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Boron Nitride Nanotubes Are Noncytotoxic and Can Be Functionalized for Interaction with Proteins and Cells

We report the discovery that boron nitride nanotubes (BNNTs), isosteres of CNTs with unique physical properties, are inherently noncytotoxic. Furthermore, we developed a biomemetic coating strategy to interface BNNTs with proteins and cells. Finally, we showed that BNNTs can deliver DNA oligomers to the interior of cells with no apparent toxicity. This work suggests that BNNTs may be superior to CNTs for use as biological probes and in biomaterials.


Part I -Supporting Figures (S1-S5)
. Structure of [G-2] Man. The dendrimers comprise synthetic carbohydrate ligands at the chain ends that enable specific binding to receptors in solution. A pyrene group at the dendrimer focal point allows adsorption to CNT surfaces through π-stacking and hydrophobic interactions.

Synthesis of BNNTs
The highly pure boron nitride nanotubes were synthesized by a chemical vapor deposition process. Briefly, a hollow cylinder of pyrolytic boron nitride was filled with equal molar quantities of Magnesium oxide (MgO) and Boron (B). Small holes were drilled into the sides of cylinder and one end of the tube was capped off. A small amount (approx. 20 SCCM) of nitrogen gas was flowed into the cylinder from the other end via a thin alumina tube. The assembly was placed in a flowing ammonia atmosphere and heated in a tube furnace to 1200 °C. After 20 min, the cylinder was removed from the furnace, and a fine coating of pure white film was scraped off the exterior of the cylinder, near the small exhaust holes. Scanning and transmission electron microscopy revealed the film to be composed almost entirely of multi-walled boron nitride nanotubes.

SEM and TEM characterization
SEM images were obtained on an FEI Sirion XL 30 SEM operated at 5 keV. TEM images were obtained on a JEOL 2011 microscope operating at electron energy of 100 keV.
In a typical preparation experiment, 0.5 mg of BNNTs were suspended in 5 ml of aqueous glycodendermer solution (0.1%~0.5% in water or in PBS buffer with pH = 7.4). The mixture was sonicated using a water-bath sonicator for 1 h. First, insoluble materials were removed by centrifugation at 1,000 x g for 30 min. Then the excess free glycodendrimer was removed by dialysis against PBS buffer for 24 h.

Functionalization of BNNTs with DNA
BNNTs were sonicated in a solution of a fluorescently labeled 20-mer oligonucleotide (FITC-ATCGATCGATCGATCTATCG; purchased from Integrated DNA Technologies, IA) for 30 min (DNA concentration 20 µM). The mixture was centrifuged at 1,000 x g for 30 min, and the resulting suspension was decanted from insoluble materials.

Lectin binding assays
Lectins, Canavalia ensiformis agglutinin (Con A) conjugated with fluorescein isothiocyanate (Con A-FITC) and Helix Pomatia agglutinin conjugated with fluorescein isothiocyanate (HPA-FITC), were obtained from EY-Laboratories. In a typical experiment, a 1-mL solution of lectin-FITC conjugates (100 µg/mL) in PBS buffer containing 1 mM Ca 2+ was added to the suspensions of glycodendrimer-coated BNNTs (1 mL). The reactions were incubated for 1 h at rt in the dark. After incubation, the solutions were all subjected to dialysis against the buffer for 48 h. The dialyzed solutions were analyzed at 517 nm using a fluorescence microplate reader (excitation wavelength 492 nm). Fluorescence intensities were corrected for background fluorescence by subtracting the fluorescence intensities of glycodendrimer-coated BNNTs alone.

Cell culture conditions
All cell lines were maintained in a 5% CO 2 , water-saturated atmosphere at 37 o C and media were supplemented with penicillin (100 unit/mL), streptomycin (0.1 mg/mL) and 10% FCS unless otherwise indicated. CHO cells were grown in Ham F12 nutrient mixture. HEK 293 cells were grown in DMEM media.

Cell binding of glycodendrimer-coated BNNTs
CHO cells were seeded onto glass slides mounted with tissue culture wells (LAB-TEK) and allowed to adhere for 2 d. The cells were washed 3 times with PBS, and then fixed in 3% paraformaldehyde in PBS (the fixation step could also be performed after cell binding to the coated BNNTs). After three washes, cells were blocked in PBS with 1% bovine serum albumin for 20 min, followed by the addition of glycodendrimer-coated BNNTs complexed with appropriate lectin-FITC conjugates as described above. After a 1-h incubation at 4 °C in the dark, the cells were washed 3 times. The cells were then mounted using Vectashield with 4,6-diamidino-2-phenylindole (Vector Laboratories). A Zeiss Axiovert 200M inverted microscope equipped with a 63 × 1.4 NA Plan-Apochromat oil immersion lens was employed for imaging. A 175W xenon lamp housed in a Sutter DG4 illuminator linked to the microscope by an optical fiber assured shuttering and illumination. Images were acquired using a CoolSNAP HQ CCD camera (Roper Scientific). Slidebook software (Intelligent Imaging Solutions) was used to control the microscope and the camera.