Transcript 274kang-pptxx

Department of Biomedical Engineering and
Environmental Sciences,
National Tsing Hua University
Hsinchu, Taiwan.
2012.8.14
2
Introduction
HIFU
Excitation
• Nanoparticles as drug delivery systems:
▫
▫
▫
▫
High stability
High carrier capacity
Specific targeting capability
Controlled release capability
Au
• Gold nanoparticles (Au NPs):
▫
▫
▫
▫
▫
Easy synthesis and bioconjugation
Plasmonic resonance oscillations
Tunable optical characteristics
Strong photothermal response
Photoacoustic imaging contrast
13 nm Au NP
Doxorubicin (Dox)-DNA
Complexes
3
Targeting
Dox-DNA Complex
• Sgc8c aptamer
Drug loading
▫ CCRF-CEM cells
▫ PTK7 proteins with a Kd ≈ 1 nM
▫ 5’-TAA CTG CTG CGC CGC CGG GAA
AAT ACT GTA CGG TTA GA-3’
• Extended double strand DNA
for Dox intercalation
▫ 5’-thiol-TTT TTT TTT TCC CTA ACC
CTA ACC CTA ACC C-3’
▫ 5’-ATC TAA CTG CTG CGC CGC CGG
GAA AAT ACT GTA CGG TTA GAT
TTT TTG GGT TAG GGT TAG GGT
TAG GG-3’
Dox:ds(sgc8c)
4
Dox:ds(sgc8c)-Au NPs
• Incubation of ds(sgc8c)-Au NPs (9.7
nM) with Dox (4 μM) for 2 h yields
the nanoconjugates loaded with 2.7
μM Dox
 68% loading efficiency
• For a single 13-nm Au NP
▫ 61 ± 10 Dox-DNA complexes
▫ 280 ± 23 Dox molecules
▫ 4-5 Dox molecules per DNA
Gold-Thiol
Bonding
• Hydrodynamic diameter change:
▫ 19.9 ±0.6 nm of citrate-stabilized Au NPs
▫ 42.1 ±4.0 nm of ds(sgc8c)-Au NPs
Au
5
PTK7
Parameters:
Sgc8c
aptamer
• Frequency: 10 MHz
• Acoustic pressure: 4 MPa,
Cells (2×105 cells/well) were incubated
• nM)
Duty
cycle: 10%
Dox-DNA
Complex
with Dox-loaded
nanoconjugates
(4.9
Au
• Treatment time: 10 min
loaded with 1.35-μM Dox in RPMI-1640
medium for 2 h.
HIFU
Treatment
CEM Cell
Au
Nucleus
http://oncozine.ning.com/profiles/blogs/new-drug-delivery-system-
Experimental Setup
Power
Amplifier
Immediately
Waveform
Generator 2
Agar
Phantom
10-MHz
HIFU Transducer
Waveform
Generator 1
Trigger
24 h later
• Flow cytometry: Mean Fluorescence Intensity (MFI)
• MTT assay: Cell Viability
7
Cellular Uptake Enhanced by Sgc8c
Dark field images of cells processed for silver enhancement.
Dox:ds(control)-Au NPs
Dox:ds(sgc8c)-Au NPs
~9000 Dox:ds(sgc8c)-Au NPs per cell determined by atomic absorption spectra.
8
Dox Release from Gold Nanoparticles
10-MHz HIFU, 4 MPa, duty cycle 10% (focal intensity ~140 W/cm2)
Pre-HIFU Treatment
Post-HIFU Treatment
Significant increase in fluorescence intensity indicates the release of Dox.
9
Dox Release from Gold Nanoparticles
10-MHz HIFU, 4 MPa, duty cycle 10% (focal intensity ~140 W/cm2)
45
40
35
30
25
20
15
10
5
0
MTT Assay
60
**
Cell Viability (%)
MFI (a.u.)
Flow cytometric analysis
**
50
15%
40
**: p < 0.01
30
20
10
0
w/o HIFU
w/ HIFU
w/o HIFU
w/ HIFU
No cytotoxicity was observed for
Dox:ds(control)-Au NPs (without specific binding affinity)
ds(sgc8c)-Au NPs (without Dox payload)
10
Comparisons of Different Cases
45
Dox release was only achieved when
both Dox-DNA complexes and Au
NPs were present.
40
MFI (a.u.)
35
30
25
Pre-HIFU
20
Post-HIFU
15
10
5
0
Pure Cells
Dox:ds(sgc8c)
PDPH-Dox-sgc8c-Au
NPs
Dox:ds(sgc8c)-Au
NPs
11
Varying HIFU Parameters
Release of Dox can be achieved by ultrasound
excitation at low acoustic pressures and duty cycles
55
50
45
Duty cycle: 10%
40
MFI (a.u.)
45
MFI (a.u.)
Pressure: 4 MPa.
40
35
30
25
35
30
25
20
20
15
15
0 2 3 4 5
Pressure (MPa)
0 0.5
1 2 3 4 5
Duty Cycle (%)
10
Possible mechanisms
Au
• Thermal effects?
• Mechanical effects?
Microcavitation
Focal intensity:
~140 W/cm2
Sonication Time (s)
(a.u.)
ΔMFI
ΔMFI (a.u.)
Temperature (˚C)
**
Normal
Degassed
13
Intracellular Reactive Oxygen Species
Cells were treated
with DCFH-DA before
HIFU treatments
30
MFI (a. u.)
25
20
ROS production was
observed in Cells with
ds(sgc8c)-Au NPs after
HIFU treatments.
15
Pre-HIFU
Post-HIFU
10
5
0
Pure Cells
ds(sgc8c)-Au NPs
Cell with H2O2
14
Summary
• Au NPs conjugated with Dox-DNA complexes have shown to
be ultrasound-responsive drug delivery systems. The drug
release mechanism was found to relate to microcavitation.
• The presence of Au NPs might provide nucleation sites for
facilitating gas nuclei on the surface of Au NPs to grow into
cavitation bubbles under HIFU excitation.
• Further investigation is required to clarify which phenomena
during microcavitation responsible for the Dox release.
•
•
•
•
ROS scavenger treatment.
DNA integrity evaluation.
Different HIFU parameters.
Different types of nanoparticles.
Thanks for
your attention.
15
16
Enhanced Dox Leakage
After HIFU Treatment
120
100
Gm (a. u.)
80
Cell
60
Cell-ultrasound
2.6-fold increase
56 nm
56 nm-ultrasound
40
20
0
0
2
4
6
Time (hr)
8
10
12
17
Varying Concentration of Au and Gas
Content (ROS measurement)
(56 nm Au NP )
35
30
Gm (a. u.)
25
20
ultrasound w/o
15
ultrasound w
10
5
0
ROS-ds-sgc8c-Au NP ROS-ds-sgc8c-Au NP ROS-ds-sgc8c-Au NP ROS-ds-sgc8c-Au NP ROS-ds-sgc8c-Au NP
(0.0625x)
(0.125x)
(0.25x)
(0.5x)
v.s. ds-sgc8c
18
Cellular uptake of ds(sgc8c)-Au NPs
determined by atomic absorption
spectra.
19
TEM image of 13-nm Au NPs
20 μm
20
MTT Assay
a) nontreated cells, and
cells incubated with
b) Dox
c) Dox:ds(sgc8c),
d) Dox:ds(sgc8c)-Au NPs
e) Dox:ds(control)-Au NPs
f) ds(sgc8c)-Au NPs
21
ROS Measurement
• Superoxide anion
(O−
2 , Lifetime: 3 us)
• Hydroxyl radical
(OH · ,Lifetime: < 1 us)
• Singlet oxygen
(1O2, Lifetime: < 1 us)
• Hydrogen peroxide
(H2O2, Lifetime: ~ms)
22
Possible Mechanisms
Au
• Pyrolysis in bubble interior.
▫ Dissociation due to high temperature.
• Hydroxylation at bubble interface.
▫ Reaction with Reactive Oxygen Species.
• Super-critical water at bubble interface.
I. Hua, R. H. Hoechemer, and M. R. Hoffmann, “Sonolytic Hydrolysis of pNitrophenyl Acetate: The Role of Supercritical Water,” The Journal of Physical
Chemistry, vol. 99, no. 8, pp. 2335-2342, Feb. 1995.