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 Selyanin2, Evgeniya Svidchenko3, Nikolay Surin3,

Alexander Zelenetskii3, and Akira Matsumura 1

1Department of Neurosurgery, Faculty of Medicine,University of Tsukuba, Tsukuba, Japan

2International research center “Martinex”, Moscow, Russian Federation3 Institute of Synthetic Polymeric Materials of Russian Academy of Sciences

University of Tsukuba筑波大学

1

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

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

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

Solid state synthesis of BHA

n

O

H

H

HO

H

H

NHH

O H

O

H

H

HOH

H

OH

H

O

O

CH 3C

O

C

O H(или Na)

O

Na2B4O7 *10H20

BHA

Pressure of 1 GPa (104 atm.) Rotation angle of 500о.

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

Boron-hyaluronic acid complex formation

1.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.0(ppm)

1

2

HOD (suppresed)

1H-NMR (300MHz)

102030405060708090100110120130140150160170180(ppm)

1

2

13C-NMR ( 75MHz )

HA

BHA

13C-HA

13C-BHA

NMR-spectrum of HA and its borate complex in an alkaline solution

13C-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 C2 (~55 ppm) and С6 (~62 ppm) atoms of the N-acetylglucosamine moiety

6

0 25 50 75 100 125 150 175 200 2250.0

0.2

0.4

0.6

0.8

1.0

1.2

12h18h24h

BHA concentration, ppm

Cell

prol

ifera

tion

ratio

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

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

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)

15 min 30 min 45 min 1 h 1,5 h 2 h0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time after IV injection

Boro

n co

ncen

trati

on, µ

g/g n=3 (ca. 150 g)

360 µg of BHA injection (2.4mg/kg)

9

6 tumors on the back of each rat

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

Gold nanoparticles for combined radiotherapy

11

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).

Radiotherapy enhancement with 50 nm GNPs

0 µg/ml

15 µg/ml

30 µg/ml

45 µg/ml

0 Gy 2 Gy 4 Gy 8 Gy

Gol

d co

ncen

trati

on

Irradiation dose

14

Colonyforming assay (2 weeksincubation )

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)

***

Zaboronok A et al, NNL, 2013.

U251 glioma cell linecolony 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

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.

10 10005

10152025303540

дол

я ча

стиц

, %

диаметр частиц, нм

1000

5

10

15

20

25

30

дол

я ча

сти

ц, %

диаметр частиц, нм

400 500 600 700 8000,0

0,1

0,2

0,3

0,4

0,5

время, мин 0 60 120 180 240 300 360 420

опти

ческ

ая п

лот

ност

ь

длина волны, нм300 400 500 600 700 800

0,0

0,5

1,0

1,5

2,0

Время, мин 0 60 120 180 240 300 360 420

опти

ческ

ая п

лот

ност

ь

длина волны, нм

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, nmwavelength, nm

optic

al d

ensi

ty

optic

al d

ensi

ty

particle diameter, nm particle diameter, nmpa

rticl

e fr

actio

n, %

parti

cle

frac

tion,

%

18

HAMG-NPs

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

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

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.

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.

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

n=3

Tumor growth analysis

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

Thank you!

University of Tsukuba筑波大学