ming-hua hsu, ph. d. nuclear science and technology development center, nthu june 16, 2014 @ icnct...
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Ming-Hua Hsu, Ph. D.Nuclear Science and Technology Development Center, NTHU
June 16, 2014
@ ICNCT 16, Helsinki, Funland
Development of Boron Containing Nanodiamonds
for Boron Neutron Capture Therapy
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2
OutlineOutline
1. Nanomedicine
2. Boron Containing Polmeric nanoparticle for
BNCT
PLA-PEOz-B-pin
PLA-PEOz-B-cage
3. Boron Containing Nanodiamonds for
BNCT
4. Boron Nanoparticles for BNCT
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Nanomedicine: Nanotechnology to Medicine
Apply Nanotechnology:
•Maintain drug activity.
• Increase water solubility.
• Delivery drug by nanoparticle
• Increase drug efficiency
• Traceable or directable drug
NanomedicineUse of nano-materials in
medicine unique medical effects
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The Advantage of Nanoparticles
• High surface/volume ratio and good solubility
• Nanometer size lead to faster movement and easy entry into cell
• Nanoparticle vector can penetrate endothelial barriers to reach tumor
sites
nanoparticlesdrug
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The enhanced permeation and retention (EPR) effect
Nature Reviews Cancer 12, 39-50 (January 2012)
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Micelle-based Drug Delivery System
The micelle must: (i) be small enough (∼10—200 nm) to effectively penetrate into tissue; (ii) be unrecognizable by the mononuclear phagocyte system (MPS) for a sufficient time to allow accumulation in target tissue; (iii) be eliminated from the organism either after degradation or dissolution;(iv) locate and interact with the target cells; (v) have tunable stability; (vi) improve the pharmacokinetic (PK) profile of the encapsulated drug cargo; (vii) possess high loading capacity; and (viii) be synthesized in a reproducible, facile method which is reasonably inexpensive
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Development of BNCT Drug Deliver System Based on Polymeric Material PLA-PEOz
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Polylactide, PLA
• Biocompatible• Biodegradable• Low immunogenicity• Good mechanical properties• FDA-approved for clinical use• Hydrophobic
OH
Ring opeing polymerization
O
O O
OLactide
O
O
OHm
PLA-OH
• Pseudopolypeptides• Adjustable• Biocompatible• Low immunogenicity• Stealth• FDA-approved as a food contact
agent• Hydrophilic
Polyoxazoline
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Strategy of Copolymers Synthesis
OO
O
ON
O CH2
OHx
y
CH3
BO
O
OH
BO
O O
O
O
O
D,L-lactide
N
OEt
2-ethyl-2-oxazoline
pinacol combined 4-Benzylphenylboronic acid (Bpin)
ROP ROP
Bpin-PLA-PEOz-OH
ROP (Ring-Opening Polymerization)
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Synthetic Scheme of Bpin-PLA-PEOz
BHO
HO OH
HO OH
MgSO4, THF
BO
O OH
O
OO O
Sn(Oct)2, toluene
BO
O
OO
O
O
OH
xMsCl
TEA,THF BO
O
OO
O
O
OMs
x
N
OEt
BO
O
OO
O
O
N
x O Et
OH
yMeCN
1.
2. KOH
4-hydroxylmethylphenylboronic acid
Bpin (Boronate ester initiator) D,L-lactide
Bpin-PLA Bpin-PLA-mesylate
2-ethyl-2-oxazoline
Bpin-PLA--PEOz
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Characterizations of synthesized polymers
Table1 Characterizations of synthesized polymers
polymer Mna Polydispersity index (PDI)b
Yield (%) CMC (wt %)
Bpin-PLA 7218 1.21 98
Bpin-PLA-PEOz 14247 1.24 62 7×10-4
a Estimated by 1H NMR . b Estimated by GPC.
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Boron Content of Bpin-PLA-PEOz micelle
Bpin-PLA-PEOz
• 6.06 ± 0.3 μg B/ml• 0.06
(boron/vehicle ratio)
Bpin-PLA-PEOz/PBAD
• 15.7 ± 0.6 μg B/ml• 0.15
(boron/vehicle ratio)
BHO
HO
PhMe, reflux, 16 h
O
BO
B
OBPTSA
Synthesis of Phenylboronic acid derivative (PBAD)
boron concentrations were determined by ICP-MS
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Micelle Formation
Bpin-PLA-PEOz
PBAD (Phenyl boronic acid derivative)
The boron bearing diblock copolymers, once form the micelles, could load additional boron compounds, PBAD in this research.
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Cell Viability by MTT assay
Cell viability after 48 h of incubation with the Bpin, PBAD, Bpin-PLA-PEOz and Bpin-PLA-PEOz/PBAD; error bars are mean ± SD (n=6). *Significantly different between PBAD and Bpin-PLA-PEOz/PBAD at the indicated concentration (p < 0.05).
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Stability of Micelle in Storage Condition
0
50
100
150
200
0 20 40 60
Time (day)
Hydro
dyn
am
ic d
iam
ete
r(n
m)
0
50
100
150
200
0 20 40 60
Time (day)
Hydro
dyn
am
ic d
iam
ete
r(n
m)
Size alteration of PLA-PEOz (left) and Bpin-PLA-PEOz micelles (right)
The encapsulated micelles were deposited in 4 °C for 10days, 30days and 50 days. Then the micelles were analyzed by DLS to investigate the alteration of the particle size.
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Nanomaterial strategies from the point-of-view of the cell
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Hypoxia
Tissue hypoxia results from:1. O2 tension
low O2 partial pressure~ pulmonary diseases, high altitude
2. anemic hypoxiareduced ability of blood to carry O2 ~ anemia, CO poisoning
3. circulatory hypoxiareduced tissue perfusion ~ generalized or local
4. diffusional hypoxiaincrease diffusion distance
5. cytotoxic hypoxiaintoxication, cyanide poisoning
a state of decreased O2 availability below critical thresholds, thus restricting function of organs, tissue, or cells.
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Tumor Hypoxia
• Solid tumor ~ highly heterogeneous~ exhibit oxygen tension, low pH, low glucose concentration
Kizaka-Kondoh et al. Cancer Sci. December, 2003
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O2 pressure (mmHg)
Normal tissues
Murine brain 60
Murine muscle 42
Bone marrow 40-50
Normal liver 55
Normal breast tissue 65
Normal cervix 48
Head and neck tissue 43
Tumors
Breast carcinoma 28
Solid tumors <2.5
Murine Fsall fibrosarcoma <5
Cancer of cervix <12
Head and neck cancers <10
Soft-tissue sarcomas <10
Oxygen Tensions in Various Tissue, Tumor
Normal tissue ~ 50-80 mmHg
Tumor < 10-30 mmHg
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Nitroimdazoles derivatives
N N OH
NO2
N N
NO2
OHO
N N
NO2
OHF
N N
NO2
OHO
Br
N N
NO2
OH I
N N
NO2
O
I
O
OH
I NN
O2N
O
F
I NN
O2N
J. Nucl. Med. 2005
Euro. J. Nucl. Med. 1995
Hypoxia-Specific Tumor Imaging with 18F-Fluoroazomycin Arabinoside
O N
N
OHOH
F18
O2N
A B C
FAZAB
O N
N
OHOH
F18
O2N
A B C
FAZAB
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Bioreductive Metabolism of Nitroimidazole
NO2
-R.
R.
R-NO2H.
R-NO2H..
oxidative
damage
MEMBRANE
entry
efflux
NO2
OUT IN
1e
reduction
. H+
protonation
1e
DNA(-T) (ox)DNA+T-
2eR-NHOH R-NH2
H+
decomposition
H2O
disproportionation
R-NO2
O2O2-
futile
cycle
.
2eR- NO2R- NO2
-R- R-NO
hypoxia
HydrophilicCan Not penetrate membrane
Stock in cell
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Size affects the biodistribution profile and therapeutical bechaviour of the system
Normal tissue
Hypoxia Tumour tissue
Dis-organized and leaky tumour endotelium
Defective lymphatic drainage
Lymph node
Normal vessels with tight endothelium
Intratumoural delivery of NPs
Nano-size penetration, cellular uptake, targeting
•active•passive
EPR-effectEnhanced permeation and retention effect
Hypoxia Targeting Nanodelivery
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BHO
HO OH
HO OH
MgSO4, THF
BO
O OH
O
OO O
Sn(Oct)2, toluene BO
O
OO
O
O
OH
x
MsCl
TEA,THF BO
O
OO
O
O
OMs
x
N
OEt
BO
O
OO
O
O
N
x O Et
OH
y
NNO2N OH
O
BO
O
OO
O
O
N
x O Et
O
y
N
O NNO2
MeCN1.
2. KOH
DMAP,DCC
Synthetic Scheme of Bpin-PLA-PEOz-NIm
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Strategy of Copolymers Synthesis
OO
OO
O
Ox N
NH2
O Et
y
O
O
ROP
O
O
O
O
N
OEt
ROP
D,L-Lactide 2-Ethyl-2-oxazoline
closo-Dodecarborate with oxonium
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Synthesis of (Bu4N)[B12H11O(CH2)2O(CH2)2OH]
= BH
O
O
OO
= B
NaH, THF
OH
2- 2-
(Bu4N) 2(Bu4N)NaOH
Reflux
Bu4NBr
Polymers Mw Mn PDI
B-PLA-OH 3175 2231 1.42
OO
OH Sn(Oct)2
D,L - Lactide
toluene
OO
OO
OH
O
O
x
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Mesyl chloride dry TEA, dry THF
OO
OO
O
O
O
x S
O
O
CH3
2-ethyl-2-oxazoline KOH
OO
OO
O
O
x NNH2
O Et
y
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O (CH2)2 OO
O
N
OX
NH2
EtO
Y
B-PLA-PEOz-NH2
O (CH2)2
Hydrophobic part
Hydrophilic part
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Nanodiamonds
• Chemically inert– however, can be surface-functionalized
• Biocompatible– low cytotoxicity
• Detectable – green fluorescence– by confocal and flow cytometry
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1. Tribology
2. Drug Delivery
3. Bioimaging
4. Tissue Engineering
5. Nanocomposites for Filler Materials
Mochalin, V. N. et al. Nat. Nanotechnol., 2011, 209, 11-23
Applications of Diamond Materials
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Functionalized Nanodiamonds
Base on Organic Chemistry ~ Create New Nanodiamonds platform for Bioconjugate
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Functionalized Nanodiamonds
Base on Organic Chemistry ~ Create New Nanodiamonds platform for Bioconjugate
O
O
O
COOH
O
O
O
OHOH
OH
OH
OH
OH
OH
SH
SH
SH
SH
SH
SH
1. HBr, HOAc, D
2. NaOH3. H2SO4
BH3, THF
NH2 NH2
S
OH+
S
NH2+
NH2
S SH OH -
H2O
- CO3-
- NH3
H+
-H2O
NH2 NH2
S
H+
Propose mechanism
N% C% H% S%
ND 3.411 84.729 1.011 0.287
ND-COOH 3.316 82.911 1.047 0.373
ND-OH 1.721 78.254 1.080 0.023
ND-SH 2.970 79.844 0.923 3.305
Accepted by ACS Applied Materials & Interfaces
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O1s
O1s
S2p
S2p
S2s
S2s
C1s
Survey CNT-SH
C1s
1200 1000 800 600 400 200 0
B.E. (eV)
Survey CNT-OH
1200 1000 800 600 400 200 0
S2pS2s
OKLL
O1s
B.E.(eV)
C1s
XPS of Thiolated Nanodiamonds and Carbon NanotubeXPS of Thiolated Nanodiamonds and Carbon Nanotube
Thiolated Nanodiamonds
180 175 170 165 160 155
-Csp3-SO2X
B.E.(eV)
-Csp3-SH
Thiolated Carbon Nanotube
Accepted by ACS Applied Materials & Interfaces
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TEM of Thiolated Nanodiamonds (100 nm) with Au-NPTEM of Thiolated Nanodiamonds (100 nm) with Au-NP
Accepted by ACS Applied Materials & Interfaces
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OH
O
O
HO
O
O
O
O
O
H
1. HNO3 / H2SO4
2. SOCl2
Bpin
Bpin
BpinBpin
Bpin
Bpin
BO
O OH
3. toluene, NEt3Bpin
Bpin :
Bpin O
O BO
O
Boron-containing NanodiamondsBoron-containing Nanodiamonds
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OH
O
O
HO
O
O
O
O
O
H
LiAlH4
THF
OHOH
OH
OH
OHHO
HO
HOO
O
= B= BH
OOB
OB
OB
OBBO
BO
BO
OO
NEt3, THF
Boron Cage-containing NanodiamondsBoron Cage-containing Nanodiamonds
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Thank you very much for your attention.Taiwan is looking forward to seeing you in 2018!
18th International Congress on Neutron Capture Therapy in Taiwan in 2018Boron Neutron Capture Therapy – Best New Concept TherapyOctober 28th – November 2nd, 2018 | Taipei, Taiwan