Effect of Single-walled Carbon Nanotubes Entry into Mammalian Cells
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Effect of Single-walled Carbon Nanotubes Entry into Mammalian Cells Howard H. Chen1, Bradly Baer2, Christopher S. Evans2, Hannah M. Ponek3, and Michelle Chen3 1 Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, U.S.A. 2 Department of Chemistry, Point Loma Nazarene University, San Diego, CA 92106, U.S.A. 3 Department of Physics and Engineering, Point Loma Nazarene University, San Diego, CA 92106, U.S.A. ABSTRACT We reported a mammalian cell-imaging paradigm to study the cellular response to singlewalled carbon nanotubes (SWCNTs). Chinese Hamster Ovarian (CHO) cells were exposed to SWCNTs resuspended in physiologically compatible buffer (phosphate buffered saline, PBS), at concentrations ranging from 0 to 50 μg/mL. Upon exposure, we optically imaged the cells in order to (1) visualize the accumulation SWCNTs in cells in real-time; (2) qualitatively and quantitatively assess the morphological changes associated with cellular stress in the presence of SWCNTs; and (3) serially quantify cell survival with highly sensitive bioluminescence-based imaging. Our results showed that the cell survival obtained from optical imaging agreed with that from CellTiter-Glo (CTG) luminescence viability assay. Acute compromise in the CHO cell’s survival rate was observed under high concentrations of SWCNT exposure. The cellular response as a function of SWCNT concentrations, and exposure time was further investigated. INTRODUCTION Research on applying carbon nanotubes for ultrasensitive detection, disease diagnosis, and drug delivery was rapidly developing [1-6]. While the fundamental and technological findings on carbon nanotubes showed great promise, the effect of the carbon nanotubes on the environment and health remained controversial [7-11]. Wide biodistribution of SWCNTs was well established [1,7] thus the importance of studying toxicity in detail. While acute toxicity was well documented in literature [8-9], whether low dose of SWCNTs confer similar toxic effect was not yet well understood. In this paper we presented an in vitro cell model to systematically determine the impact of SWCNTs on CHO cells’ survival as a function of SWCNT concentration and exposure time. Our experimental design took advantage of: (1) the ease of visualizing SWCNTs intake in realtime; (2) the well characterized morphological changes associated with cellular stress in CHO cells; (3) serial quantification of cell survival with highly sensitive bioluminescence-based imaging. EXPERIMENTAL DETAILS Carbon nanotubes Purified SWCNTs in sterile PBS solution of pH 7 was obtained from Nano-Lab. SWCNTs were grown by chemical vapor deposition which produced median diameter of 1nm
and length of 3 m. The nanotubes came COOH functionalized to allow good dispersion without surfactants. Series of acid purification and filtration removed Ni-Co-Fe catalyst particles to achieve > 97% of SWCNTs. Prior to experiments, SWCNT solutions were sonicated for about 10 minutes before each dilution with PBS and before the
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