In Vitro Cytotoxicitiy of Silica Nanoparticles at High Concentrations

Feb 15, 2007 - In Vitro Cytotoxicitiy of Silica Nanoparticles at High Concentrations Strongly Depends on the Metabolic Activity Type of the Cell Line...
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Environ. Sci. Technol. 2007, 41, 2064-2068

In Vitro Cytotoxicitiy of Silica Nanoparticles at High Concentrations Strongly Depends on the Metabolic Activity Type of the Cell Line JENQ-SHENG CHANG, KE LIANG B. CHANG, DENG-FWU HWANG, AND ZWE-LING KONG* Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, Republic of China

Amorphous silica is increasingly used in diagnostic and biomedical research because of its ease of production and relatively low cost. It is generally regarded as safe and has been approved for use as a food or animal feed ingredient. Recent literature reveals that amorphous silica may present toxicity concerns at high doses. In anticipation of potential human exposure to silica, it is advisable to examine its toxicity to cells of different organs. Consequently, we investigated the response of several normal fibroblast and tumor cells to varying doses of amorphous silica or composite nanoparticles of silica and chitosan. A cell proliferation assay indicates that silica nanoparticles are nontoxic at low dosages but that cell viability decreases at high dosages. A lactate dehydrogenase (LDH) assay indicates that high dosages of silica induce cell membrane damage. Both assays reveal that fibroblast cells with long doubling times are more susceptible to injury induced by silica exposure than tumor cells with short doubling times. In contrast, silica-chitosan composite nanoparticles induce less inhibition in cell proliferation and less membrane damage. This study suggests that the cytotoxicity of silica to human cells depends strongly on their metabolic activities but that it could be significantly reduced by synthesizing silica with chitosan.

Introduction The rapid development of nanotechnology in recent years has created a myriad of engineered nanomaterials. Nanoscaled particulates of metal (gold and silver nanoparticles), semiconductors (quantum dot), carbon (nanotube and buckyball), and oxides (iron oxide, titanium dioxide, and silica) are increasingly being used in industrial production as well as scientific, biological, and medical research. To prevent potential hazards originating from accidental exposure of nanomaterials, their toxicological effects have been reviewed from the environmental (1), health (2), toxicological (3, 4), and scientific (5) perspectives. Although the toxicology of carbon nanomaterials (4, 6-8) and quantum dots (9-15) has become a subject under intensive investigation, other * Corresponding author phone: +886 2 24622192 ext. 5130; fax: +886 2 24632179; e-mail: [email protected]. 2064

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engineered nanomaterials have not received as much attention. Human exposure to crystalline silica dust occurs in the course of mining operations, foundry work, mineral processing, and construction sites. Inhaled crystalline silica induces chronic obstructive pulmonary disease, silicosis, or even lung cancer (16, 17). In contrast, amorphous fumed or precipitated silica is generally considered to be safe and approved for use as a food or animal feed ingredient by FDA. This is because of the fact that traditional routes of exposure, inhalation/oral ingestion, to amorphous silica have been found to pose minimal or no health-related risks in prior toxicological studies. The potential applications of amorphous silica in nanobiotechnology have encompassed areas such as diagnostics (18, 19), bioanalysis and imaging (20, 21), drug delivery (22, 23), and gene transfer (24, 25). As a consequence, it is conceivable that amorphous silica may someday be administered into the human body through all possible routes of entry, including oral ingestion, inhalation, intravenous injection, and transdermal delivery. Information regarding the absorption, biodistribution, retention, degradation, clearance, and safety of silica in different tissues and organs is therefore of vital importance to the future of silica as a biomaterial. Silica is commonly prepared in the solution by the Sto¨ber (sol-gel) process that produces silica by the hydrolysis and polycondensation of silicon alkoxide (26, 27). Biomimetic processes (26) that utilize an amine-containing compound such as polypeptide or polyamine have inspired many biologists, chemists, and material scientists to propose novel procedures for silica synthesis. We have recently demonstrated that a naturally derived polysaccharide, chitosan, is capable of forming composite nanoparticles with silica (28). Although the toxicity of silica and chitosan is well-known, the cytotoxicity of chitosan-modified silica nanoparticles has never been reported. It is therefore of interest to investigate the effects of chitosan modification on the cytotoxicity of these nanoparticles to the cells that may come in contact with silica. In this study, we present an in vitro study that examines the cytotoxicity of amorphous and composite silica nanoparticles to different cell lines. These cell lines include normal pulmonary and dermal fibroblast cells as well as tumor cells of the colon, gastric system, and lung. It is hoped that by examining the response of multiple cell lines to silica nanoparticles, more basic information regarding the cytotoxicity of silica in potential dermal and pulmonary contacts or future oncological applications could become available.

Materials and Methods See Supporting Information for details of sample preparation and analysis. Silica nanoparticles (27) and silica-chitosan nanoparticles (28) were dispersed in deionized water by sonication, filtrated through 0.22 µm membranes, and characterized for particle size, morphology of nanoparticles, and chemical states of elements. These nanoparticles at low concentrations dispersed well and did not aggregate in deionized water. For cytotoxicity tests, more concentrated silica nanoparticles and silica-chitosan nanoparticle dispersions of ca. 4 mg of particles/mL were quickly diluted with cell culture medium prior to experiment. The cultured selected human fibroblast and cancer cell lines (Table 1) were exposed to silica or silicachitosan composite nanoparticles and incubated for 48 h. The cytotoxicity of different silica nanoparticles and silicachitosan nanoparticles to selected human fibroblast cells and cancer cells (Table 1) was subsequently examined by 10.1021/es062347t CCC: $37.00

 2007 American Chemical Society Published on Web 02/15/2007

TABLE 1. Name and Types of Cell Lines Tested in This Study cell namea

origin

tissue

cell type

WS1 CCD-966sk MRC-5 A549 MKN-28 HT-29

Homo sapiens (human) H. sapiens (human) H. sapiens (human) H. sapiens (human) H. sapiens (human) H. sapiens (human)

skin skin lung lung gastric colon

adherent fibroblast adherent fibroblast adherent fibroblast adherent epithelial adherent epithelial adherent epithelial

disease

doubling time (h)

normal carcinoma adenocarcinoma colorectal adenocarcinoma

128.4 128.4 128.5 22.9 28 19.5

normal

a CCD-966sk, WS1, MRC-5, A549, HT-29, and MKN-28 cell lines came from the American Type Culture Collection (ATCC) or Dr. Murakami’s Research Laboratory (Kyushu University, Japan).

FIGURE 1. SEM picture of silica nanoparticles (a) from sodium silicate and (b) from TEOS. two different assays. The MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay was used to measure the succinate dehydrogenase mitochondrial activity as an indicator of cell viability/proliferation (29). The amount of viable cells after each treatment was expressed as a percentage of control. The release of lactate dehydrogenase (LDH) was used as a measure of plasma membrane leakage (30). LDH released into the medium was shown as a percentage of total cellular LDH. MTT and LDH measurement data were expressed as the mean ( SD from three independent experiments. The Pearson product-moment correlation coefficient (r) was used to analyze correlations. One-way analysis of variance (one-way ANOVA) and Duncan’s Test were used for significance testing, using a p value of 0.05 (SPSS 11, SPSS Inc., Chicago, IL).

Results and Discussion The average size of nanoparticles was determined on a scanning electron microscope (SEM) by measuring the size of about 100 particles in the micrograph. The scanning electron micrographs (Figure 1) show that freeze-dried silica nanoparticles are mostly spherical and aggregated. The aggregation results mainly from the freeze-drying and sample treatment procedures before SEM observations. The average size of individual silica nanoparticles prepared from sodium silicate, silica (silicate), were 21.58 ( 4.36 nm (Figure 1a), and silica nanoparticles prepared from TEOS, silica (TEOS), were 80.21 ( 14.43 nm (Figure 1b). The particle size of the silica-chitosan composite nanoparticles increased slightly from ca. 10 to 14 nm with a reaction time of 15-360 min (Figure 2). The silica-chitosan nanoparticles prepared after different hours of reaction are designated as silica-chitosan 1H to 6H (nanoparticles) in the rest of the discussion. The hydrodynamic particle size of silica (from silicate) was 188.3 ( 11.5 nm, and silica (from TEOS) was 236.3 ( 6.85 nm. The hydrodynamic particle size of the silica-chitosan composite nanoparticles increased slightly from ca. 153 to 177 nm with a reaction time of 15-360 min. By comparing the results from SEM and light scattering measurements, it is likely that agglomerates of three to eight silica nanoparticles are present

FIGURE 2. Average diameters of silica-chitosan composite nanoparticles after different reaction times. in the solution. No further aggregation was observed during the cytotoxicity experiment. The results of the MTT assay, as a measure of metabolic competence of the cells following 48 h of contact with the silica nanoparticles or silica-chitosan 6H nanoparticles, are shown in Figure 3. The difference among the cytotoxicity of silica-chitosan 1H to 6H was insignificant (Figures S2 and S3). Consequently, only 6H has been compared to silica prepared from silicate or from TEOS. The cytotoxicity of silica nanoparticles increased with increasing particle mass concentration in all of the cell cultures. At a concentration of 667 µg/mL of silica nanoparticles, the cell viability decreased to a level between 60 and 80% of control. If we use IC20 (20% inhibitory concentration, µg/mL) to represent the concentrations that cause the cell proliferation rate to drop to 80% of control, then the IC20 of silica to the cultured cells increases in the following order: MRC-5 (138 to 155 µg/mL), WS1 (171 to 221 µg/mL), CCD (224 to 310 µg/mL), A549 (386 µg/mL), HT-29 (508 to 510 µg/mL), and MKN-28 (443 to 572 µg/mL). Cancer cell lines (A549, HT-29, and MKN-28) have a higher cell viability than lung and skin fibroblast cell lines (MRC-5, WS1, and CCD-966sk) when incubated with silica nanoparVOL. 41, NO. 6, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Effect of silica and silica-chitosan composite nanoparticles (667 µg/mL) on the cell viabilities of different cell lines (MTT assay) after 48 h. (White) silica-chitosan 6H, (black) silica from silicate, and (gray) silica from TEOS. Values are mean ( SD from three independent experiments. Significance indicated by p < 0.05. ticle suspensions in culture media. This indicates that cancer cells are more resistant to silica nanoparticles. In contrast, the decrease of cell viability was negligible (mostly >85% of control at 667 µg/mL exposure) for cells treated with silicachitosan composite nanoparticles. Apparently, the synthesis of silica in the presence of chitosan helps to produce nearly nontoxic silica nanoparticles with improved biocompatibility. These results correspond with the report that chitosan (MW ) 213 000, DD ) 88%) entities exhibited significant cytotoxicity only at concentrations higher than 0.741 mg/mL in A549 cell lines (31). Brunner et al. (32) found that silica nanoparticles were nontoxic to human mesothelioma cells and mouse embryonic fibroblast cells within the concentration range of 0-15 ppm (µg/g). Limbach et al. (33) noticed that oxide nanoparticle size, nanoparticle number density, or total particle surface area would affect the transport and uptake of oxide nanoparticles into human lung fibroblasts. Nevertheless, our results revealed that silica nanoparticles may become slightly cytotoxic at high concentrations (gca. 138 µg/mL), even though they were nontoxic at low concentrations. It has been reported that surface modification with aminosilanes increased the 50% lethal concentration of silica nanoparticles of Cos-1 cells to well above 1 mg/mL (34). By comparison, polylysine modified silica (35) showed cytotoxic effects (cell viability dropped to ca. 60% of control) to HNE1 and HeLa cells at concentrations g500 µg/mL. In view of the fact that silica-chitosan nanoparticles maintain a cell viability above 85% of control in most test conditions, it appears that the presence of chitosan is more effective than polylysine in reducing the cytotoxicity of silica. The effects of silica nanoparticles or silica-chitosan composite nanoparticles on cell membrane integrity were determined by the LDH assay (Figure 4). None of the silicachitosan composite nanoparticle treatments (Figure S3) caused a significant (>3.7% LDH released) membranolytic effect up to 48 h for all the cultured cells even at a concentration of 667 µg/mL. In contrast, pure silica nanoparticles were found to cause significant (5.6 to 11.3% LDH released) membrane damage at the same condition. The membrane damage caused by 48 h exposure to silica nanoparticles results in LDH release from the cultured cells in the following order: WS1 (9.8 to 11.3%), CCD-966sk (8.7 to 9.1%), MRC-5 (8.7 to 8.8%), HT-29 (6.6 to 7.7%), A549 (5.6 to 7.1%), and MKN-28 (5.6 to 5.7%). Thus skin and pulmonary fibroblast cells are more susceptible to membrane injury 2066

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FIGURE 4. Effect of silica and silica-chitosan composite nanoparticles (667 µg/mL) on the membrane damage of different cell lines (LDH assay) after 48 h. (White) silica-chitosan 6H, (black) silica from silicate, and (gray) silica from TEOS. Values are mean ( SD from three independent experiments. Significance indicated by p < 0.05. caused by silica nanoparticles than tumor cells. These results are in agreement with those obtained from the MTT assay (Figure 3). Because a loss of membrane integrity is one of the physiological features of necrotic cell death (36), the LDH assay reveals that increasing silica exposure induces more cells to die of necrosis. The slight difference between the cell viability sequence and its susceptibility to membrane damage could result from the relative susceptibility to concurrent apoptosis and necrosis (15, 37) in nanoparticle induced cell death. However, it may be more informative to relate the cytotoxicity data of different cell lines to their metabolic activity or their rate of proliferation. As a consequence, the cell population doubling time could be an important indicator of the tolerance of cells exposed to silica or silica-chitosan nanoparticles. The population doubling times of different cell lines used in this study were shown in Table 1. The cell population doubling times of normal human skin and lung fibroblast cells were 128.4-128.5 h (38), and those of the human lung carcinoma, human colon carcinoma, and human gastric adenocacinoma were 22.9, 19.5, and 28 h (39, 40), respectively. The cancer cells had much shorter cell population doubling times than normal human fibroblast cells because of their higher metabolic activities. Figure 5a plots the cell viability values versus the population doubling times of different cell lines exposed to silica or silica-chitosan nanoparticles. There was a negative correlation between the cell viability and the population doubling time of different cell lines exposed to silica or silica-chitosan nanoparticles (r values of silica from silicate, silica from TEOS, and silicachitosan nanoparticles were -0.68, -0.79, and -0.97, respectively). Most human tumor cell lines are capable of maintaining their cell proliferation after they are exposed to a high concentration of silica nanoparticles. However, the proliferation of A549 cells was inhibited by silica nanoparticles nearly as much as that of normal fibroblast cells. This result indicated that the exposure to high concentrations of silica nanoparticles might be toxic to lung cells no matter if they are normal or tumorgenic. Lin et al. (41) evaluated the in vitro toxicity of silica nanoparticles to A549 cells. They demonstrated that the exposure of A549 cells to silica nanoparticls induced dose- and time-dependent cytotoxicity, increased ROS levels, and reduced glutathione levels. It appears that the A549 cell line is a unique type of tumor cell that is unable to tolerate exposure to nanoparticles through

FIGURE 5. Correlation between (a) cell viability after 48 h exposure to 667 µg/mL nanoparticles (r values of silica from silicate, silica from TEOS, and silica-chitosan nanoparticles were -0.68, -0.79, and -0.97, respectively), (b) 20% inhibitory concentration (IC 20) (r values of silica from silicate and silica from TEOS nanoparticles were -0.88 and -0.95, respectively), and the population doubling time of different cell lines. (b) Silica from silicate, (O) silica from TEOS, and (1) silica-chitosan 6H nanoparticles. Values are mean ( SD from three independent experiments. high metabolic activity during in vitro cytotoxicity tests. In comparison, silica-chitosan nanoparticles could maintain cell viability above 85% of control for both tumor and normal cells of widely different doubling times under similar test conditions. This suggests that chitosan is effective in reducing the cytotoxicity of silica to normal human cells that have a much slower metabolic activity than tumor cells. Another way to examine the cell specific response to nanoparticles is to look at the relationship between IC20 and the population doubling time. The IC20 showed a similar negative correlation with the population doubling time of different cell lines exposed to silica nanoparticles (Figure 5b) with higher correlation values (r values of silica from silicate and silica from TEOS nanoparticles were -0.88 and -0.95, respectively). IC20 values were relatively high for tumor cells with short population doubling times, but they were much lower for normal fibroblast cells with long doubling times. It is interesting to note that A549 cells behave more in line with other tumor cells when we group the cells by how their population doubling times affect their IC20 values. Currently, human exposure to amorphous silica is mostly within the regulatory and safety limit. Recent studies have demonstrated that SiO2 nanoparticles may cause aberrant clusters of topoisomerase I (topo I) in the nucleoplasm in cells and fibrogenesis in Wistar rats (42-44). Noting the rapid progress in silica nanotechnology and active research in its future applications, it is predictable that more and more people may someday be exposed to much higher concentrations of silica during their production process or biomedical applications. For instance, amorphous silica nanoparticles may someday be used as carriers of functional food ingredients or antitumor pharmaceuticals in cancer prevention or therapeutic applications. What are the dosage-dependent responses of human cells to silica nanoparticles if their accumulation level in any tissue is comparable to the 0.52.0 (45) or 0.04-2.0 mg/mL (46) range that may cause hemolytic threats? Is there any economical means of ameliorating the potential toxicity of silica nanoparticles? With limited current knowledge about their health effects on human organs besides gastrointestinal tracts and pulmonary organs, it is probable for us to underestimate the potential risk of accidental overexposure of silica. This study may therefore provide the basis for future improvements on the cytotoxicity amelioration and safety precautions of silica nanoparticles. In conclusion, the MTT assay and LDH assay revealed that silica nanoparticles may become slightly toxic to cultured human cells at high concentrations. High dosages of silica

nanoparticles were more toxic to normal human fibroblast cells than to cancer cells. At high dosages, amorphous silica nanoparticles may retard cell proliferation, damage the cell membrane, and possibly induce cell apoptosis/necrosis. Fibroblast cells with long doubling times are more susceptible to injury induced by silica exposure than tumor cells with short doubling times. The cytotoxic effects of silica nanoparticles were significantly reduced by synthesizing the nanoparticles with chitosan. These results were the first investigation regarding the relationship between the cytotoxicity of silica and the population doubling time of different cell lines as well as the benefits of using chitosan to prepare composite silica nanoparticles. They are relevant to future applications of silica nanoparticles in drug delivery and controlled release applications.

Acknowledgments This research is partially supported by the National Science Council, Taiwan, Republic of China (Grants NSC 94-2120M-019-001 and NSC 93-2313-B-019-023); Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University; and University Grant NTOU-AF94-04-03-01-01. We thank Prof. Chung-Yuan Mou, Che-Chen Chang, and Shi-Chang Tsai (Instrumentation Center, National Taiwan University) for their assistance in the X-ray photoelectron spectroscopy analysis.

Supporting Information Available Detailed Materials and Methods; binding energies and spectra from X-ray photoelectron spectroscopy (XPS) measurements (Tables S1 and S2 and Figure S1); MTT assay of cell viabilities (Figure S2) and the LDH assay of membrane damage (Figure S3) taken after a 48 h exposure period of different cell lines to silica made of silicate, TEOS, or silicate and chitosan with varying reaction time; and TEM picture of silica-chitosan composite nanoparticles (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review October 2, 2006. Revised manuscript received December 26, 2006. Accepted January 5, 2007. ES062347T