ultrasound contrast agents: from imaging to therapysfp.in2p3.fr/expo/conf2008/vivant/bouakaz.pdf ·...

Ultrasound contrast agents:Ultrasound contrast agents:From imaging to therapyFrom imaging to therapy

Ayache BOUAKAZAyache BOUAKAZAyache BOUAKAZAyache BOUAKAZ

‐ UMR Imagerie et cerveau

‐ Inserm U930 – FRE CNRS 2448

‐Université François RabelaisUniversité François Rabelais

Tours, France

Contrast agent for ultrasound

UCA : Suspensions of tiny gas microbubbles for intravenous injection

Cross the capillary bedCross the capillary bed

• Diameter 1‐10 μm (Φ=3 μm)shell

Diameter 1 10 μm (Φ 3 μm)• Persistence: minutes to hours

• Gas: air or high molecular weight gas Gas• Shell: albumin/lipid/polymer

• Shell thickness 5 ‐ 500 nm

C i 1 5 108 b bbl / l• Concentration 1‐5 x 108 bubbles/ml• Sonovue Europe 2001

• Definity USA & Europe (Luminity 2007)Definity USA & Europe (Luminity 2007)

• Sonazoid Japan 2007

RBC6–8 µm

Contrast agent for ultrasound

What do we need contrast agent for?

• “Boost” image quality

• Enhance diagnostic confidence

Principle :‐ Amplify the amplitude of backscattered signal (echo)G i b bbl

• Clinical applications

‐ Gas microbubbles

Clinical applications– Estimation of myocardial perfusion– Detection and characterization of tumors– Amplification of Doppler signal

Microbubble – Ultrasound interaction

Linear resonator (harmonic oscillator)Linear resonator (harmonic oscillator)

Incident acoustic pressurePositive pressure

Negative pressure

Bubble size changes with acoustic pressurepressure

0 1 100.1 – 10 μs


3 P

Resonance frequencyResonance frequency

fr =1

2πR

3 γ Po

ρFree bubble : fr[MHz]= 3/R [μm ]

9

10

f

μm]

6

7

8Φ = 3 μm, fr=2.5MHz

Dia

mèt

re [μ

4

5

6

D

2

3

0 1 2 3 4 5 6 7 8 9 10Fréquence [MHz]

1


Scattering cross sectionScattering cross section

22

22

2

1

4

δ

π

+⎟⎟⎞

⎜⎜⎛

−

==Σf

RIP

ri

ss

10-2

m2 ]

2 1 δ+⎟⎟⎠

⎜⎜⎝ f

10-3

diffu

sion

[c

10-4effic

ace

de

R=1μm

10

Sec

tion

e

0 1 2 3 4 5 6 7 8 9 1010-5

Fréquence [MHz]


US: f=1.8MHz, 50kPa (MI=<0.1)Optic: Frame rate 16MHz

Linear and nonlinear scatteringLinear and nonlinear scattering

Soft-shelled agent (Bracco)

p

-10

0

7.5

7.7

dB

-30

-20

77.1

7.3

Dia

met

er [μ

m]

Frequency [MHz]0 1 2 3 4 5 6 7 8-50

-40

0

6.9

1 2 3 4 5 6 7 8Time [μs]


US: f=1.8MHz, 50kPa (MI=<0.1)Optic: Frame rate 16MHz

US: f=1MHz, 200kPaOptic: Frame rate 16MHz

Linear and nonlinear scatteringLinear and nonlinear scattering

Soft-shelled agent (Bracco)

p Optic: Frame rate 16MHz

4 5

5

10

0fondamentale

d

Harmonic Imaging

3.5

4

4.5

Dia

met

er [μ

m]

-30

-20

-10

dB

2nd H3rd HH

0 1 2 3 4 5 6 7 82.5

3

D

Time [μs]0 1 2 3 4 5 6-50

-40

Frequency [MHz]


MicrobubbleMicrobubble destruction (Triggered intermittent imaging)destruction (Triggered intermittent imaging)

PB127 (POINT Biomedical CA USA) Hard shelled agent

MI=1.3, f=1.7MHzOptic frame rate≈15Mfps

1st US pulse

PB127 (POINT Biomedical, CA, USA) Hard shelled agent


MicrobubbleMicrobubble destruction (Triggered intermittent imaging)destruction (Triggered intermittent imaging)

PB127 (POINT Biomedical CA USA) Hard shelled agent

MI=1.3, f=1.7MHzOptic frame rate≈15Mfps

1st US pulse

PB127 (POINT Biomedical, CA, USA) Hard shelled agent

2nd US pulse (100ms later)

Ultrasound and Ultrasound and microbubblesmicrobubbles

ImagingImagingi f i d i i l i• Tissue perfusion and microcirculation

• « Boost » sensitivity and specificity of US imaging (Harmonic – PI – PM ‐ Coded, …)

ExampleExample

• Detection and characterization (benign/malign) of lesions (e.g. liver)

• Exploration of cardiac function (estimation of myocardial perfusion)Exploration of cardiac function (estimation of myocardial perfusion)




ExampleExample



The video shows CEUS ultrasound in a kidney.




ExampleExample






ExampleExample



TherapyTherapy• Modulation of cell membrane permeability (sonoporation)

• Transport and release into targeted tissue of active principles (sonorelease)

• Targeted microbubbles for specific attachment (targeting)

Sonoporation

• Ultrasound creates transient permeabilization of cell membrane

in the presence of gas microbubbles

Sonoporation: In vitro

•• Experimental setExperimental set upup•• Experimental setExperimental set‐‐upup

Transducer Water (37°C)

5.105 cells

+ microbubbles+ microbubbles

DNADNA+ pDNA+ pDNAFocal distance

Magnetic stirrer

AmplifierWaveform generator

Confocal Fluorescent microscopy: In‐vitro

Transfection: Cy3Cy3-labelled DNA encoding for eGFPeGFPObservation T6H

Transmission eGFPExc laser: 488 nm en mode META, Obj x63, coupe optique de 0.8 µm d’épaisseur

%)

Transfection: DNA incorporation•Cellular model

20253035

cted

cel

ls (%•Cellular model

Human glioblastoma cells (U87‐MG)

5101520

r of

tran

sfec US + DNA

US+BR14+DNAUS parameters

•Frequency: 1 MHz05

18 24 48

Time (h)

num

be

eque cy:

•Pressure: 300 kPa

•Duty cycle: 40 % Time (h)

•Exposure time : 2 min

•20 bubbles per cell

•DNA: 30 μg/ml

25

•DNA: 30 μg/ml

15

20

xici

ty (%

)US + DNA

5

10cy

toto

xUS+BR14+DNA

018 24 48

Time (h)

FITC dextran incorporationCell line Hela

% cell uptake, FI120 1400

Cell line Hela

Dextran 70 kDa

60

80

100

percen

t [%

]

600

800

1000

1200

fluorescence

ensity [a

u]

1MHz, DC: 40 %

20 bubbles per cell0

20

40

200 400 600cell p

0

200

400

Mean inte

200 400 600

Pressure [kPa]

120

60

80

100

rcen

t [%]

0

20

40

cell pe

r

FITC‐dextran introduction

0Before insonation Immediately after insonation 1 min after insonation

Transfection: DNA incorporation

60

30

40

50

rcen

t (%

)

10

20

30

cell

per

0 GFP expression DNA-FITC

Transfection: DNA incorporation•Cellular model

50

60

70

%)

•Cellular modelHuman glioblastoma cells (U87‐MG) Transfection

Mortality

20

30

40

cell

perc

ent (

%

US parameters

•Frequency: 1 MHz

0

10

electroporation sonoporation lipofection

frequency (MHz)

eque cy:

•Pressure: 300 kPa

•Duty cycle: 40 % frequency (MHz)

1,82

atio

•20 bubbles per cell

11,21,41,61,8

mor

talit

y ra

te r

a

00,20,40,60,8

tran

sfec

tion-

m

0electroporation sonoporation lipofection

t

Transmembrane release of GFP with sonoporation

1MHz, 400 kPa, DC 40 %

Exposure time: 1 min

20 bubbles per cell

Not treatedHela GFP cell

US+ bubbles

%GFP

FI100

120

1200

1400

1600

ity [a

u]

FI

%PI

60

80

erce

nt [%

]

800

1000

1200

ence

inte

nsi

20

40cell

pe

200

400

600

ean

fluor

esce

0not treated cells US alone US+ BR14

0

200

Me

GFP recoveryGFP recovery

GFP %, FI120

1200

1400

e

Frequency: 1MHz

k60

80

100

P cell %

600800

1000

1200

luorescence

nsity [aU]

Pressure: 400 kPa

Duty cycle: 75 %

Exposure time: 2 min 0

20

40GFP

0

200

400

600

Mean f

inten

Nbr of bubbles per cell: 200

control T0 T4 T48TIme [hr]

0

152025

I %

PI %

120

140160

x vs

cont

rol

120

140160

x vs

cont

rol

05

10PI

2040

6080

100

olife

ratio

nin

dex

2040

6080

100

olife

ratio

nin

dex

control T0 T4H T48HTIme [hr]

020

Control US US+BR14

Pro

020

Control US US+BR14

Pro

In‐vivoIntramuscular transfectionf

Mouse (8w)Plasmid lucifirase

25000

Polymer/DNA complexesInjection: interior tibial muscle

20000

otei

n)

US1=400kPa, DC=40%

15000

(cps

/mg

pro

US2=500kPa, DC=40%US3=400kPa, DC=20%

US 00 a, C 0%

10000

ase

activ

ity (

5000

Luci

fera

0CTRL BR14+US1 BR14+US2 BR14+US3

ULTRASOUND ULTRASOUND ‐‐MICROBUBBLE MICROBUBBLE ‐‐ CELL INTERACTION CELL INTERACTION Optical observationsOptical observations

“Cellular Massage”“Cellular Massage”

Jet speeds V> 100’s m/s

http://www.scs.uiuc.edu

ll f dUS=0.5MHz, MI=0.9, Frame rate≈3Mfps

BR14

10 million frames per second

SonoVueTM, endothelial cell 1 burst 1 MHz Ultrasound MI 0.9

V<100m/s

Brandaris EMC Rotterdama b

Patch clamp technique“Whole cell” configuration

Seal Patch

Whole cell configuration

Em recordings- Seal - Patch - Em recordings

- A glass pipette gently pressed on the cell membrane (few microns)Electrical access « Seal » between the glass pipette and the membrane- Electrical access « Seal » between the glass pipette and the membrane

- Suction is applied to the pipette - Membrane breaks

C l d i l i i

- Measurement of cell membrane potential

- Cytoplasm and pipette solution mix up- Control of the intracellular medium

Measurement of cell membrane potential- Estimation of ion exchange through the cell membrane

ULTRASOUND ULTRASOUND ‐‐MICROBUBBLE MICROBUBBLE ‐‐ CELL INTERACTION CELL INTERACTION

Cell membrane potential

US alone US and Sonovue

iel [mV]

ne potent

Mem

bran

US=1MHz, 200kPa US 150kPa US 200kPa

Hyperpolarization of the cell membraneyp p



US 0.2MPa, 1MHz



US 0.2MPa, 1MHz

‐ Hyperpolarization: ‐ amplitude: 25 ± 1,4 mV (n=16)‐ function of the bubble‐cell adherence‐ function of ultrasound parameters


Mechanical stress10 μm


Glass rod

cell

Pipette de patch

rod

p

Involvement of stretch activated channels“SAC”IbTx PSS 2PSS 1 “SAC”

US

US+BR14 US+BR14


Intracellular calcium concentration measurement (fluorescent dye fura‐2 )

Mechanical Pressure

[Ca]i measurefitting curve

Cascade of events initiated by microbubble oscillationsCascade of events initiated by microbubble oscillations


Microbubble oscillations

Cell membrane deformation

Opening of SAC channels

Hyperpolarization

[Ca2+] concentration increase

Important role in the increased permeability

Trigger for different pathways e.g. endocytosis

Conclusions

• Imaging:

• CA has an established utility in improving accuracy of diagnostic

ultrasound

• CA provides information that makes better diagnoses

• Therapy:

• Ultrasound and microbubbles enhance drug internalization.

• Microbubbles are able to achieve drugs /genes to regions of interest,

and eventually recognize their target (e.g., thrombus).

• Ultrasound can be used to control the drug release from outside and onUltrasound can be used to control the drug release from outside and on

demand.

Acknowledgments

[email protected]

• K Kaddur MSc (PhD program) (Biologist) • Prof C Pichon CBM Orléans

Research team:

• K. Kaddur, MSc (PhD program), (Biologist)

• T. Tran, MSc (PhD program), (Biologist)

• Dr. P. Palanchon, PhD (PostDoc)

• Prof. C. Pichon CBM Orléans

• A. Delalande, MSc (PhD program)

• Dr. P. Midoux, CBM OrléansDr. P. Palanchon, PhD (PostDoc)

• A. Novell, MSc (PhD program)

• Prof. F. Tranquart, MD – PhD

Dr. P. Midoux, CBM Orléans

• Dr. B. Pitard, Inserm Nantes

Financial support

ultrasound contrast agents: from imaging to therapysfp.in2p3.fr/expo/conf2008/vivant/bouakaz.pdf ·...

Documents