Environ. Sci. Technol. 2007, 41, 3018-3024
Simple and Easy Method to Evaluate Uptake Potential of Nanoparticles in Mammalian Cells Using a Flow Cytometric Light Scatter Analysis HIROSHI SUZUKI, TATSUSHI TOYOOKA, AND YUKO IBUKI* Laboratory of Radiation Biology, Institute for Environmental Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka-shi 422-8526, Japan
Many classes of nanoparticles have been synthesized and widely applied, however, there is a serious lack of information concerning their effects on human health and the environment. Considering that their use will increase, accurate and cost-effective measurement techniques for characterizing “nanotoxicity” are required. One major toxicological concern is that nanoparticles are easily taken up in the human body. In this study, we developed a method of evaluating the uptake potential of nanosized particles using flow cytometric light scatter. Suspended titanium dioxide (TiO2) particles (5, 23, or 5000 nm) were added to Chinese hamster ovary cells. Observation by confocal laser scanning microscopy showed that the TiO2 particles easily moved to the cytoplasm of the cultured mammalian cells, not to the nucleus. The intensity of the side-scattered light revealed that the particles were taken up in the cells dose-, time-, and size-dependently. In addition, surfacecoating of TiO2 particles changed the uptake into the cells, which was accurately reflected in the intensity of the side-scattered light. The uptake of other nanoparticles such as silver (Ag) and iron oxide (Fe3O4) also could be detected. This method could be used for the initial screening of the uptake potential of nanoparticles as an index of “nanotoxicity”.
Introduction Nanotechnology is a highly promising molecular technology that spans many areas of science and has numerous potential technological applications (1). Nanomaterials, which range in size from 1 to 100 nm, have been used to create unique nanoscale devices possessing novel physical and chemical functional properties. Nanotubes, nanowires, fullerene derivatives, and quantum dots have recently received enormous attention. Although they are currently being considered for use in modern technology, there is a serious lack of information concerning their effect on human health and the environment. Therefore, concerns about the use of nanomaterials are being increasingly expressed by the public and in the media. One major toxicological concern is that nanomaterials are easily taken up in the human body (2-4). It is a particular concern that nanomaterials are similar in size to major classes * Corresponding author phone/fax: +81-54-264-5799; e-mail:
[email protected]. 3018
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 8, 2007
of biologically active materials like DNA, RNA, membranes, and microtubules. Notably, nanoparticles are considered to be inhaled and distributed in the body. For example, when inhaled, airborne nanoparticles are efficiently deposited via diffusional mechanisms in all regions of the respiratory tract. Inhaled poorly soluble particles have been demonstrated to cause lung cancer in rodents (5). The small size facilitates uptake into cells and transcytosis across epithelial and endothelial cells into the blood and lymph nodes, spleen, and heart (3). Access to the central nervous system has also been observed (6). Furthermore, nanoparticles penetrating the skin distribute via uptake into lymphatic channels (2). In addition to size-dependency, the potential to translocate to biological tissues and cells is largely dependent on the surface chemistry of nanoparticles, and in vivo surface modifications (2, 7, 8). Therefore, evaluations of translocation potential in biological tissues and cells corresponding to particle size and surface charge are essential for the safe use of manufactured nanoparticles (9). Titanium dioxide (TiO2) is an important material used in the paint, pharmaceutical, and cosmetic industries. Especially, TiO2 particles have come to be used as a physical UV filter in sunscreen formulations. The penetration of TiO2 microparticles into the horny layer and the orifice of hair follicles is less than 1% of the total amount of sunscreen applied, having indicated that microparticles of TiO2 are toxicologically inert, at least under nonoverload conditions (10-12). However, oily dispersions of TiO2 nanosized particles penetrate deeper than aqueous dispersions (13, 14). In addition, chronic inhalation of very small TiO2 particles can have a pulmonary toxicological effect. Ultrafine particles, defined as having a diameter of less than 100 nm, have been shown to have a greater capacity to induce inflammation of the lung than larger fine particles (15, 16). An increased surface area of particles due to a small size and oxidative stress were hypothesized to be important factors (17). Ultrafine TiO2 particles have been reported to induce micronuclei and apoptosis in embryo fibroblasts in vitro (18). Considering the increasing concerns about “nanotoxicology” accompanying the growing usage of nanotechnology, the use of nanomaterials which can spread easily in the human body and have a high translocation potential should be avoided. The uptake potential of nanomaterials depends on size, surface charge, and behavior (e.g., disperse or aggregate); however, accurate, sensitive, and cost-effective measurement techniques for characterization do not exist. The International Life Sciences Institute Research Foundation/Risk Science Institute Nanomaterial Toxicity Screening Working Group reported the elements of a screening strategy for characterizing the potential effects on human health of exposure to nanomaterials, in which they demonstrated that in vitro assays need to be used to determine important parameters that drive toxicity including the uptake potential of nanoparticles, e.g., size, surface area, surface reactivity, etc. (9). In this study, we developed a method to evaluate the uptake potential of nanoparticles using cultured mammalian cells. The analysis using flow cytometric light scatter accurately reflected the change in amounts of TiO2 taken up into cells according to particle size and surface coating. This method would be available for the initial screening of uptake into cells as an index of “nanotoxicity”.
Experimental Section Materials. TiO2 particles (5 nm: anatase, 5 nm average particle size; 23 nm: mixture of anatase and rutile 23 nm 10.1021/es0625632 CCC: $37.00
2007 American Chemical Society Published on Web 03/20/2007
FIGURE 1. Incorporation of TiO2 nanoparticles into CHO cells. CHO-K1 cells were treated with several doses (10, 100, 300, and 1000 µg/mL) of TiO2 nanoparticles (23 nm). (a) Real images taken under optical microscope. The cells were treated with TiO2 nanoparticles (23 nm) for 24 h. Red arrows point to the accumulation of TiO2 nanoparticles. Two cells were enclosed by white lines. (B) Fluorescence images taken under a confocal laser scanning microscope. The cells were treated with TiO2 nanoparticles (23 nm, 300 µg/mL) for 3 h. Left: Green fluorescence (505-550 nm) from TiO2 on excitation at 488 nm with an argon laser beam; middle: red fluorescence (>650 nm) from mitochondria stained with Mitotracker Red CMXRos on excitation at 543 nm with a helium-neon laser beam; right: merge. (C) Bleaching on exposure to the 488 nm laser. The cells were treated with TiO2 nanoparticles (23 nm, 300 µg/mL) for 3 h. Photobleaching was carried out by exposing a pre-enclosed area (white square) to the 488 nm laser 20 times using a LSM510 photobleaching system. As a positive control of photobleaching, cells treated with NBD-PS vesicles were used.
average particle size; 5000 nm: anatase,
