The Use of Magnetic Nanoparticles to Tag Boron Compounds in Boron Neutron Capture Therapy

1. The Use of Magnetic Nanoparticles to Tag Boron Compounds in Boron Neutron Capture Therapy M. Bonora1, M. Corti1, F. Borsa1, S. Bortolussi2, D. Santoro2, M.A. Gadan2, S. Altieri2, C. Zonta3, A.M. Clerici3, L. Cansolino3, C. Ferrari3, A. Marchetti4, G. Zanoni4, G. Vidari4. • Physics Department “A. Volta”, University of Pavia, Via Bassi 6, 27100 Pavia, Italy. • Theoretical and Nuclear Physics Department, University of Pavia, INFN, Section of Pavia, Via Bassi 6, 27100 Pavia, Italy. • Laboratory of Experimental Surgery, University of Pavia, Piazza Botta 10, 27100 Pavia, Italy. • Organic Chemistry Department, University of Pavia, Via Taramelli 10, 27100 Pavia, Italy. E.Mail: Bonora@fisicavolta.unipv.it Introduction The expertise of four different groups of the University of Pavia, involving physics, chemistry, biology and medicine, is combined to the determination of a standard protocol for the BNCT therapy of widespread tumors that affect a whole vital organ and that are not surgically removable. Optimisation of BNCT requires that the concentration and the spatial distribution of the 10B nuclide in tumoral and healthy tissues is precisely known. An  spectroscopy method has been introduced to evaluate the boron concentration in tissue samples. On the other hand, one of the main goals is the synthesis of molecular nanomagnets, that is, molecules that would contain both the BNCT essential chemical moieities and an ion carrying a magnetic moment. Such magnetic moment act as a contrast agent in order to detect both concentration and distribution of boron in the tissue under exam via proton MRI, a well-established and non-invasive technique. The synthesis of molecular nanomagnets and the use of them in NMR/MRI are thus essential for the improvement of the BNCT therapy of inoperable tumors.

2. Synthesis of the material Preparation of Test Animals with Tumors and Infused Substance Magnetic Characterization and Relaxometry of the synthesized material Measurements of Boron Concentration and Imaging by Thermal Neutrons MRI Monitoring of the Distribution of Nuclides Thermal Neutron Irradiation Post Treatment Tests and Assessment of Results General Outline: a Multidisciplinary Project Boron Neutron Capture Therapy (BNCT) The concept of Boron Neutron capture Therapy has been introduced by G.L. Locher in 1936 [1]. The 10B nucleus (natural abundance 19.8%) has a very large nuclear cross section of thermal neutrons capture. After the neutron absorption, a 7Li nucleus, an  particle and (with a probability of 94%) a  ray are produced. Hence, a large quantity of energy is released in a little spherical volume (typical diameter of 12 m). If the 10B nucleus is contained in a tumoral cell, the diseased cell is severely damaged and inactivated in the process. This means that a precise knowledge of the 10B distribution between the healthy and the tumoral tissues should be attained for a correct determination of the BNCT therapy plan (neutron irradiation time and total absorbed dose). Fig.1. BNCT: General scheme. 10B Concentration and Distribution NMR-MRI on 10B in Boron Compounds The molecule that has been used to carry the boron inside tumoral cells Is 10B-enriched [(L)]-4-dihydroxy-borylphenylalanine, also known as BPA [4]. 10B has nuclear spin 1 and can be studied by Nuclear Magnetic Resonance-Magnetic Resonance Imaging (NMR-MRI). NMR-MRI is a powerful, non-invasive technique [5] that can quickly establish 10B concentration, and, potentially, its spatial distribution in living tissue. Thin slices of tissue are analysed by  spectroscopy (fig.2) [2] and subjected to neutron radiography and histological exams (fig.3) to determine the 10B concentration and its spatial distribution. 10B is then shown to be preferentially absorbed in tumoral tissues and this makes BNCT feasible as a therapy. Fig.4. Cartoon of the BPA molecule. BPA is absorbed much more easily in tumoral cells than in healthy ones. Fig.2. The average concentration of 10B in a tissue is determined through observation of number and energy of  particles. Fig.3. Comparison between histological and neutron radiography exams[3]: 10B is much moreconcentrated in tumoral areas. Fig.5. Determination of 10B Relaxation Time T1 in a tumoral rat liver with a 10B average concentration of 12 ppm. We can detect 10B NMR signal with a sensitivity down to 1 ppm. Fig.6.10B MRI of a rat section. Whiter zones of the image denote a higher uptake of boron [6].

3. Nanomagnets as Contrast Agents in 1H-MRI In Vivo Models The introduction of a so-called contrast agent (CA) alters the 1H relaxation times and can greatly improve the resolution of the MRI image. A popular CA is the complexed Gd3+ ion, its effect in MRI can be seen in the figure 7 example. A molecule that can introduce in tissues both Gd3+ CA and 10B- enriched BPA is the BPA-Conjugated Gd-DTPA shown in figure 8. A slightly modified version (fig. 9) of this molecular nanomagnet is currently synthesised by the Prof. Vidari group. Fig.7. Effect of the CA Gd3+ ion. Left: normal brain MRI. Right: Brain MRI in presence of Gd3+ as CA; resolution is highly improved and the tumoral zone is much more evident. Fig.10. Cells are grown in vitro and are then injected in rats to induce widespread metastases in the liver (left) and in the lung (right). Fig.8. Cartoon of BPA-Conjugated Gd-DTPA as it was proposed in [7]. The CA Gd3+ ion and the 10B-carrying BPA are linked together. BNCT-feasibility and high MRI resolution are thus provided in a single molecular unit. Fig.9. A new synthetic path for the BPA-Gd ensemble. The production of this molecular nanomagnet has been recently optimized and it only requires five synthetic steps.

4. Conclusions BPA in Healthy and Tumoral Rat Liver References Cooperation between four different groups of the University of Pavia has been established to optimize the BNCT therapy of unoperable, disseminated tumors. Preliminary  spectroscopy and NMR experiments have been carried on in order to determine the BPA uptake in rat livers. 10B Relaxation times < 1 ms have been determined in the same rat liver samples through NMR. Central to this project is the synthesis and the use of BPA-Conjugated Gd-DTPA, a molecular nanomagnet that contains both the 10B-carring BPA and a complexed Gd3+ ion. While the first is essential to introduce 10B in the cell and to undergo the BNCT reaction, the paramagnetic ion will act as a CA and it will greatly enhance the resolution in proton MRI experiments. A new, more efficient and quick synthetic method for a molecular nanomagnet BPA-Gd3+ has been developed. The 1H-MRI boosted by the presence of the BPA-Gd3+ nanomagnet will allow the accurate measure of the in vivo distribution and local concentration of 10B and will therefore be a decisive factor in planning an adequate neutron irradiation scheme in BNCT-based therapy of tumors. [1]G.L. Locher, Biological Effects and Therapeutical Possibilities of Neutrons, Am. J. Roentgenol. Radium Ther. 36, 1-13 (1936). [2] A. Wittig, J. Michel, R.L. Moss, F. Stecher-Rasmussen, H.F.Arlinghaus, P. Bendel, P.L. Mauri, S. Altieri, R. Hilger, P.A. Salvadori, L. Menichetti, R. Zamenhof, W.A.G. Sauerwein, Boron Analysis and Boron Imaging in Biological Materials for Boron Neutron Capture Therapy (BNCT), Critical Reviews in Oncology/Hematology 68, 66-90 (2008). [3] S. Altieri, S. Bortolussi, P. Bruschi, P. Chiari, F. Fossati, S. Stella, U. Prati, L. Roveda, A. Zonta, C. Zonta, C. Ferrari, A. Clerici, R. Nano, T. Pinelli, Neutron Autoradiography Imaging of Selective Boron Uptake in Human Metastatic Tumours, Appl. Rad. And Isotop. 66, 1850-1855 (2008). [4] R.F. Barth, J.A. Coderre, M.G.H. Vicente, T.E. Blue, Boron Neutron Capture Therapy of Cancer: Current Status and Future Prospects, Clin. Cancer Res. 11(11), 3987-4002 (2005). [5] A. Lascialfari, M. Corti, Basic Concepts of Magnetic Resonance Imaging in NMR-MRI, SR and Mössbauer Spectroscopies in Molecular Magnets, P. Carretta and A. Lascialfari Eds., Springer-Verlag (2007). [6] P. Bendel, Biomedical Applications of 10B and 11B NMR, NMR Biomed. 18, 74-82 (2005). [7] K. Takahashi, H. Nakamura, S. Furumoto, K. Yamamoto, H. Fukuda, A. Matsumura, Y. Yamamoto, Synthesis and In Vivo Biodistribution of BPA-Gd-DTPA Complex as a Potential MRI Contrast Carrier for Neutron Capture Therapy, Bioorg. Med. Chem. 13, 735-743 (2005). [8] L. Roveda, U. Prati, J. Bakeine, F. Trotta, P. Marotta, P. Valsecchi, A. Zonta, R. Nano, A. Facoetti, P. Chiari, S. Barni, T. Pinelli, S. Altieri, A. Braghieri, P. Bruschi, F. Fossati, P. Pedroni, How to Study Boron Biodistribution in Liver Metastases from Colorectal Cancer, Journal of Chemotherapy (Florence, Italy), 16 suppl. 5, 15-18 (2004). Fig.11. Boron concentration in healthy (empty red squares) and tumoral (full red squares) rat liver [8] is reported in function of the lag time from BPA injection to tissue sampling (measured by  spectroscopy [2]). The blue points indicate the ratio between the tumoral and the healthy BPA concentration at the studied times. The best ratio values are obtained after 2-4 hours from BPA injection.