Tuning the Cellular Uptake and Cytotoxicity of Carbon Nanotubes by Surface Hydroxilation

Tuning the cellular uptake and cytotoxicity of carbon nanotubes by surface hydroxilation

Xiaoyong Zhang1,2,1, Ying Zhu1, Jing Li1, 2, Zhiyong Zhu1, Jingye Li1, Wenxin Li1,*, and Qing Huang1,*

1 Laboratory of Physical Biology, Shanghai Institute of Applied Physics, CAS,

Shanghai 201800, China

2 Graduate School of Chinese Academy of Sciences, Beijing 100049, China

1.1 Synthesis and characterization of MWCNTs

1* Corresponding authors. Tel: +86-21-39194530; Fax: +86-21-59553021. Xiaoyong Zhang, E-mail address: [email protected]; Wenxin Li, [email protected]; Qing Huang, E-mail address: [email protected] The CNT samples with different surface properties was prepared according to previous reports (Fenoglio et al., 2008; Guo et al., 2005). First, to obtain the purified

CNT (P-CNT), the pristine CNT was first oxidized by concentration HNO3 for 24 h at the temperature of 80 °C (method 1). A fraction of P-CNT was further oxidized by

mix acid solution (volume of 98% H2SO4: volume of 68% HNO3=3:1) for 48 h at the temperature of 80 °C (method 2). Then the oxidant CNT was washed with distilled water until the pH>6. The solid was collected and dried under vacuum at 60 °C overnight, and the resultant powder was named as CNTox. CNTan was obtained

through high temperature annealing of CNTox at 600 °C for 4 h under the N 2 flow

(method 3), which could remove the amorphous carbon and functional groups on the surface of CNTox. To obtain the CNTir, P-CNT was irradiated at the dosage of 250

KGay in the present of concentration H2SO4 (method 4) to induce much more defects on the surface of CNT. And then irradiated CNT was further oxidized by mix acid solutions as described above (method 2). After washed by distilled water and dried under vacuum, the finally resultant of CNT was obtained and named as CNTir. Fig. S1 Schematic showing three different CNT preparations compared in this study.

The number 1, 2, 3, 4 represent the treatment method 1, method 2, method 3, and method 4, respectively. CNT samples were prepared by the procedures as follow.

CNTox: 1-2, CNTan: 1-2-3, and CNTir: 1-4-2.

1.2 Characterization of CNTs

All of the MWCNT samples were thoroughly characterized by a series of characterization techniques. The microstructure of all the CNT samples was carried out by transmission electron microscopy (TEM; JEOL, JEM-2010). CNT samples were prepared by placing a droplet of MWCNT aqueous suspension on 300 mesh copper carbon grid. All of these MWCNT samples were dried overnight at 60 °C under vacuum before observation. Raman spectroscopy was employed for the determination of the presence of defects and amorphous carbon. The characterization of the materials was carried out in a Confocal Nikon Modified Raman Micro-probe with laser source at 514.5 nm (2.41 eV), readily obtainable from an argon ion laser. Thermal Gravimetric Analysis (TGA) was carried out on NETZSCH STA 429C thermal analyzer under argon flow at the heating rate of 10 °C/min to obtain information on the decomposition and burning properties of CNT samples. The temperature of the sample was varied from room temperature to 600 °C. The percentage of residual catalyst of the CNT samples was determined by high resolution inductively coupled plasma mass spectroscopy (ICP-MS;

Thermo Elemental, X-7), For the ICP-MS analysis, CNT powders were dispersed with the mixture 2.5

mL HNO3 and 0.5 mL H2O2 (30%), and these solutions were then digested at 160 °C for 12 h. The surface wettability of the modified CNTs was evaluated by water contact angle measurement. Briefly,

CNT powders were cast on glass slides to form films by using double-side tape. The CNT films were at the dimension of 2×4 cm2, and tens of millimeters in thickness. The glass slides were put on a flat platform and then 50 μl of water droplets were dripped onto the CNT films. The contact angle and the shape of water on the CNT films were measured with an optical tensionmeter (THETA, KSV

Instruments Ltd.).

1.3 Observation of interaction by optical microscopy

The interaction between cells and the CNT samples was investigated by optical microscope. Hela cells were seeded at 1×105 mL–1 cells in 6 well cell culture plates and incubated overnight to allow for cell adherence. Cells were washed twice with phosphate buffered saline (PBS) and then incubation with 40 μg mL–1 of CNT at 37

°C for 2 h. Before optical observation, cells were washed thrice with PBS to remove the uninternalized CNTs. Then cells were observed and recorded using an optical microscope (Motorized inverted system microscope IX81/IX81–ZDC, Japan) to evaluate the interaction between cell and CNTs. The overall magnification was 400×.

Results

2.1 Characterization of CNTs

The metal content of CNTan, CNTox, CNTir was measured by ICP-MS and list in Table S1. Results showed that No Co we detected by ICP in all the CNT samples. The metal content in pristine CNT is

0.6305% for Fe and 0.14% for Ni, compared with the pristine CNT, only limited Fe and Ni was found in CNT samples, suggesting that almost all the metal contaminants have been removed after surface modification.

Table S1 metal contaminant in CNT samples determined by ICP-MS

The metal content was determined by ICP-MS, and the * indicated that the concentration of Co was not detected.

2.2. Interaction between cells and MWCNT

The interaction between CNTs and cells was observed by optical microscopy. Fig.

S2 shows the optical microscopy images of different CNT samples incubation with

Hela cells for 2 h. It can be seen that only a few CNTan was adhered to cell surface or internalized by cells (Fig. S2A). However, a large number of CNTox and CNTir aggregates with their size at the micrometer-scale were observed on the surface of cells (as shown in Fig. S2B and Fig. S2C). Furthermore, to identify CNTs were indeed internalized into cell interior; TEM observation was performed previously (Zhu et al., 2009). We found that the MWCNT aggregates as large as several microns could also enwrapped by the elongated cell synapses and internalized by Hela cells.

Fig. S2. Optical microscope images of Hela cells after cells incubated with three kinds of CNT samples in complete cell culture medium for 2 h. (A) CNTan, (B) CNTox, (C)

CNTir. The concentration of CNTs was 40 μg mL–1 (magnification =400).

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