1. Hyaluronic acid- and melanin-based boron compounds for combined neutron capture therapy Alexander Zaboronok 1, Kei Nakai 1, Tetsuya Yamamoto 1, Fumiyo Yoshida 1, Sergej Uspenskii 2, Mikhail Selyanin 2, Evgeniya Svidchenko 3, Nikolay Surin 3, Alexander Zelenetskii 3, and Akira Matsumura 1 1 Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan 2 International research center “Martinex”, Moscow, Russian Federation 3 Institute of Synthetic Polymeric Materials of Russian Academy of Sciences University of Tsukuba 筑波大学 1

2. Presentation outline Hyaluronic acid as a boron carrier Solid state synthesis of HA compounds Boron-hyaluronic acid complex formation analysis Cytotoxicity in tumor and normal cells Accumulation in subcutaneous rat tumor model Gold nanoparticles for combined radiotherapy Photon capture therapy concept Glioma cells radiosensitization with gold nanoparticles Hyaluronic acid- and melanin-based gold nanoparticles Cytotoxicity, X-ray contrasting and tumor radiosensitization 2

3. Why hyaluronic acid? Found in nearly all vertebrate tissues Biocompatible and nonimmunogenic Can imbed a large number of boron atoms A liquid or a gel depending on its molecular weight – applied IV or locally Can cover nanoparticles reducing their toxicity CD44 receptors to HA are overexpressed in a variety of tumors 3

4. CD44-mediated cellular uptake K.Y. Choi, et al., Hyaluronic acid-based nanocarriers for intracellular targeting: Interfacial interactions with proteins in cancer. Colloids Surf. B: Biointerfaces, 99 (2012) 82–94 CD44-mediated signaling transduction and cellular HA uptake. 4

5. Solid state synthesis of BHA Na 2 B 4 O 7 *10H 2 0 BHA Pressure of 1 GPa (10 4 atm.) Rotation angle of 500 о. 1 – Cylinder of hydraulic press; 2 – Anvils; 3 – Spring element; 4 – Mechanotron; 5 – Supporting ball-bearing; 6 – Radial ball bearing; 7 – Constant-temperature jacket; 8 – Position for thermocouples; 9 – Step-bearing of heat insulator to thermostat Bridgman anvils HA 5

6. Boron-hyaluronic acid complex formation HA BHA 13 C-HA 13 C-BHA NMR-spectrum of HA and its borate complex in an alkaline solution 13 C-HA spectrum and its borate complex in an alkaline solution Borate formation features: spectrum broadening and splitting of the signals of the acetyl group protons (~2 ppm) and C 2 (~55 ppm) and С 6 (~62 ppm) atoms of the N-acetylglucosamine moiety 6

7. Time-dependent cytotoxicity in C6 cells BHA cytotoxicity in C6 glioma cells, 12, 18 and 24 h incubation (30 kDa HA, MTS-assay: Cell Titer 96® AQueous One Solution, Promega, USA) 7

8. Cytotoxicity in C6 and V79 cells BHA cytotoxicity in C6 glioma and V79 fibroblast-like cells, 24 h incubation (30 kDa HA, MTS-assay: Cell Titer 96® AQueous One Solution, Promega, USA) 8

9. Accumulation in tumor model in vivo BHA accumulation in C6 glioma subcutaneous tumor models after tail vein injection in Wistar rats – rapid decrease in BHA concentration (30 kDa HA, ICP-AES: ICP-8100, Shimadzu, Kyoto, Japan) n=3 (ca. 150 g) 360 µg of BHA injection (2.4mg/kg) 9 6 tumors on the back of each rat

10. Preliminary report BHA complex formation analysis shows the efficacy of the solid state synthesis BHA shows certain degree of cytotoxicity Cytotoxicity is time-dependent in C6 cells Similar toxicity in normal and tumor cells (at 24h) Rapid decrease in BHA concentration in subcutaneous tumors in Wistar rats (15min-2h) Further functionalization might be necessary to achieve sufficient boron accumulation in tumor tissue 10

11. Gold nanoparticles for combined radiotherapy 11

12. Why gold nanoparticles? Gold is expected to be relatively inert and biologically compatible material Nanoparticles are characterized by enhanced permeability and retention (EPR) effect X-ray absorption by gold in keV-range is several times more beneficial than in other compounds Gold can enhance the radiation dose by 1.2 to >5 times Gold-produced photons can go over 100 µm, providing the influence on several cells Gold nanoparticles are characterized by low osmolality (Hainfeld et al 2004, 2008, Cai et al 2008, Kong et al 2008, Jelveh and Chithrani 2011, Rahman et al 2009, Butterworth et al 2010). 12

13. Photon capture therapy concept Direct and indirect damage of tumor cell DNA is caused by initial X-rays and free radicals and particles-emitted photons and produced free radicals. †Sample DNA helix is designed with the Pymol software (www.pymol.org) using 2L8Q code (Julien et al 2011).www.pymol.org 13

14. Radiotherapy enhancement with 50 nm GNPs 0 µg/ml 15 µg/ml 30 µg/ml 45 µg/ml 0 Gy2 Gy4 Gy8 Gy Gold concentration Irradiation dose 14 Colony forming assay (2 weeks incubation )

15. Radiotherapy enhancement with 50 nm GNPs Irradiation with 2,4 and 8Gy X-rays after 24 hours incubation with 15, 30 and 45µg/ml of 50 nm GNPs (*p=0.006, **p=0.006) 15 * ** Zaboronok A et al, NNL, 2013. U251 glioma cell line colony forming assay

16. HA-Melanin-Gold nanoparticles Hyaluronic acid combined with melanin-gold, 15~ nm sized GNPs. Particle core Gold+ melanin HA surface (BHA for ICNCT) 15~ nm 16

17. Gold nanoparticles formation analysis Growth of nanoparticles according to the peaks of the surface plasmon resonance and DLS. The size of the particles depends on the Au/Melanin ratio. Au/Melanin/HA=0.01%/0.01%/0.5%Au/Melanin/HA=0.02%/0.01%/0.5% Average d=30 nm Average d=150 nm 17 wavelength, nm optical density particle diameter, nm particle fraction, %

18. HAMG-NPs TEM: GNPs are clustered in U251 glioma cells in vacuoles and localized in the cell cytoplasm (15µg/ml, 24 h incubation). 18

19. Cytotoxicity for U251 glioma cells HAMG-NPs were not toxic within the studied concentration range. Ionized gold shows high toxicity at 30 µg/ml (ppm) and over. The control cell proliferation equaled 1 (or 100%), p-values by ANOVA. n/s p=0.006 p<0.001 19

20. Contrasting properties of HAMG-NPs GNPs injected intratumorally, examined at CT at the injection and 6h after the injection; contrast distribution within the tumor tissue was observed. 20

21. Light microscopy and TEM Light microscopy (toluidine blue stain) and TEM of U251MG tissue from the animal model 6 h after the injection of HAMG-NPs. 21

22. Radiosensitization of U251 glioma model p=? GNPs injection Irradiation 2 mice died in radiation group Aspects: local injection – insufficient gold distribution; 8Gy - insufficient irradiation; slow growth of U251 cells in vivo; flank model – is not a real glioma in brain. n=5 n=4 n=3 22 Tumor growth analysis

23. Preliminary report 23 1.HAMG-NPs presented in a stable colloidal solution and showed optical properties of spherical nanoparticles 2.HAMG-NPs were captured by U251MG cells in vitro, clustered in vacuoles in the cytoplasm of glioma cells 3.HAMG-NPs provided contrasting of the tumor tissue in vivo right after the injection with further redistribution 4.The study is ongoing and further development of the compounds is necessary to succeed in combined NCT

24. University of Tsukuba 筑波大学