Nanoparticles before and after

Jul 15, 2014 - Organically modified rectorite (OREC) micro/nanoparticles can be synthesized by organic modification from calcium rectorite (Ca2+-REC o...
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Cytotoxicity of Calcium Rectorite Micro/Nanoparticles before and after Organic Modification Yin Liu,† Hongbing Deng,‡ Chunlian Xiao,† Chengfeng Xie,† and Xue Zhou*,† †

Key Laboratory of Environment and Health, Ministry of Education & Ministry of Environmental Protection, and State Key Laboratory of Environmental Health (Incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan, Hubei 430030, China ‡ School of Resource and Environmental Science, Wuhan University, Wuhan, Hubei 430079, China S Supporting Information *

ABSTRACT: Organically modified rectorite (OREC) micro/nanoparticles can be synthesized by organic modification from calcium rectorite (Ca2+-REC or REC), a common form of rectorite in nature. Although REC and OREC have potential applications in food packing and drug delivery, their cytotoxicity is not clear. In the present study, we investigated and compared the cytotoxicity of REC and OREC micro/ nanoparticles in Chang liver cells, the human normal hepatic cells, and human hepatoma HepG2 cells. The interlayer spacing of OREC was enlarged after organic modification. After treatment with REC or OREC for 24 h at 1 and 5 μg/mL, they were taken up by Chang liver cells. REC and OREC induced cytotoxicity in Chang liver and HepG2 cells at almost all doses (1, 2.5, 5, 7.5, and 10 μg/mL) after 6, 24, and 48 h of treatment (P < 0.05 or P < 0.01). Compared with REC, OREC was more cytotoxic. However, there was no difference in the cytotoxicity of REC and OREC between the two cell lines. After treatment with REC or OREC at 7.5 and 10 μg/mL for 24 h, the apoptotic and necrotic percentages of Chang liver cells were increased (P < 0.05 or P < 0.01). The levels of apoptosis-related proteins Bax, Bcl-2, and pro-caspase-3 were all decreased in Chang liver cells after 24 h of exposure to REC or OREC at 5, 7.5, 10 μg/mL. There was no change in the relative ratio of Bax/Bcl-2 after treatment, indicating that REC or OREC-induced apoptosis was not associated with Bax-related mitochondria-mediated apoptotic pathway. Our results suggested that OREC was more cytotoxic than REC, but the underlying mechanisms need further investigation.

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INTRODUCTION The European Food Safety Authority (EFSA) has reported that bentonite, also called dioctahedral montmorillonite (MMT), a kind of layered silicate, is safe and effective to be used as a food additive to reduce the contamination of feed by aflatoxin for ruminants at the recommended level.1 Rectorite, another kind of layered silicate, has the structure similar to that of MMT. It has alternate pairs of dioctahedral mica-like layers (nonexpansible) and MMT-like layers (expansible) at the ratio of 1:1.2 The MMT-like layer of rectorite contains exchangeable cations. As a result, it can be expanded and exfoliated by intercalating cations or polar molecules.3,4 The absorption behavior of rectorite toward cationic surfactants is similar to that of MMT due to the presence of the MMT component.5 Other properties of rectorite, such as suspension, adhesion, plasticity, gelling, and UV-blocking, are also similar to those of MMT. Therefore, rectorite may also be a considerable candidate for food additive and even food processing and packaging. Calcium rectorite (Ca2+-REC or REC), a common form of rectorite in nature, can be transformed into organically modified rectorite (OREC) through ion-exchange with hexadecyl trimethylammonium bromide (CTAB).2 The average diameter of OREC particles © 2014 American Chemical Society

transformed with this method could be well distributed in nanoscale.6 After organic modification, the interlayer spacing would be enlarged and the specific surface area is increased. In recent years, REC and OREC have been used to prepare polymer martix nanocomposites to enhance the absorption capacity. Deng et al.7 found that layer-by-layer film modified nanofibrous cellulose mats with the addition of OREC could be used as antibacterial membranes by increasing the degree of inhibition on Escherichia coli. A more recent study by Xu et al.8 showed that CS-OREC/alginate (ALG) composite beads can be used in drug delivery since OREC could avoid the burst release phenomenon of the drug in the first burst period and improve drug utilization in the later sustained release period. Despite the potential applications of REC and OREC, few studies have investigated their cytotoxicity. Recently, it has been reported that the content of OREC in nanofibrous mats has a significant impact on the viability of mouse lung fibroblasts.9 However, the risks of REC and OREC in the form of particles to human health still remain unknown. Received: March 25, 2014 Published: July 15, 2014 1401

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Tokyo, Japan) with a Cu target and Kα radiation (λ = 0.154 nm), and was also characterized by selected area electron diffraction (SAED) on a TEM. Uptake of REC and OREC by Chang Liver Cells. After treatment, cells were fixed with glutaric dialdehyde, double stained with uranium (U) acetate and lead (Pb) citrate, and then photographed by TEM at an acceleration voltage of 200 kV. Silicon (Si), the characteristic element of REC and OREC, was detected by energy-dispersive X-ray (EDX) spectroscopy (Model, S-4800; Hitachi Ltd., Tokyo, Japan). MTT Assay. The cytotoxicity of REC and OREC was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich Inc., St. Louis, MO, USA) assay. Cells were seeded into 96-well plates and treated with REC or OREC. At the end of treatment, the medium was replaced by 200 μL of MTT solution, which was dissolved in serum-free medium at the concentration of 0.5 mg/mL. After 4 h of incubation at 37 °C, the MTT solution was carefully aspirated, and formazan crystals were dissolved with 100 μL of dimethyl sulfoxide (Sigma-Aldrich Inc.). The plate was covered with aluminum foil and agitated on an orbital shaker for 10 min. The absorbance was measured with a multidetection microplate reader (Model, Synergy 2; BioTek Instruments Inc., Winooski, VT, USA) at 570 nm. Data were expressed as the means ± SD from three independent experiments. Relative cell viability was calculated as follows: relative cell viability = (A570treatment − A570blank)/(A570control − A570blank). The blank represents the samples that were added with only medium without cells. Assessment of Apoptosis and Necrosis Induced by OREC in Chang Liver Cells. The percentages of the cells undergoing apoptosis and necrosis were determined using an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. The cells were double supravitally stained with recombinant FITC-conjugated annexin-V and propidium iodide (PI), and examined by a flow cytometer (BD Biosciences, San Jose, CA, USA) at 570 nm. The percentages of apoptotic and necrotic cells were calculated by ModFit software (Verity Software House, Topsham, ME, USA). Preparation of Whole Cell Protein. Whole cell lysates were extracted by incubation with ice-cold radioimmunoprecipitation assay buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Nadeoxycholate, 150 mM NaCl, and 1 mM ethylenediaminetetraacetic acid) supplemented with a protease inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany) for 20 min on ice. The lysates were centrifugated at 14,000g for 15 min at 4 °C, and the supernatant was collected. Western Blotting. The protein concentration was determined using Bio-Rad DC (detergent-compatible) protein assay (Bio-Rad, Hercules, CA, USA). The proteins were separated by 12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Immunoblotting was performed using primary antibodies including anti-β-actin (1:5000, Abmart, Shanghai, China), Bax (1:8000, Epitomics, Burlingame, CA, USA), Bcl-2 (1:8000, Epitomics), caspase-3 (1:8000, Epitomics) and horseradish peroxidaseconjucated (HRP) antirabbit (1:5000, Jackson ImmunoResearch, West Grove, PA, USA) or HRP-conjucated antimouse secondary antibodies (1:5000, Jackson ImmunoResearch). Protein signals were detected by chemical fluorescence following an enhanced chemiluminescence (ECL) Western blotting protocol (Multisciences Biotech Co., Ltd., Hangzhou, China). The immunoblots were scanned using a GeneGnome chemiluminescent imaging system (Syngene, Frederick, MD, USA). The relative densities of the protein bands were analyzed using ImageJ software. Data were expressed as relative units. Statistical Analysis. Statistical analysis was performed using SPSS for Windows statistical package (version 18.0, SPSS Inc., Chicago, IL, USA). The data, presented as the means ± SD, were analyzed by a two-tailed Student’s t-test. P values 0.05) (Figure 4C). The viability of HepG2 cells was significantly decreased after treatment with OREC (P < 0.05 or P < 0.01 for all), except 1406

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Figure 5. Flow cytometric analysis of apoptosis and necrosis induced by REC (A) or OREC (B) in Chang liver cells. Panels a to f represent the apoptotic and necrotic percentages of cells after treatment with REC or OREC at 0, 1, 2.5, 5, 7.5, and 10 μg/mL for 24 h, respectively. The apoptotic and necrotic percentages of Chang liver cells were quantified. The experiments were repeated at least twice and the results were expressed as the means ± SD in the histograms. As shown in the histograms, the asterisk symbols denote significance between treated and untreated groups: *P < 0.05. The pound symbols denote significance between treated and untreated groups: #P < 0.01.

in the cells treated with 1 μg/mL OREC for 6 h (P > 0.05) (Figure 4D). These results indicated that REC and OREC induced an increase in cytotoxicity in both Chang liver and HepG2 cells. However, there was no significant difference in the cytotoxicity induced by REC and OREC between the two cell lines. Compared with REC, OREC was more toxic in the two cell lines, which might be caused by the following reasons. First, compared with REC, OREC is smaller in particle diameter. It is easier to be transported into the cells. Second, negatively charged cells tend to absorb OREC with positive potential rather than REC with negative potential. More OREC could be transported into the cells. Thus, smaller size and

positive potential would result in a higher biological effective dose of OREC. Finally, CATB is a chemical compound detrimental to cell survival according to Russo et al.24 As the TGA results suggested that OREC still contained residual CTAB, which might be released into the culture medium and taken up by the cells, CTAB may be one of the factors contributing to the cytotoxicity of OREC. The cytotoxicity of unmodified and organically modified MMT has been investigated by Lordan et al.21 They have been shown to significantly decrease the viability of HepG2 cells at the concentrations of 1, 5, 10, 50, 100, 500, and 1000 μg/mL after 24 h of treatment. Compared with untreated cells, the 1407

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Figure 6. Western blotting analysis of Bax, Bcl-2, and pro-caspase-3 proteins in Chang liver cells treated with REC for 6 h (A) and 24 h (B), or OREC for 6 h (C) and 24 h (D) at 0, 1, 2.5, 5, 7.5, and 10 μg/mL. The same membranes were stripped and reprobed with β-actin antibody as a loading control. The results shown are representative of three independent experiments. The intensity of the bands was quantified, and values were normalized to that of the untreated samples.

cytotoxicity between Chang liver and HepG2 cells, we used Chang liver cells in the following study. The cells were treated with 0, 1, 2.5, 5, 7.5, and 10 μg/mL (a to f, respectively) REC (Figure 5A) or OREC (Figure 5B) for 24 h. In Figure 5A and B, the upper left quadrant (Q1) represents the percentage of cell debris from mechanical deterioration and necrotic cells, upper right quadrant (Q2) represents the percentage of necrotic cells and late apoptotic cells, the lower left quadrant (Q3) denotes the percentage of live cells, and the lower right quadrant (Q4) denotes the percentage of early apoptotic cells. After treatment with 1 and 2.5 μg/mL REC or OREC, there was almost no change in the percentages of necrotic and apoptotic cells. At the concentration of 5 μg/mL, REC and OREC slightly increased the percentage of early apoptotic cells (Q4) (P < 0.05 for both). The percentages of early apoptotic cells (Q4), late apoptotic cells and necrotic cells (Q2) were significantly increased at the concentrations of 7.5 and 10 μg/ mL for both REC and OREC (P < 0.05 or P < 0.01), and the percentages were higher as the concentration increased. It is noticeable that at 10 μg/mL after treatment with OREC, the percentage of necrotic and late apoptotic cells (Q2) was almost six times, and the percentage of early apoptotic cells (Q4) was approximately 21 times that of untreated cells. The result indicated that OREC induced more apoptosis and necrosis in Chang liver cells at the concentrations of 7.5 and 10 μg/mL

viability of the cells was approximately 68% to 23% after treatment with unmodified MMT, and 77% to 37% after treatment with organically modified MMT. In comparison with our results, unmodified MMT was more cytotoxic than REC, whereas organically modified MMT was less cytotoxic than OREC. The apparent discrepancies in the cytotoxicity between their and our studies were most likely due to the difference in the interlayer structure and particle size of MMT and REC. The separable layer thickness and layer aspect ratio of REC are larger than those of MMT, which may be favorable for forming the intercalated structure.25,26 Particle size plays an important role in the effective dose by influencing adhesion forces, amounts taken up, and biophysical interactions with the cells.17 In Lordan’s study, the air drying procedure to sterilize unmodified and organically modified MMT resulted in the formation of microsized agglomerates. Apart from this, there was no appreciable difference in the cytotoxicity between unmodified and organically modified MMT, which was contrary to our results. It might be due to different organically modified groups. Organic MMT was modified with a ternary ammonium salt, whereas OREC was modified with CTAB. Effects of REC and OREC on Apoptosis and Necrosis in Chang Liver Cells. The modes of cell death (apoptosis or necrosis) induced by REC and OREC were next examined by flow cytometry. Since there was no difference in the 1408

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more cytotoxic than REC, precautions should be made when OREC is implemented in food and medical applications. Further investigations are required to uncover how REC and OREC are taken up into the cells and the mechanisms of the cytotoxic effects.

than REC, which was in accordance with the MTT result that OREC was more cytotoxic than REC. Effects of REC and OREC on the Mitochondria-Related Apoptotic Proteins in Chang Liver Cells. To investigate the underlying mechanisms of REC- and OREC-induced apoptosis in Chang liver cells, the protein levels of Bax, Bcl-2, and procaspase-3 were measured by Western blotting. The cells were exposed to 0, 1, 2.5, 5, 7.5, and 10 μg/mL REC or OREC for 6 and 24 h. After treatment with REC for 6 h, there was almost no change in the protein levels of Bax, Bcl-2, and pro-caspase-3 (Figure 6A). However, after treatment with REC for 24 h, Bax and Bcl-2 were down-regulated, especially at 10 μg/mL for Bax (Figure 6B). Pro-caspase-3 was slightly decreased at 5, 7.5, and 10 μg/mL (Figure 6B). Similar to REC, the protein levels of Bax, Bcl-2, and pro-caspase-3 were not significantly changed after treatment with OREC for 6 h (Figure 6C). However, a down-regulation of these proteins was observed after 24 h exposure to OREC (Figure 6D). After treatment with OREC at 10 μg/mL for 24 h, the protein levels of Bax, Bcl-2, and procaspase-3 were approximately half of those of the untreated cells (Figure 6D). However, after treatment with REC or OREC for 6 h and 24 h, there was no change in the relative ratio of Bax/Bcl-2, which up-regulates caspase-3 and modulates cell apoptosis27 (Figure S1, Supporting Information). Our results suggested a correlation between REC- or ORECinduced apoptosis and decrease of Bax, Bcl-2, and pro-caspase-3 proteins. However, the molecular events of apoptosis may not be regulated by the Bax-related mitochondria-mediated apoptotic pathway. In addition to this pathway, other mechanisms by which nanoscale materials induce cytotoxicity have been proposed. Lesniak et al.28 reported that “corona”, a protein molecule layer contacted on the surface of silica nanoparticles in biological medium, controlled the interactions at the interface between nanoparticles and cells. In the absence of corona, nanoparticles had stronger adhesion to the cells and higher internalization efficiency compared with nanoparticles that had a preformed corona on their surface. The intracellular location of nanoparticles and their impact on cells were different in the presence or absence of a preformed corona. In our present study, cells were treated with REC or OREC in the culture medium supplemented with 10% fetal bovine serum, forming corona on their surface, thus greatly reducing direct physical damage to the cells. As reported by Wang et al., amino modified polystyrene nanoparticles were trafficked to the lysosome, where the corona was degraded and lysosomal damage was induced, leading to cytosolic release of lysosomal content, and ultimately apoptosis.29 In another study conducted by the same group, morphological changes in lysosomes and mitochondria, activation of caspases 3/7 and 9, and cleavage of poly(ADP-ribose) polymerase (PARP)-1 were observed in human brain astrocytoma 1321N1 cell line treated with the same nanoparticles.30 Apart from this, evidence has shown that intracellular nanoparticles induced cytotoxicity in the reactive oxygen species (ROS)-dependent pathway.31−33 Nanoparticles also potentially induce cell death through autophagy and lysosomal dysfunction.34 The underlying mechanisms of REC- and OREC-induced cytotoxicity still need further investigation.

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ASSOCIATED CONTENT

S Supporting Information *

The ratio of Bax/Bcl-2 in Chang liver cells treated with REC or OREC at 0, 1, 2.5, 5, 7.5, and 10 μg/mL for 6 and 24 h. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-27-83693280. Fax: +86-27-83693280. E-mail: xue. [email protected]. Funding

This work was supported by grants from the National Natural Science Foundation of China (81202237), the Specialized Research Fund for the Doctoral Program of Higher Education (20110142120027), Hubei Health Department (QJX2012-01), and the Fundamental Research Funds for the Central Universities, HUST: 2011QN206. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ALG, alginate; C, carbon; CS, chitosan; CTAB, hexadecyl trimethylammonium bromide; DMEM, Dulbecco’s modified Eagle’s medium; ECL, chemiluminescence; EDX, energydispersive X-ray; EFSA, The European Food Safety Authority; FITC, fluorescein isothiocyanate; FT-IR, Fourier transform infrared; GI, gastrointestinal; HRP, horseradish peroxidase; MMP, mitochondrial membrane potential; MMT, dioctahedral montmorillonite; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; O, oxygen; OREC, organic rectorite; Pb, lead; PI, propidium iodide; PVDF, polyvinylidene difluoride; REC, rectorite; ROS, reactive oxygen species; SAED, selected area electron diffraction; SAXRD, small angle X-ray diffraction; SDS−PAGE, sodium dodecyl sulfate− polyacrylamide gel electrophoresis; Si, silicon; TEM, transmission electron microscope; TGA, thermogravimetric analysis; U, uranium

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REFERENCES

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CONCLUSIONS The results in the present study contributed to our understanding of the cytotoxicity of REC and OREC micro/ nanoparticles. As indicated by our results that OREC was 1409

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