Gianni Ciofani1, Serena Danti2, Vittoria Raffa1, Mario Petrini2, Arianna Menciassi1,3

Potenziali applicazioni biomediche dei nanotubi di nitruro di boro, con speciale

attenzione alla terapia a cattura neutronica

Gnosca (TI), CH, 27 novembre 2009

1 CRIM Lab, Scuola Superiore Sant’Anna, Pisa, Italy – g.ciofani@sssup.it2 School of Medicine, University of Pisa, Pisa, Italy – s.danti@med.unipi.it3 IIT Network, Genova, Italy – arianna@sssup.it

Outline of the presentation

• Introduction: Nanomedicine and BNNTs

• Biomedical applications of BNNTs: Preliminary results

• The boron neutron capture therapy

• BNNT as boron carriers

• Conclusions and perspectives

Outline of the presentation

• Introduction: Nanomedicine and BNNTs

• Biomedical applications of BNNTs: Preliminary results

• The boron neutron capture therapy

• BNNT as boron carriers

• Conclusions and perspectives

• Boron nitride nanotubes (BNNTs) are of significant interest to the scientificcommunity because of their unique and important properties ideal forstructural and electronic applications.

• They are structural analogues of carbon nanotubes (CNTs) in nature: alternating B and N atoms entirely substitute for C atoms in a graphite-likesheet with almost no change in atomic spacing.

• However, despite this similarity, carbon and boron nitride nanotubes exhibitmany different properties.

What are BNNTs? – 1/2

Chopra N G, Luyken R J, Cherrey K, Crespi V H, Cohen M L, Louie S G and Zettl A 1995 Boron-nitride nanotubes. Science 269 966

Terrones M, Romo-Herrera J M, Cruz-Silva E, L´opez-Urıas F, Munoz-Sandoval E, Vel´azquez-Salazar J J, Terrones H, Bando Y and Golberg D 2007 Pure and doped boron nitride nanotubes. Mater. Today 10 30

What are BNNTs? – 2/2

• In addition to high Young’s modulus similar to that of CNTs, BNNTs exhibit superior chemical and thermal stability.

• In contrast to the electronic behaviour of CNTs which varies in accordance with their chirality, BNNTs have more stable electronic properties, with a uniform band gap of 5.5 eV, because they preferentially form zig-zag rather than armchair or chiral structures because of the polar nature of the B–N bond.

• Recent theoretical and experimental studies have confirmed that BNNTs have excellent piezoelectric properties, superior to those of piezoelectric polymers.

Golberg D, Bai X, Mitome M, Tang C, Zhi C and Bando Y 2007 Structural peculiarities of in situ deformation of an individual boron nitride nanotube inside a high-resolution transmission electron microscope Acta Mater. 55 1293

Nakhmanson S M, Calzolari A, Meunier V, Bernholc J and Buongiorno Nardelli M 2003 Spontaneous polarization and piezoelectricity in boron nitride nanotubes Phys. Rev. 67 235406

First investigations in nanomedicine• Biomedical applications of BNNTs remain largely unexplored, and just Zhi et

al. investigated the interaction between BNNTs and various protein species and between BNNTs and DNA.

• The first study of the interactions between BNNTs and living cells was performed by our group. BNNT suspensions obtained by using PEI as a dispersing agent, when tested in vitro with human neuroblastoma SH-SY5Y cells in concentrations of up to 5.0 �g/ml, demonstrated no adverse effects on the viability of these tumor cells.

• Other experiments demonstrated efficient cellular uptake of BNNTs by an energy-dependent process.

Ciofani G, Raffa V, Menciassi A and Cuschieri A 2008 Cytocompatibility, interactions with and uptake of polyethyleneimine-coated boron nitride nanotubes by living cells: confirmation of their potential for biomedical applications. Biotechnol. Bioeng. 101 850

Ciofani G, Raffa V, Menciassi A and Cuschieri A 2009 Boron nitride nanotubes: an innovative tool for nanomedicine. Nano Today 4 8

Outline of the presentation

• Introduction: Nanomedicine and BNNTs

• Biomedical applications of BNNTs: Preliminary results

• The boron neutron capture therapy

• BNNT as boron carriers

• Conclusions and perspectives

BNNT preparation

BNNT samples are prepared using a ball-milling and annealing method. Amorphous boron powder with purity of 95-97% is used as the base material and anhydrous NH3 as the reaction gas for both milling and annealing processes. Ball-milling treatment was conducted at room temperature using a steel rotating ball mill with four hardened steel balls within a stainless steel cell. The annealing was carried out under NH3 gas at a flow rate of 500-1000 ml/min up to 1300°C in a tube furnace for 16 h.

Chen Y, Fitz Gerald J, Williams J S and Bulcock S 1999 Synthesis of boron nitride nanotubes at low temperatures using reactive ball milling. Chem. Phys. Lett. 299 260

Yu J, Chen Y, Wuhrer R, Liu Z and Ringer S P 2005 In situ formation of BN nanotubes during nitriding reactions. Chem. Mater. 17 5172

Samples are prepared by weighing 5 mg of BNNTs and then mixing them with 10 ml of a 0.1% PLL solution in PBS. This mixture is initially heated over a hot plate with stirring up to a temperature of 70°C for 6 h. This treatment is then followed by sonication for 12 h with a Branson sonicator 2510 at 20 W. Finally, the sample is centrifuged at 1,100 g for 10 minutes to remove non-dispersed residuals and impurities. Excess PLL is removed by ultracentrifugation (three times, 30 min, 30,000 g). Quantification of dispersed BNNTs is performed spectrophotometrically.

Sample preparation

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Ciofani G, Raffa V, Menciassi A, Dario P 2008 Preparation of boron nitride nanotubes aqueous dispersions for biological applications. J. Nanosci. Nanotechno. 8 6223

Cytocompatibility on C2C12 cellsC2C12 mouse myoblasts (ATCC CRL-

1772) were cultured in Dulbecco’s

modified Eagle’s medium supplemented

with 10% fetal bovine serum, 100 IU/ml

penicillin, 100 �g/ml streptomycin and 2

mM L-glutamine. Cells were maintained at

37°C in a saturated humidity atmosphere

containing 95% air / 5% CO2. This cell

line, widely used as muscle cells model,

differentiates rapidly, forming contractile

myotubes and producing characteristic

muscle proteins.

Differentiation into myotubes was induced by switching 80–90% confluent cells to medium

supplemented with 1% of fetal bovine serum and 1% of Insulin-Transferrin-Selenium (ITS)

supplement.

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BNNT concentration (�g/ml)

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24 h 48 h 72 h p < 0.05

C2C12 cells maintain good viability up to 72 h of treatment with 5, 10 and 15 µg/ml of PLL-BNNT: no differences in cell morphology were observed between k- and BNNT tests without significant differences in cell counts at 24, 48 and 72 h of culture: in BNNT tests cells reached confluence as in the control test. MTT assay provided clear evidence for satisfactory metabolic activity at 24, 48 and 72 h of treatment, statistically different from the control only after 72 h for the 15 µg/ml test

Viability assay: MTT test

Investigation of differentiation: Reverse transcription-polymerase chain reaction (RT-PCR) for detection of Connexin 43 and MyoDin differentiating C2C12 cells

MyoD is a protein that plays a key role in regulating myoblast differentiation. It is one of the earliest markers of myogenic commitment, being expressed in activated satellite cells and in their progenitors. Connexins, such as Cx43, are proteins present in myoblasts that are involved in the formation of gap junctions, composed of connexin-based connexons.

RT-PCR results highlighted that the expression of Cx43 and MyoD genes is not affected by the administration of BNNT at a concentration up to 10 µµµµg/ml.

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Investigation of differentiation: Myotube formationQualitative differentiation is assessed by phase contrast microscopy. Images of myotubes after 4 days of incubation in differentiating medium with 0 (a), 5 (b) and 10 (c) µg/ml of BNNTs. Samples were fixed with 1% buffered-formalin and stained with hematoxylin and eosin (H&E) according to standard histological procedures.

C2C12 cells treated with BNNTs developed myotubes without qualitative differences from the control culture

a b c

Quantitative analysis (fusion index evaluated with Hoechst staining) confirmed qualitative results

Outline of the presentation

• Introduction: Nanomedicine and BNNTs

• Biomedical applications of BNNTs: Preliminary results

• The boron neutron capture therapy

• BNNT as boron carriers

• Conclusions and perspectives

What is BNCT? - 1/2

Boron neutron capture therapy (BNCT) is based on the nuclear capture and fission reactions that occur when non-radioactive boron-10 is irradiated with low energy thermal neutrons to yield high linear energy transfer (LET) alpha particles (4He) and recoiling lithium-7 (7Li) nuclei.

For BNCT to be successful, a sufficient number of 10B atoms (about 109 atoms/cell) must be selectively delivered to the tumor and enough thermal neutrons must be absorbed by them to sustain a lethal 10B(n, �)7Li capture reaction.

BNCT primarily has been used to treat patients with brain tumors, and more recently those with head and neck cancer.

The large cross section of thermal neutron interactions with B-10 isotope causes high probability of a slitting of boron nucleus onto He and Li. As ionization capability of He and Li ions is high, and their runs are short, then the cells, preferably enriched by boron, are killed and the healthy cells are damaged much less.

What is BNCT? - 2/2

How does BNCT work? - 1/2

It uses a boron-containingcompound that preferentiallyconcentrates in tumor sites

BPA Boronophenylalanine

BSH Sulfhydryl borane

The tumor site isirradiated by a neutron

beam

Neutrons interact with the boron in the tumor

Damage is doneto the tumor cell

Boron Neutron Capture Therapy is a binary formof cancer therapy

How does BNCT work? - 2/2

Emission of high LET and low range ions:4He2+ (9 µm) 7Li3+ (6 µm)

• These ranges are comparable with the average cell diameter.

• The ionizations are responsible of the DNA double strand break and thisthiseffecteffect isis lethallethal at at leastleast in 95% of in 95% of casescases..

The procedureAn epithermal beam of neutronsis directed towards a patients' head, during their passagethrough tissue these neutronsrapidly lose energy by elasticscattering (a process calledthermalization) until they end up as thermal neutrons. The thermalneutrons thus formed, are captured by the 10B atoms whichbecome 11B atoms in the excitedstate for a very short time (~ 10-12

seconds). The 11B atoms thenfissions producing alpha particles, 7Li recoil nuclei and in 94% of the reactions, gamma rays. Tumorcells are killed selectively by the energetic alpha particles and 7Li fission products

Outline of the presentation

• Introduction: Nanomedicine and BNNTs

• Biomedical applications of BNNTs: Preliminary results

• The boron neutron capture therapy

• BNNT as boron carriers

• Conclusions and perspectives

Our idea: exploitation of BNNT as “drug”

The low content of boron targeting species in glioblastoma multiforme accounts for the difficulty in selective targeting of this very malignant cerebral tumour by this radiation modality. In the present study, we have used for the first time BNNTs as carriers of boron atoms to overcome this problem and enhance the selective targeting and ablative efficacy of BNCT for these tumors.

Following their dispersion in aqueous solution by non-covalent coating with biocompatible poly-L-lysine solutions, boron nitride nanotubes were functionalized with a fluorescent probe to enable their tracking, and with folic acid as selective tumor targeting ligand.

Initial in-vitro studies have confirmed substantive and selective up-take of these nano-vectors by glioblastoma multiforme cells, an observation which confirms their potential clinical application for BNCT therapy for these malignant cerebral tumors.

PLL coating

Folate group

Quantum dots

Folate receptor

Cell

The proposed nanovector

Our preliminary in vitro testing: the protocol• Two kinds of cell lines: Human glioblastoma multiforme T98G cells (ATCC CRL-1690) and,

as control, healthy human primary fibroblasts• After adhesion (at 6 h), the cells were treated for 90 min with the Qdots labelled PLL-BNNT

and F-PLL-BNNT modified culture medium (final concentration 10 �g/ml of BNNTs). In a control experiment, folic acid was added to the cell culture medium to compete with the F-PLL-BNNTs. For this competitive inhibition experiment, folic acid was first added to the medium at a concentration of 3.5 mM and cells were incubated for 30 min. Subsequently, F-PLL-BNNTs were added and the cells were incubated for a further 90 min.

• Qdots labelling of PLL-BNNTs allowed the direct visualization of the up-take by glioblastoma and healthy (control) fibroblast cells.

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Mixture Folic Acid + PLL-BNNT

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Folate conjugation is achieved by linking the amino-groups of PLL with the � carboxylic-

groups of the folate molecules. Folic acid, 3.5 mM, and 5 mM EDC are added to a

dispersion of 50 µg/ml of PLL-BNNT. After reaction, the dispersion is ultracentrifuged to

remove unbound folic acid and EDC, resulting in stable dispersions of folate conjugated PLL-

BNNT (F-PLL-BNNT).

UV-Vis spectra of folic acid, PLL-BNNTs, a mixture of folic acid and PLL-BNNTs and F-PLL-BNNTs show the successful reaction.

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Fibroblasts

Cytocompatibility: MTT assay

• The investigated cultures confirmed the complete cytocompatibility of PLL-BNNTswith both glioblastoma and primary fibroblast cells. No differences in cell morphology were observed between control (k-), PLL-BNNT and F-PLL-BNNT treatment culture.

• Cell viability assessed with Trypan blue assay was >95 % in each case.

MTT assay provided clear evidence for excellent metabolic activity after 24 h of treatment. With both PLL-BNNT and F-PLL-BNNT treatments, metabolic activity

exceeded 80 % of the control for both the cell populations, with no significant difference between the experiments (n = 6; p > 0.1).

Selective up-take investigation – 1/3The fluorescence image of glioblastoma cells after 90 min incubation in the presence of F-PLL-BNNTs resulted in an intensive fluorescence was clearly observed, indicating significant cellular up-take of nanotubes.

In sharp contrast, fluorescence of glioblastoma cells was considerably weaker when the cells were incubated for 90 min in the presence of not folate-functionalized PLL-BNNTs.

It was also found that the folate-mediated cellular up-take of F-PLL-BNNTs could be blocked by competitive inhibition if a large amount of free folate (3.5 mM) is added into the culture medium for 30 min before the addition of F-PLL-BNNTs.

From left to right: fluorescence images of glioblastoma cells incubated with 10 µg/ml of Qdot conjugated F-PLL-BNNTs; PLL-BNNTs; F-PLL-BNNTs with pre-

incubation of 3.5 mM free folate

Selective up-take investigation – 2/3

From left to right: fluorescence image of fibroblast cells incubated with 10 �g/ml of Qdot conjugated F-PLL-BNNTs (a); PLL-BNNTs (c); F-PLL-BNNTs with pre-

incubation of 3.5 mM free folate.

The same experiments were carried out on normal human gingival fibroblasts which acted as controls. In these experiments, similar but much weaker fluorescence intensity was detected in cultures treated with 10 µg/ml of F-PLL-BNNTs, with 10 µg/ml of non folate-functionalized PLL-BNNTs, and finally treated with F-PLL-BNNTs preceded by a pre-incubation of 3.5 mM free folate.

In summary, these results indicate that after 90 min of incubation with labeled PLL-BNNTs, the presence of the folate molecule on the BNNT coating strongly enhances the up-take by glioblastoma cells, but no appreciable improvement in uptake of the nanotubes is observed with normal cultured fibroblasts).

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Quantitative analysis of the fluorescence intensity, confirm these data. The up-take of F-PLL-BNNTs by glioblastoma multiforme cells, in folate depleted medium, is statistically different (p < 0.001) and results in a 2-fold improvement with respect to all the other treatments, which show no statistically significant differences (p > 0.05).

Receptor mediated endocytosis of F-PLL-BNNTs by glioblastoma multiforme cells is confirmed by the following picture. This microphotograph consists of a high magnification image of a glioblastoma cell following up-take of Qdot conjugated F-PLL-BNNTs and lysosomal tracking. Yellow fluorescence arises from merging of Qdot (red) and LysoTracker probe (green) and demonstrate therefore a strong accumulation of the nanovectors in the acidic compartments (lysosomes) of the cells.

Selective up-take investigation – 3/3

Conclusions and perspectives

� The results of recent and ongoing clinical trials which confirm the safety of BNCT for the

treatment of cerebral GBM highlight the immediate need for truly effective boron target

agents

� In the present study, we have investigated the use of boron nitride nanotubes as boron

atom carriers. After their dispersion in biocompatible poly-L-lysine solutions, boron nitride

nanotubes were functionalized with a fluorescent probe and with folic acid for tumor

targeting.

� In sharp contrast to proposed applications of CNTs in biotechnology in recent years, the

biomedical applications of BNNTs have received scant attention despite their unique and

impressive chemical and physical properties.

� For the first time, we have carried out extensive cytocompatibility investigations on many

different cell lines.

� The results obtained indicate that folate-functionalized BNNTs have the potential to act as

a boron delivery agents to malignant glioblastoma cells as these preliminary in vitro

studies have confirmed a strong and selective up-take of these nanotubes vectors by

glioblastoma multiforme cells, but not by normal human fibroblasts.

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