High-grade glioblastoma multiforme is uniformly fatal and largely unresponsive to all available treatments. Patients with these tumors usually survive for <1 year from the time of first diagnosis. Conventional surgical excision, generally limited to the main tumor mass, does not remove the microscopic foci of neoplastic cells that invade the surrounding normal brain substance beyond the main tumor mass, and are responsible for the inevitable tumor recurrence . Conventional radiotherapy cannot ablate completely these tumors , since this would require unacceptably high radiation doses that result in severe brain damage . Boron neutron capture therapy (BNCT) is a binary modality therapy that has the potential for effective treatment of many forms of cancers, including cerebral glioblastoma multiforme brain and melanomas [4–6]. BNCT is based on the neutron capture reaction, 10B(n, α)7Li, where a 10B atom captures a low-energy thermal neutron and spontaneously decays to produce the linear recoiling particles 4He (α particle) and 7Li. In tissues, these particles have short penetration ranges, approximately the width of a single cell (5 μm for 7Li and 9 μm for 4He). As the average linear energy transfer is high (7Li, 162 keV/μm; 4He, 196 keV/μm), this results in densely ionizing radiation restricted to the track of each particle [7, 8]. Thus, the essential requirement for effective BNCT is selective targeting of tumor cells with sufficient quantities of 10B atoms (15–30 μg/g or more) and their irradiation with low-energy thermal neutrons. In theory, BNCT is potentially capable of killing individual cancer cells while sparing the healthy normal parenchyma. Consequently, knowledge of the micro distribution of 10B from boronated drugs in cells of cancerous and normal tissues is of critical importance . The ideal drug would provide boron selectively to tumor cells and would be nontoxic to normal cells. Experimentally, cell culture tumor models provide a useful approach for the investigation and understanding the mechanisms of boron delivery by BNCT agents to cancer and normal cells [10, 11]. The problem, however, is that there is currently no truly satisfactory 10B target agent for any human tumor type  although several boron agents have been proposed and synthesized, but many of these have either one or more disqualifying features . The ability of BNCT to fulfill its clinical therapeutic potential thus clearly rests on the discovery and availability of effective and safe boron transport agents.
In the present preliminary study, we have investigated the use of boron nitride nanotubes (BNNTs) as boron atom carriers. BNNTs are structural analog of carbon nanotubes (CNTs) with alternating B and N atoms which entirely substitute for C atoms in a graphitic-like sheet with almost no change in atomic spacing. Thus, BNNTs are composed of a substantial number of boron atoms (about 50%) equivalents to hundreds to thousands per each nanotube. Hence our hypothesis underlying the present investigation is that BNNTs may serve as effective boron targets for BNCT. BNNTs have generated considerable interest within the scientific community by virtue of their unique properties. As with CNTs, they have various potentially useful structural and electronic applications [14, 15] and share good flexibility with CNTs with a Young modulus of 1.22 ± 0.24 TPa [16, 17] but have superior chemical and thermal stability. Together with their high resistance to oxidation and stability , these properties mark BNNTs as attractive potential candidates for a wide range of applications. Nevertheless, in sharp contrast to the many proposed biomedical applications of CNTs in recent years, e.g., biosensors, DNA chip, nanovectors for drug, protein and gene delivery , etc., the use of BNNTs in this field has been largely unexplored . Cytocompatibility studies and the interaction between BNNTs and living cells have only been reported recently [21, 22] by our group and other biomedical applications concerning their magnetic properties are actively under investigation in our laboratory.
In the present study, we realized stable dispersions of BNNTs employing a positively charged protein (ploy-l-lysine, PLL) as dispersion agent. Poly-l-lysine coated BNNTs were then conjugated with fluorescent molecules (quantum dots) to enable their tracking in living cells. Folic acid was used as a targeting ligand to functionalize BNNTs against tumor cells, because of the ease of the reaction procedure and the small size of the folate molecule, enabling a stable binding with the nanotube coating, thus permitting folate receptor-mediated endocytosis of PLL-BNNTs. To date, folate has been used as a targeting ligand for both tumor imaging and cancer chemotherapy [23–26].
The functionalized BNNTs were characterized by imaging, Z-potential, size distribution, and UV–Vis spectra. In vitro uptake tests were carried out on human glioblastoma multiforme cells and on healthy human primary fibroblasts as controls.