Preparation and functionalization of boron nitride containing carbon nanohorns for boron neutron capture therapy

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Preparation and functionalization of boron nitride containing carbon nanohorns for boron neutron capture therapy Yoko Iizumi a, Toshiya Okazaki a,*, Minfang Zhang a, Ryota Yuge b, Toshinari Ichihashi b, Maki Nakamura a, Yuzuru Ikehara a, Sumio Iijima Masako Yudasaka a,* a b c

a,b,c

,

National Institute of Advanced Industrial Science and Technology, Higashi, Tsukuba 305-8565, Japan Smart Energy Research Laboratories, NEC Corporation, Tsukuba 305-8501, Japan Faculty of Science & Technology, Meijo University, Nagoya 468-8502, Japan

A R T I C L E I N F O

A B S T R A C T

Article history:

Boron neutron capture therapy requires boron carriers that can deliver abundant boron to

Received 5 February 2015

tumors. To obtain such a carrier, we have prepared boron nitride (BN) containing carbon

Accepted 24 May 2015

nanohorn aggregate (CNH) by heating CNHs with ammonia borane at 800 C. The obtained

Available online 29 May 2015

BN-CNH had a C:B:N mole ratio of 5:2:2, as estimated from X-ray photoemission spectroscopy. The nanohorn tubule walls had graphene–BN double layers or BN–graphene–BN triple layers. The graphite-like sheets constituting the CNHs together with the nanohorn tubules also had BN layers on their surfaces. BN release from BN-CNH was small in cell culture medium, and about 70–80% of the BNs remained on the CNHs. For selective accumulation in tumor cells, the BN-CNH was coated with phospholipid polyethylene glycol having folate (BN-CNH/PLPEG-FA). In primary cell culture experiments, human tumor KB cells overexpressing FA receptors ingested more BN-CNH/PLPEG-FA than those without FA, while normal human FHs 173We cells did not show preferential uptake due to FA. The quantity of boron per cell exceeded the criteria required for boron neutron capture therapy by more than 100 times in the cell experiments. These results suggest that BN-CNH is potentially a good carrier for boron neutron capture therapy.  2015 Elsevier Ltd. All rights reserved.

1.

Introduction

In boron neutron capture therapy (Boron-NCT), a neutron beam is used to irradiate malignant brain tumors, i.e., glioblastoma. The nuclear reaction between boron and low energy neutrons generate a beams and 7Li, which have short trajectories, about 10 lm, comparable to tumor cell sizes.

The neutron beam is harmless to cells having no boron in them [1]. Thus, Boron-NCT can selectively kill tumor cells even where tumor and normal cells coexist, if the boron is only in the tumor cells. Boron-NCT is feasible to use for the control of not only glioblastoma, but also other solid tumors with dissemination, massive invasion and metastasis by properly delivering the boron to cancer cells.

* Corresponding authors. E-mail addresses: [email protected] (T. Okazaki), [email protected] (M. Yudasaka). http://dx.doi.org/10.1016/j.carbon.2015.05.085 0008-6223/ 2015 Elsevier Ltd. All rights reserved.

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Boron-NCT studies started in the 1930s [1], and it has been used clinically since the 1950s [1–3]. Much of the effort has been placed on the development of nuclear reactors suitable for Boron-NCT, and there has been remarkable progress over the years. On the other hand, there has not been much research on boron carriers that can deliver boron selectively to tumor cells. Boron-NCT must accumulate a large amount of boron in a tumor (109 B atoms per cell, or 10–30 ppm B in tissue) [1,4]. The boron carriers of borocaptate sodium mercapto undecahydrododecaborato (BSH) [2] and p-boronophenylalanine (BPA) [3] are applied for Boron-NCT, which need high dose levels in the body tens mg/kg body [1], because their selective accumulation in tumor cells is difficult [1,4,5]. To solve this problem, various boron compounds have been tried, including, boron-containing compounds such as porphyrin [6], dendrons [7], nanomaterials [8], carbide [9], BN nanotubes [10], and carborane conjugated to singlewalled carbon nanotunes [11], but none of them has shown low enough toxicity and high enough accumulation at tumor sites for clinical use. In this context, we have prepared boron nitride (BN) loaded single-walled carbon nanohorns. Single-walled carbon nanohorn tubules are pseudo cylindrical materials with horn shaped-tips having diameters of 2–5 nm and lengths of 40–50 nm, and thousands of them form a spherical aggregate with a diameter of about 100 nm [12,13]. In this report, this aggregate is designated as ‘‘CNH’’, and individual single-walled carbon nanohorn tubules, ‘‘nanohorn tubules.’’ The CNH is robust aggregate and individual nanohorn tubules cannot be separated. CNH has low toxicity [14,15] and is modifiable with a tumor-targeting molecule of folic acid [16]. A method that uses ammonium borane for the creation of BN films [17] and BN tubes inside singlewalled carbon nanotubes (SWCNTs) [18] has been adapted to load BN on CNHs. The obtained BN-loaded CNH (BN-CNH) was functionalized with phospholipid polyethylene glycol (PLPEG) conjugated with folate (PLPEG-FA). Preferential uptake of BN-CNH/PLPEG-FA by tumor cells was confirmed in in vitro experiments.

2.

Experimental

2.1.

BN-CNH preparation

CNHs were prepared by CO2 laser ablation of graphite [12,13]. Since no metal catalysts were used in the preparation, the CNHs did not contain any metal [12,13]. To make the inner space of the nanohorn tubules accessible, holes were opened by oxidation in dry air by increasing the temperature at a rate of about 1 C/min up to 500 C [19]. The oxidized CNH (10 mg) and ammonium borane (10 mg) were vacuum-sealed in quartz glass ampoules. The background pressure in the vacuum system was 5 · 104 Pa being evacuated with a diffusion pump. The obtained ampoule was slowly heated up to the target temperature for 24 h, kept at that temperature for 24 h, and subsequently naturally cooled. During the heating, the chemical reaction H3BNH3 ! BN + 3H2 occurred, and BN was generated on the CNH. The BN-CNH was taken out of the ampoule by pouring in and drawing off ethanol. The obtained BN-CNH in

ethanol was filtered (pore size 1 lm), washed 3 times by filtration with ethanol (10 mL), and dried. The structure of BN-CNH was studied with transmission electron microscopy (TEM; JEOL2100F, JEOL Ltd.) at 120 kV. Xray diffraction analysis (XRD) was performed with the capillary method (R-AXIS IV, Rigaku Co.). The existence of boron and nitrogen in BN-CNHs was examined with an electron energy loss spectroscopy (EELS) device (Enfina, Gatan Inc.) equipped on the scanning transmission electron microscope (STEM; HD2300, Hitachi Co.) operated at 120 kV. The elements were also examined with X-ray photoemission spectroscopy (XPS; SSX-100, Surface Science Instruments) and induction coupled plasma atomic emission spectroscopy (ICP; ULTIMA 2, Horiba Co.). Thermogravimetric analysis (TGA; TGA-50, Shimadzu Co.) was performed in air to confirm the structure stability and for the quantity estimation of boron. The stability of the BN attachment to CNH in cell culture medium was examined prior to the cell culture experiments. BN-CNH (1 mg) was mixed with 2 mL of cell culture medium (RPMI 1640 (Gibco) with 10% fetal bovine serum and 0.02% P enicillin–Streptomycin) in Eppendorf (5 mL) and bathsonicated for 5 min. After the sonication, the BN-CNH dispersion solution was left still at 37 C in air for 1–10 days, and thereafter, the BN-CNH was separated by filtration (pore size 1 lm). To quantify the boron in the separated BN-CNH by using ICP, the separated BN-CNH was dispersed with a 1% aqueous solution of SDBS (2 mL) by using bath-type sonication for 5 min, and 0.5 mL of this dispersion solution was diluted with 4.5 mL of a 1% aqueous solution of SDBS in glass bottles (10 mL). This was followed by ultrasonic homogenization with a tip-type sonicator (SONICS VCX130, Labotal Scientific Equipment Ltd.) operated at 130 W for 3 min. The resultant dispersion solutions were subjected to the ICP measurements.

2.2. Preparation of BN-CNH coated with PLPEG-FA (BNCNH/PLPEG-FA) PLPEG having an NH2 group and folic acid was purchased from Avanti Polar Lipids, Inc. and Tokyo Chemical Industry Co., Ltd., respectively. Their molecular structures are shown in Fig. 1. PLPEG-FA was prepared by following the previously reported method [20]. FA (50 mg) was dissolved in dimethyl sulfoxide (4 mL) and mixed with PLPEG (200 mg), pyridine (2 mL), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Tokyo Chemical Industry Co., Ltd.) (38 mg). This mixture was stirred for 4 h at room temperature. The reaction product of PLPEG-FA was confirmed by the appearance of a new spot (Retention factor = 0.57) in thin layer chromatography using silica gel GF (chloroform:methanol:water = 75:36:6). The disappearance of PLPEG (Retention factor = 0.76) from the reaction mixture was confirmed by using ninhydrin spray. Pyridine was removed by rotary evaporation. Water (50 mL) was added to the reaction mixture, and centrifuged to remove trace amounts of insoluble materials. The supernatant was dialyzed in the tube (MWCO: 25,000) against water (5 · 2000 mL). The dialysate was lyophilized.

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Fig. 1 – Molecular structures of PLPEG and FA.

In coating BN-CNH with PLPEG-FA, BN-CNH (3 mg) and PLPEG-FA (4 mg) were mixed using bath sonication in phosphate buffered saline (PBS) (2 mL) for 20 min at room temperature. High dispersion and high dispersion stability in PBS are required for adding BN-CNH/PLPEG-FA to the cell culture medium. To confirm that this was the case, the particle sizes were measured with dynamic light scattering equipment (DLS) (FPAR-1000, Otsuka Electronics Co., Ltd.) and the time course of light transmittance at 700 nm was measured with a light absorption spectrometer (UV-3100PC, Shimadzu Co.) for 5 days. As a control material, BN-CNH was similarly coated with PLPEG (BN-CNH/PLPEG), and the dispersion stability was examined by the measurements of DLS and the time course of 700-nm light absorbance.

Bradford protein assay. For the quantification of BN-CNHs ingested by the cells, the centrifuged sediment was redispersed with the remaining supernatant and 0.01 mL PBS, which was returned to the remaining lysates-PBS solution. The resultant lysate-PBS solution was sonicated with a tiptype sonicator (SONICS VCX130, 130 W) for 10 min to improve the dispersion homogeneity of BN-CNHs. The CNH quantity in the obtained BN-CNH dispersion solution was estimated by measuring the 700-nm light absorbance (Lambda 1050, Perkin Elmer Co., Ltd.) [16]. Here, the quantity-absorbance calibration line for CNH obtained previously [16] was used.

2.3.

It is reported that chemical vapor deposition of ammonium borane produces hexagonal BN sheets via a reaction intermediate of borazine (B3N3H6) [17,22]. If the ammonium borane is heated with SWCNT, hexagonal BN nanotubes form inside the SWCNTs [18]. As estimated from these studies, the BN sheets become deposited on CNHs when they are heated with ammonia borane, evidence of which is shown below.

Cell experiments

KB cells (ATCC), i.e., a cell line of human epidermal carcinoma of the mouth over-expressing FA receptors [21], and normal human embryo cells, i.e., FHs173We cells (ATCC), were cultured with RPMI 1640 (Gibco) with 10% fetal bovine serum and 0.02% penicillin–streptomycin. They were incubated at 36.5 C in an atmosphere containing 5% CO2 for 24 h. After this, the culture medium was changed to one not containing FA (RPMI 1640 Medium, no folic acid with 10% FBS and 0.02% penicillin–streptomycin) to which BN-CNH/PLPEG or BNCNH/PLPEG-FA were added (CNH: 10 lg/mL) and incubated for 24 h. After that, the culture medium was replaced with PBS, when the BN-CNH/PLPEG or BN-CNH/PLPEG-FA that were not ingested by the cells was removed. To estimate the quantities of BN-CNH/PLPEG and BNCNH/PLPEG-FA that were ingested by the cells, the cells were lysed and separated from BN-CNHs by centrifuge. The CNH quantity of BN-CNH was estimated from 700-nm light absorbance, which was normalized by the protein quantities of the cells as reported in reference [16]. In detail, the cells were detached from the cell dishes and lysed by the addition of CelLytic M (Shigma) and protease inhibitor cocktail (Nacalai) (99:1) 1 mL, followed by incubation at 36.5 C in 5% CO2 atmosphere for 20 min. The lysates were mixed with PBS 1 mL, being designated as lysate-PBS solution. The lysate-PBS solution 0.1 mL was centrifuged for 50 min at 1800·g, and the protein quantity in the 0.01 mL supernatant was evaluated by

3.

Results

3.1.

Structure analysis of BN-CNH prepared at 800 C

3.2.

Electron microscopy

TEM observation of BN-CNH prepared by heating CNH with ammonia borane at 800 C revealed that the sphericalaggregate form of CNH was largely maintained (Fig. 2a); however, several structure changes occurred. The higher magnification images showed that the tubule walls changed from a single graphene layer of the starting nanohorn tubules to BN–graphene double layers or BN–graphene–BN triple layers in BN-CNH (Fig. 2b and c, red arrows). There were BN layer fragments attached to the outside walls of the nanohorn tubule walls (Fig. 2d, a white arrow head). In addition, there were BN fragments inside the nanohorn tubules (Fig. 2c, a black arrow head). The BN layers were also deposited on graphite-like thin sheets (GLSs) in the CNH. Here, the GLSs constructed the CNH together with nanohorn tubules. In the original CNHs, about 60% of the GLSs were double layers, and about 20% were quadruple layers, as previously reported (Fig. 2f) [23]. However, the layer number increased as a result of the BN

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Fig. 2 – TEM images of BN-CNH prepared at 800 C (a–d) and layer number histogram of graphite-like thin sheet in BN-CNH (e). Red arrows indicate 2 and 3 layers, and black and white arrow heads indicate the fragments of BN layers localized inside the nanohorn tubules and on the outside walls of the nanohorn tubules, respectively. A magenta circle denotes the graphite-like thin sheets. Layer number histogram of graphite-like thin sheets in as-grown CNH is also shown (f) [23]. (A color version of this figure can be viewed online.)

layer deposition (Fig. 2b, a magenta circle), and the corresponding histogram (Fig. 2e) became very different from that for the CNH before the BN deposition (Fig. 2f), reaching a maximum at a layer number of four. This means that hexagonal BN sheets deposited on GLSs were located on the surface of the GLS, resulting in the changes of double layers into quadruple layers.

3.3.

X-ray diffraction

The XRD analysis of the CNHs showed diffraction peaks at 26.5 (0.342 nm) and 25.2 (0.353 nm) (Fig. 3, black line). The XRD data on the BN-CNH showed the same peaks but the intensity of the 25.2 peak became much stronger (Fig. 3, red

Fig. 3 – XRD patterns of CNH (black line) and BN-CNH (red line). (A color version of this figure can be viewed online.)

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line). The 26.5 peak corresponds to the micrometer-sized graphitic (GG) particles, which is a 5% impurity of CNHs [24]. The 25.2 peak is due to diffraction from GLSs because the layer distance, 0.353 nm, coincides with the GLS layer distances, as estimated from the TEM images in the previous study [23]. The intensity increase of 25.2 peak of BN-CNH probably corresponded to the increased layer numbers as a result of BN layers deposition on GLSs and nanohorn tubules (see the TEM images in Fig. 2b–d and histograms in Fig. 2e–f). Here, we presume that the distance between the BN layer and the surface layer of the GLS or nanohorn tubule wall was similar to the GLS layer distances. h-BN has a (0 0 2) diffraction peak at about 27 [25], which was not obvious in the XRD data of Fig. 3, suggesting bulk BN crystals were not likely included in the BN-CNHs. The layer number increase could occur on GG; however, it did not increase the 26.5 peak intensity, because the layer number increase caused by the deposition of BN-layers was not significant for GG with 105 or more layers.

3.4.

Electron energy loss spectroscopy

The EELS measurements confirmed the existence of boron and nitrogen. Boron and nitrogen were detected at all measurement points on the BN-CNHs. Fig. 4 shows a typical EELS (the inset is a TEM image indicating the measurement point). The figure shows boron and nitrogen peaks characteristic of hexagonal BN [26]. The carbon peaks were similar to those of graphenes [27]. The peak intensities indicated that C:B:N mole ratio was 51:27:22.

3.5.

X-ray photoemission spectroscopy

The elements and their quantity ratio were further analyzed with XPS. The peaks corresponding to B1s, N1s, C1s, and O1s were observed in the wide scan spectrum (Fig. 5). The mole ratio of C:B:N estimated from the XPS peak intensities was about 5:2:2. In the narrow scan spectra, the C1s, B1s, and N1s peaks seem to have certain weak components at the higher energy side, which probably correspond to the carbon, boron, and nitrogen binding to oxygen.

Fig. 4 – EELS of BN-CNH prepared at 800 C. A yellow point in the inset TEM image indicates the EELS measurement point. (A color version of this figure can be viewed online.)

Fig. 5 – XPS spectra of BN-CNH prepared at 800 C.

3.6.

Thermogravimetric analysis

The TGA analysis performed in air showed that CNH combusted at about 600 C, while BN-CNH combusted at about 680 C and the combustion temperature range became broader (Fig. 6). This is reasonable because a multiple-layer structure is more combustion resistive than a single-layer structure. In addition, the bulk BN combustion temperature was about 1000 C [28], higher than that of graphite, 750– 800 C [24,29]. Assuming that the TGA residue at 1000 C (Fig. 6) was B2O3 and B:N = 1:1, the C:B:N mole ratio can be estimated to be 5:1.6:1.6, which largely coincides with the EELS and XPS results. There was a 10% weight increase at ca. 830 C (Fig. 6), which could be due to the change of BN to B2O3 [28]. The Raman spectra of CNH and BN-CNH similarly showed G (1590 cm1) and D (1340 cm1) bands (Supplementary Information 1). The peak characteristic of BN (1366 cm1 [30]) was not clearly visible, perhaps because the D band of

Fig. 6 – TGA results of CNH and BN-CNH measured in air. (A color version of this figure can be viewed online.)

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CNH appeared near this wavenumber (Supplementary Information 1).

3.7.

Cell experiments of BN-CNH/PLPEG-FA

We carried out the primary biological study for BNCNH/PLPEG-FA with an eye to its possible application to Boron-NCT. The stability of the BN attachment to CNH in cell culture medium was examined. Then, the dispersion stability of BN-CNH/PLPEG-FA in PBS was examined. Finally, preferential uptake of BN-CNH/PLPEG-FA in tumor cells was studied.

3.7.1.

Stability of BN attachment on CNH

BN should not be detached or released from the CNH during the cell experiments. To evaluate the stability of the attachment, BN-CNH was immersed in cell culture medium at 37 C for various periods, and the amount of boron remaining on the CNH was estimated by ICP. The remaining boron was 70–80% of the original amount after immersion for 10 days and otherwise there were no obvious changes (Fig. 7). This stability was good enough for the primary cell test to examine the effect of functionalization with DSPEPG-FA. Here, the amount of boron fell from 100% to 70–80% during the dispersion process in the culture medium with sonication for 3 min. The results for the BN-CNHs prepared at different temperatures are summarized in Supplementary Information 2 and show that the optimum preparation temperature was 800 C in terms of loaded BN quantities and stability of BN attached to the CNH.

3.7.2.

Fig. 8 – Particle size distributions of (a) BN-CNH/PLPEG and (b) BN-CNH/PLPEG-FA dispersed in PBS. (A color version of this figure can be viewed online.)

Dispersion of BN-CNH/PLPEG-FA

The dispersion state of BN-CNH coated with PLPEG-FA in PBS at room temperature was examined to find out if it was suitable for use in the cellular experiments. PLPEG and PLPEG-FA were excellent dispersants; the CNH and BN-CNH dispersed with them had particle sizes of about 130 nm (Fig. 8a–b), as measured by DLS. These sizes were close to those observed with TEM (about 100 nm); thus, the PLPEG and PLPEG-FA coating was effective at dispersing the BN-CNH individually. The dispersion stability of BN-CNH/PLPEG and BNCNH/PLPEG-FA in PBS was further evaluated from the time course of the 700 nm light transmittance measurements. The 700-nm light transmittance did not change much over the course of 5 days for all the dispersion solutions (Fig. 9),

Fig. 7 – Quantities of boron remaining in BN-CNH after immersion in cell culture medium at 36.5 C for various periods.

Fig. 9 – Optical absorbance at 700 nm of BN-CNH/PLPEG and BN-CNH/PLPEG-FA dispersed in PBS.

meaning again that PLPEG and PLPEG-FA were good at dispersing BN-CNH in PBS.

3.7.3. cells

Preferential uptake of BN-CNH/PLPEG-FA by KB tumor

The results of the primary cellular experiments using BNCNH/PLPEG-FA and BN-CNH/PLPEG are shown here. KB cells are a cell line of human epidermal carcinoma of the mouth, and they overexpress FA receptors on their surfaces [21]. After KB cells were incubated with BN-CNH/PLPEG-FA for 24 h, they were observed with confocal microscopy. BNCNH/PLPEG-FA, appearing as black particles in Fig. 10b, seemed to be mainly located inside KB cells whereas some was outside or on the surface of the cells. When BNCNH/PLPEG was used, black agglomerates internalized in the cells were rarely observed (Fig. 10a). The cellular uptake quantity of BN-CNHs was evaluated using the 700-nm light absorbance of the CNHs contained in the cell lysates. The 700-nm light was adopted because the biological materials well transmit the light at 700 nm, while CNHs absorb. The BN, colorless material, do not absorb this wavelength light. The quantity of BN-CNH/PLPEG-FA ingested by the KB cells was much larger than that of BN-CNH/PLPEG (Fig. 10c). This is because KB cells have a lot of FA receptors

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Fig. 10 – Uptake of BN-CNH/PLPEG and BN-CNH/PLPEG-FA by KB (a–c) and FHs173We (d–f) cells. Confocal microscopy images (a, b, d, e) and 700-nm light absorbance of CNHs in cell lysates (c and f). (A color version of this figure can be viewed online.)

on the cell surfaces. Similar experiments using normal human whole embryo FHs173We cells that do not overexpress FA receptors were carried out, in which the preferential uptake due to FA was not observed (Fig. 10d–f). The results of quantity estimation of BN-CNH ingested by the cells coincided with the optical microscopy observation, indicating the effect of FA attached to BN-CNH via PLPEG induced the preferential uptake by the tumor KB cells, while such tendency was not observed for the normal cells.

3.7.4.

Toxicity

CNHs have been reported to have low toxicity [14,15,37]. Megascopic tests of cultured cells in dishes did not reveal any abnormal death of KB and FHs173We cells caused by BN-CNHs. The cytotoxicity of BN-CNHs was examined with RAW264.7 macrophages, showing that the cytotoxicity was a little higher than that of CNHs (Supplementary Information 3) [37]. The reason why we chose RAW264.7 macrophage in the cytotoxicity test was that the CNHs, and maybe also BNCNH, are engulfed by macrophages in animal bodies [38,39]. The toxicity of BN-CNH should be studied in more detail for the future possible application of BN-CNH in the boron neutron capture therapy.

4.

Discussion

The potential advantages of using nanocarbon for medical therapy and diagnosis are numerous. Loading drugs on nanocarbons and delivering them to the diseased sites has been shown to have strong therapeutic effects in in vivo animal experiments [31,32]. Some nanocarbons have photothermal conversion properties, absorbing near infrared light and warming up their surroundings to more than 42 C, high enough to kill tumor cells [33]. Moreover, by attaching image contrast agents in addition to tumor targeting molecules, nanocarbon could be useful for tumor diagnosis. SWCNTs

also absorb and emit near infrared light, suggesting that they themselves could be imaging reagents [34]. In contrast with the above-described advances, using nanocarbon for boron delivery is still a challenging task. It has been reported that o-carborane can be incorporated inside SWCNTs [35], and BN tubes can be formed inside SWCNTs [18]. The attachment of boron compounds to the outside of carbon nanotubes has also been reported [11]. These pioneering studies apparently indicate that nanocarbon would have great potential as a boron carrier for BoronNCT. Placed in this context, our results would be valuable, as they show that boron quantities were high and little BN became detached or released from the CNH. To deliver boron to tumor cells, BN-CNH should have a stealth effect, i.e., they should avoid being engulfed by macrophages, and be selectively captured by tumor cells. These two points could be achieved by coating BN-CNH surface with PLPEG-FA in the cell experiments. Alkyl chains of PLPEG strongly attach to the CNH surface, while PEG groups do not attach to it; thus, PLPEG carries with it a hydrophilic property and in turn, likely, the stealth effect to BN-CNHs. The tumor cells often overexpress FA receptors at the cell surfaces; therefore, BN-CNH/PLPEG-FA might be preferentially ingested by tumor cells. Since CNH has about 8 · 107 carbon atoms as estimated from its diameter of about 100 nm and density of 1.25 g/cm3 [36], BN-CNH would have about 3.2 · 107 boron atoms. Thus, an uptake of about thirty BN-CNHs per cell meets the Boron-NCT criteria of 109 B atoms per cell [1,4]. It is reportedly shown that 1000 or more CNHs per cell are ingested by KB or FHs163We cells under similar cell experimental conditions [16], suggesting that the control for reducing the uptake quantity, for example, by decreasing the dosage, might be important for the Boron-NCT application. Besides this, the biological responses of BN-CNHs and their functionalized forms should be indeed studied in vitro

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and in vivo in more detail for Boron-NCT applications. Especially, the issues of toxicity and clearance from the body are important, By clarifying them, BN-CNHs could be practically useful in the therapies.

5.

[10]

Conclusion [11]

BN was successfully loaded on CNHs by heating CNH with ammonia borane at 800 C. The BN sheets formed on the outside and inside of the nanohorn tubules walls and in the inside space of the nanohorn tubules. BN sheets were also formed on graphite-like thin sheets that are the constituent material of the CNHs together with nanohorn tubules. The C:B:N ratio was 5:2:2 (atom number). 70–80% of BN did not become detached from the CNHs during the immersion in cell culture medium for 10 days. By coating their surface with PLPEG-FA, the BN-CNHs were preferentially ingested by tumor cells, and the cellular uptake quantity of B was estimated to be much higher (hundreds of times higher) than the BoronNCT criteria of 109 B per cell in the cell culture experiments. These results suggest that BN-CNH could potentially be applied to Boron-NCT, though the biological responses to BN-CNHs should be studied in detail in vitro and in vivo.

[12]

[13]

[14] [15]

[16]

[17]

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbon. 2015.05.085.

[18]

[19]

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