1

2012.8.14

DNA-Conjugated Gold Nanoparticles as

Ultrasound-Responsive Drug Delivery

Systems

Department of Biomedical Engineering and Environmental Sciences,

National Tsing Hua UniversityHsinchu, Taiwan.

Shih-Tsung Kang, Yun-Ling Luo, Yu-Fen Huang, Chih-Kuang Yeh

2

Introduction

• Nanoparticles as drug delivery systems:

▫ High stability▫ High carrier capacity▫ Specific targeting capability▫ Controlled release capability

• Gold nanoparticles (Au NPs):

▫ Easy synthesis and bioconjugation▫ Plasmonic resonance oscillations▫ Tunable optical characteristics▫ Strong photothermal response▫ Photoacoustic imaging contrast

Au

13 nm Au NP

Doxorubicin (Dox)-DNAComplexes

HIFUExcitation

3

Dox-DNA Complex• Sgc8c aptamer

▫ 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’

Targ

etin

gD

rug

load

ing

Dox:ds(sgc8c)

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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

• Hydrodynamic diameter change: ▫ 19.9 ±0.6 nm of citrate-stabilized Au NPs▫ 42.1 ±4.0 nm of ds(sgc8c)-Au NPs Au

Gold-Thiol Bonding

5

http://oncozine.ning.com/profiles/blogs/new-drug-delivery-system-gold

PT

K7 CEM Cell

Nucleus

HIFUTreatment

Au

Sgc8c aptamer

Dox-DNA Complex

Au

Cells (2×105 cells/well) were incubated with Dox-loaded nanoconjugates (4.9 nM) loaded with 1.35-μM Dox in RPMI-1640 medium for 2 h.

Parameters:• Frequency: 10 MHz• Acoustic pressure: 4 MPa, • Duty cycle: 10%• Treatment time: 10 min

10-MHz HIFU Transducer

AgarPhantom

Waveform Generator 2

Power Amplifier

Waveform Generator 1

Trigger

Experimental Setup

• Flow cytometry: Mean Fluorescence Intensity (MFI)

• MTT assay: Cell Viability

Immediately

24 h later

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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.

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Dox Release from Gold Nanoparticles10-MHz HIFU, 4 MPa, duty cycle 10% (focal intensity ~140 W/cm2)

Significant increase in fluorescence intensity indicates the release of Dox.

Pre-HIFU Treatment Post-HIFU Treatment

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Dox Release from Gold Nanoparticles

Flow cytometric analysis MTT Assay

w/o HIFU w/ HIFU05

1015202530354045

MF

I (a

.u.)

**

w/o HIFU w/ HIFU05

101520253035404550

Cell

Via

bil

ity (

%)

**

10-MHz HIFU, 4 MPa, duty cycle 10% (focal intensity ~140 W/cm2)

No cytotoxicity was observed for Dox:ds(control)-Au NPs (without specific binding affinity)

ds(sgc8c)-Au NPs (without Dox payload)

**: p < 0.0115%

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Pure

Cells

Dox:d

s(sg

c8c)

PDPH-D

ox-sgc

8c-A

u N

Ps

Dox:d

s(sg

c8c)

-Au

NPs

05

1015202530354045

Pre-HIFUPost-HIFU

MF

I (a

.u.)

Comparisons of Different Cases

Dox release was only achieved when both Dox-DNA complexes and Au NPs were present.

Varying HIFU Parameters11

Release of Dox can be achieved by ultrasound excitation at low acoustic pressures and duty cycles

0 2 3 4 515

20

25

30

35

40

45

50

55

Pressure (MPa)

MF

I (a

.u.)

0 0.5 1 2 3 4 5 1015

20

25

30

35

40

45

Duty Cycle (%)

MF

I (a

.u.)

Pressure: 4 MPa.Duty cycle: 10%

Sonication Time (s)

Tem

pera

ture

(˚

C)

ΔM

FI

(a.u

.)Δ

MFI

(a.u

.)

Normal Degassed

**

Au

Possible mechanisms

• Thermal effects?• Mechanical effects?

Microcavitation

Focal intensity: ~140 W/cm2

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Pure Cells ds(sgc8c)-Au NP Cell with H2O20

5

10

15

20

25

30

Pre-HIFUPost-HIFUM

FI

(a. u

.)

Intracellular Reactive Oxygen Species

Cells were treated with DCFH-DA before HIFU treatments

ROS production was observed in Cells with ds(sgc8c)-Au NPs after HIFU

treatments.

s

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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.

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16

Enhanced Dox LeakageAfter HIFU Treatment

0 2 4 6 8 10 120

20

40

60

80

100

120

CellCell-ultrasound56 nm56 nm-ultrasound

Time (hr)

Gm

(a.

u.)

2.6-fold increase

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Varying Concentration of Au and Gas Content (ROS measurement)

ROS-ds-sg

c8c-Au N

P (0.0

625x)

ROS-ds-sg

c8c-Au N

P (0.1

25x)

ROS-ds-sg

c8c-Au N

P (0.2

5x)

ROS-ds-sg

c8c-Au N

P (0.5

x)

ROS-ds-sg

c8c-Au N

P v.s. d

s-sg

c8c

0

5

10

15

20

25

30

35

(56 nm Au NP )

ultrasound w/o

ultrasound w

Gm

(a. u

.)

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Cellular uptake of ds(sgc8c)-Au NPs determined by atomic absorption spectra.

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TEM image of 13-nm Au NPs

20 μm

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MTT Assay

a) nontreated cells, and cells incubated with

b) Doxc) Dox:ds(sgc8c), d) Dox:ds(sgc8c)-Au NPse) Dox:ds(control)-Au

NPsf) ds(sgc8c)-Au NPs

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ROS Measurement

•Superoxide anion (, Lifetime: 3 us)•Hydroxyl radical (OH

· ,Lifetime: < 1 us) •Singlet oxygen (1O2, Lifetime: < 1 us)•Hydrogen peroxide (H2O2, Lifetime: ~ms)

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Au

Possible Mechanisms

•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 p-Nitrophenyl Acetate: The Role of Supercritical Water,” The Journal of Physical Chemistry, vol. 99, no. 8, pp. 2335-2342, Feb. 1995.