ming-hua hsu, ph. d. nuclear science and technology development center, nthu june 16, 2014 @ icnct...
TRANSCRIPT
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
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
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
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
The enhanced permeation and retention (EPR) effect
Nature Reviews Cancer 12, 39-50 (January 2012)
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
Development of BNCT Drug Deliver System Based on Polymeric Material PLA-PEOz
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
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)
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
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.
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
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.
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).
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.
Nanomaterial strategies from the point-of-view of the cell
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.
Tumor Hypoxia
• Solid tumor ~ highly heterogeneous~ exhibit oxygen tension, low pH, low glucose concentration
Kizaka-Kondoh et al. Cancer Sci. December, 2003
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
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
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
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
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
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
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
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
O (CH2)2 OO
O
N
OX
NH2
EtO
Y
B-PLA-PEOz-NH2
O (CH2)2
Hydrophobic part
Hydrophilic part
Nanodiamonds
• Chemically inert– however, can be surface-functionalized
• Biocompatible– low cytotoxicity
• Detectable – green fluorescence– by confocal and flow cytometry
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
Functionalized Nanodiamonds
Base on Organic Chemistry ~ Create New Nanodiamonds platform for Bioconjugate
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
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
TEM of Thiolated Nanodiamonds (100 nm) with Au-NPTEM of Thiolated Nanodiamonds (100 nm) with Au-NP
Accepted by ACS Applied Materials & Interfaces
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
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
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