell lines from various origins but also on primary cultures of neurons and astrocytes. Tumor cell lines as highly proliferating cells were used, while both neurons and astrocytes with almost no proliferation were used in vitro. Experimental procedures Silver containing nanosized zeolite crystals, synthesis and properties: EMT-type zeolite nanocrystals were prepared from organic template-free precursor mixture with a molar composition 5.15SiO2:1Al2O3:18.45Na2O:240.3H2O; the precursor mixture was aged for 14h at room temperature prior hydrothermal treatment at 38°C for 36 hours in conventional oven17. The EMT-type zeolite nanocrystals with a diameter of 10 - 20 nm were stabilized in water suspensions (sample EMT, solid concentration of 2 wt.%). The encapsulation of silver in the EMT-type zeolite was performed via ion exchange in aqueous medium to avoid agglomeration. The ion-exchange process was performed as follows: 20 ml silver perchlorate solutions with a concentration of 0.05 M were added to EMT zeolite suspensions (7.5 wt.%, 12 ml) and kept for 4h, followed by purification using a high-speed centrifugation, and finally re-dispersed in water (sample Ag+-

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EMT). The reduction of silver cations (Ag+) to silver nanoparticles (Ag0) in the EMT zeolite suspensions (2 wt.%, 7 ml) were obtained under microwave reduction (120 °C, 10 min, 1000 W) in the presence of a triethylamine (N(C2H5)3, 2 ml) as a reducing agent. The reduced suspensions were additionally purified and dispersed in water. The silver containing zeolite suspensions, before and after reduction, were abbreviated as Ag+-EMT and Ag0-EMT, correspondingly. The crystalline structure of nanosized zeolite was confirmed by recording the Powder X-ray Diffraction (XRD) patterns using a PANalytical X’Pert Pro diffractometer with Cu Kα monochromatized radiation (λ = 1.5418 Å). The crystal size, morphology and crystallinity of solids were determined by a transmission electron microscopy (TEM) using a Titan 80-300 operating at 300 kV. Energy Dispersive Spectrometer (EDS) coupled with the microscope was used to characterize the chemical composition of the EMT zeolite nanocrystals. In addition, the chemical composition of the zeolites was determined by inductively coupled plasma (ICP) optical emission spectroscopy using a Varian ICP-OES 720-ES. TABLE 1. ICP results of silver containing EMT zeolite samples. Sample

Ag

Si

Al

Na

(wt.%)

(wt.%)

(wt.%)

(wt.%)

Pure EMT

0

13.4

11.9

12.1

Ag+-EMT

9.0

15.0

12.9

7.5

Ag0-EMT

9.0

14.3

12.9

7.5

Cell lines: A human glioblastoma cell line, U87-MG purchased from American Type Culture Collections (ATCC, Manassas, VA, USA) and HEK 293 cells (Human Embryonic Kidney cells) were used. Cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine

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serum (Eurobio), 2 mM glutamine (Sigma-Aldrich), and 100 U/ml penicillin/streptomycin (Sigma4

Aldrich). Cells were seeded in 24-wells plates at a concentration of 5.10 cells/ml, and maintained in culture at 37°C with 5% CO2 and 95% humidity. Ethical approval and animal issues: Animal investigations were performed under the current European directive (2010/63/EU) as incorporated in national legislation and in authorized laboratories (B14118001). Ethical approval was sought and obtained by the principal investigators (CA and SV) from the French Ministère de l’éducation nationale, de l’enseignement supérieur et de la recherche (study authorization: N/04-01-13/04/01-16). The animals were obtained from an in-house breeding stock at the Centre Universitaire de Ressources Biologiques (CURB, A14118015). Primary culture of astrocytes: Cerebral cortices were isolated from neonatal (1- to 3- day old) mice (Swiss, CURB) carefully stripped of the meninges and dissociated to generate a single-cell suspension as described18. Cultures were allowed to grow in a humidified 5% CO2 incubator at 37°C to confluence (15-20 days) prior to use in DMEM, supplemented with 10% fetal bovine serum (Eurobio), 10% horse serum (Eurobio), 2 mM glutamine (Sigma-Aldrich) and 100 U/ml penicillin/streptomycin (Sigma-Aldrich). At about 80% confluence, the growth medium was replaced by the same medium. Primary cultures of cortical neurons/astrocytes: Cultures were prepared from E15–E16 mouse embryos (Swiss mice, CURB). Micro-dissection of cortices was followed by a dissociation of the tissue in DMEM at 37°C (Sigma-Aldrich). Cells grew on plates coated with poly-d-lysine (0.1 mg/ml) and laminin (0.02 mg/ml) in DMEM, supplemented with 5% fetal bovine serum, 5% horse serum (Eurobio), and 2 mm glutamine (Sigma-Aldrich). Cells were maintained in a humidified 5% CO2 atmosphere at 37°C. Neurons/astrocytes were used after 12 d in vitro.

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Cells treatment: Suspensions of pure EMT, Ag+-EMT, Ag0-EMT and silver perchlorate were diluted in culture medium in order to obtain concentrations of 50, 100 or 400 µg/ml. Cells were exposed to these suspensions for 24h and 48h. Cells viability: Cell viability was analyzed for 24h or 48h following exposure to zeolites with the WST-1 assay (Roche) according to manufacturer's instructions. Bacterial culture: Frozen glycerol stock (-80°C) of E.Coli was inoculated in a 15 ml tube containing 10 ml of liquid LB culture medium (Sigma-Aldrich) and grown overnight on a rotating shaker (220 rpm) at 37°C. The culture was transferred into fresh LB medium containing 50, 100 or 400 µg/ml zeolites to obtain 5% solutions (v/v) of the overnight culture. These solutions were distributed in a 96 wells plate (100 µl per well), and incubated at 37°C under constant shaking (220 rpm) during the time of the experiment. Bacterial growth was assessed by measuring the OD600 every 15 minutes for 3.5 hours using a spectrophotometer (Asys UVM 340, Biochrom Ltd.). Blank wells contained the fresh LB medium with zeolites. Time Lapse microscopy: The experiments were conducted on U87-MG cells. 24h after seeding, 20 mM of HEPES buffer (Sigma-Aldrich) was added into the wells, and 2h later cells were exposed to 25 µg/ml of Ag+-EMT; water was used as a control sample. The acquisition started immediately after exposure to zeolites. Time-lapse images were obtained for 2h, every minute an image was taken with a Leica DMi8 videomicroscope equipped with an i8 Incubator (Pecon). Statistical analyses: Data were presented as mean ±SD. Statistical analyses were obtained with JMP programs (SAS Institute) and detailed on each caption.

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Results and discussion General characteristics of silver containing nanosized EMT type zeolite: Pure EMT and silver containing zeolite samples (Ag+-EMT and Ag0-EMT) are highly stable in water suspensions at ambient conditions for two years. The pictures of suspensions containing pure EMT and silver containing zeolites with a concentration of 2 wt.% are presented in Figure 1. As can be seen, the suspensions have colloidal appearance, no sedimentation, and a slight color change from white to yellow and gray is observed for pure EMT, Ag+-EMT and Ag0-EMT, respectively. The particle size distribution curves of the three suspensions are similar, and only one peak centered at 50 nm is present (Fig. 1a). The chemical compositions of the samples are summarized in Table 1. The silver concentration for the sample subjected to ion exchange is 9 wt.% according to the ICP analysis. The same chemical composition is measured for the reduced sample (Ag0-EMT). The high crystallinity of the ion-exchanged and reduced samples is confirmed by recording the XRD patterns. Only the Bragg peaks corresponding to the EMT type zeolite are present and no signature for silver oxides was observed. The morphology and size of the zeolite crystals do not change under ion exchange and reduction in the suspensions. The highly crystalline EMT zeolite nanoparticles preserve their identity; no agglomeration and amorphization are observed after encapsulation of silver (Fig. 1b). The presence of silver nanoparticles is clearly seen in the TEM pictures, the black spots correspond to silver encapsulated in the sodalite cages of the EMT zeolite. The presence of silver in the zeolite nanocrystals is also confirmed by EDS, and the results are consistent with the ICP (SI, Fig. S1). The dispersion of the silver nanoparticles is homogeneous. The color change of the Ag0-EMT suspension is the first evidence for the presence of silver nanoparticles encapsulated in the zeolite

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crystals, and this color is preserved thus confirming the high crystallinity of the samples (Fig. 1a, inset).

Figure 1. (a) Dynamic light scattering (DLS) curves of pure EMT, Ag+-EMT and Ag0-EMT suspensions. Inset: photographs of the three suspensions with solid concentration of 7.5 wt. %. TEM pictures of (b) pure EMT, (c) Ag+-EMT, and (d) Ag0-EMT samples. Scale bar = 20 nm. Antibacterial properties of silver containing nanosized EMT type zeolite: E.Coli growth was unaffected by the presence of pure EMT zeolite independently on concentrations used (Fig. 2a). In contrast, a very acute toxicity toward E.Coli cells exposed to Ag+EMT was observed. Indeed, bacteria were unable to grow in the medium containing Ag+-EMT, even at the lowest concentration used, (50 µg/ml) (Fig. 2b), The Ag0-EMT sample was found to be significantly less toxic. A loss in E.Coli cells growth of 20.2% and 40.2 % for Ag0-EMT with a concentration of 50 and 100 µg/ml, respectively in comparison to the control sample was obtained. However, at high concentration of Ag0-EMT (400 µg/ml), bacteria were also unable to grow (Fig. 2c). These results confirm the anti-bacterial efficacy of silver containing EMT zeolites

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toward E.Coli as previously reported16. In the current experiment the cationic silver (Ag+-EMT) was more toxic than the reduced silver (Ag0-EMT), as expected.

Figure 2. Growth quantification of bacteria exposed to various concentrations of (a) pure EMT, (b) Ag+-EMT, and (c) Ag0-EMT samples for 3.5 hours. Bacterial growth was assessed by

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measuring the OD600. Mean ±SD, n=3; HSD Tuckey post-hoc test after a significant ANOVA (*p