University of Tsukuba
筑波大学
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 Federation
3 Institute of Synthetic Polymeric Materials of Russian Academy of Sciences
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
CD44-mediated
signaling transduction
and cellular HA
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
4
Solid state synthesis of BHA
HA
H
H
H3C
HO
O
Na2B4O7 *10H20
O
H
C
H
O
O
H
NH
C
H
OH
O
HO
H
BHA
H
H OH
H
O
O H(или Na)
n
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
5
Boron-hyaluronic acid complex formation
NMR-spectrum of HA and its borate
complex in an alkaline solution
13C-HA
spectrum and its borate complex in
an alkaline solution
BHA
13C-BHA
HA
13C-HA
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
Time-dependent cytotoxicity in C6 cells
1.2
Cell proliferation ratio
1.0
0.8
12h
0.6
18h
24h
0.4
0.2
0.0
0
25
50
75
100
125
150
175
200
225
BHA concentration, ppm
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
3.0
360 µg of BHA injection (2.4mg/kg)
Boron concentration, µg/g
2.5
n=3 (ca. 150 g)
2.0
1.5
1.0
6 tumors on the back of each rat
0.5
0.0
15 min
30 min
45 min
1h
1,5 h
2h
Time after IV injection
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)
9
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
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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).
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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).
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Radiotherapy enhancement with 50 nm GNPs
Irradiation dose
0 Gy
2 Gy
4 Gy
8 Gy
Gold concentration
0 µg/ml
15 µg/ml
Colony
forming
assay
(2 weeks
incubation )
30 µg/ml
45 µg/ml
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Radiotherapy enhancement with 50 nm GNPs
U251 glioma cell line
colony forming assay
Zaboronok A et al,
NNL, 2013.
*
**
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)
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HA-Melanin-Gold nanoparticles
Particle core
15~ nm
Gold+ melanin
HA surface
(BHA for ICNCT)
Hyaluronic acid combined with melanin-gold, 15~ nm sized GNPs.
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Gold nanoparticles formation analysis
Au/Melanin/HA=0.01%/0.01%/0.5% Au/Melanin/HA=0.02%/0.01%/0.5%
0,5
1,5
оптическая
плотность
optical density
оптическая
плотность
optical density
2,0
Время, мин
0
60
120
180
240
300
360
420
1,0
0,5
0,0
300
400
500
600
700
длина волны,
wavelength,
nmнм
40
35
30
25
20
15
10
5
0
10
0,3
0,2
0,1
0,0
время, мин
0
60
120
180
240
300
360
420
400
500
600
700
длина волны, нм
100
диаметр частиц, нм
particle diameter, nm
800
wavelength, nm
Average d=150 nm
fraction, %
particle
доля частиц, %
доляfraction,
частиц, % %
particle
Average d=30 nm
800
0,4
30
25
20
15
10
5
0
100
диаметр частиц, нм
particle diameter, nm
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.
17
HAMG-NPs
TEM: GNPs are clustered in U251 glioma cells in vacuoles and
localized in the cell cytoplasm (15µg/ml, 24 h incubation).
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Cytotoxicity for U251 glioma cells
n/s
p=0.006
p<0.001
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. 19
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
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
Radiosensitization of U251 glioma model
Tumor growth analysis
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
Irradiation
GNPs
injection
2 mice died in
radiation group
n=3
p=?
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Preliminary report
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
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University of Tsukuba
筑波大学