Innovative Imaging Probes - Encite

The European Network for Cell Imaging Traking Expertise
ENCITE Teaching File
Chemistry Department,
University of Turin, Italy
Contribution of Prof. Silvio Aime,
edited by Simona Baroni
For more information pleae visit: www.encite.org or contact EIBIR (office@eibir.org).
ENCITE is co-funded by the European Commision within the 7° Framework Programme and co-ordinated
by the European Institute for Bio-medical Imaging Research (EIBR).
Innovative Imaging Probes
CEST agents
Hyperpolarized molecules
1

CEST (Chemical Exchange Saturation Transfer) agents
Dw (rad . Hz) = w WAT - w CEST
If Dw > k ex then….
CEST
Agent
w CEST
w WAT
water
molecule
CEST agent
mobile protons
-40 -30 -20 -10 0 10 20 30 40
Dw
KHz
The MR-CEST experiment
Aqueous solution of
a CEST agent
ON resonance
irradiation
I S
rf field
Bulk
water
I WAT = 16.9 a.u.
I S = 7.8 a.u.
-40 -30 -20 -10 0 10 20 30 40
KHz
CEST agent
-40 -30 -20 -10 0 10 20 30 40
KHz
Dw Dw
Drawback: the irradiation may decrease I WAT even
in the absence of the CEST agent
- direct saturation of bulk water signal
- presence of immobilized mobile protons (in
biological samples, ca. 100 KHz broad)
Both these effects only depend on the absolute
value of the irradiation frequency
2

ST %
The MR-CEST experiment
Aqueous solution of
a CEST agent
ON resonance
irradiation
I S
rf field
Bulk
water
I WAT = 16.9 a.u.
rf field
I S = 7.8 a.u.
-40 -30 -20 -10 0 10 20 30 40
KHz
I 0
CEST agent
-40 -30 -20 -10 0 10 20 30 40
KHz
Dw Dw
OFF resonance
irradiation
I 0 = 14.5 a.u.
-40 -30 -20 -10 0 10 20 30 40
KHz
ON-OFF
difference image
ST % = (1-I S /I 0 )*100
46.2 %
ST-weighted
image
CEST agents: the sensitivity issue
The ST efficiency is proportional to the number of mobile protons
100
Simulated profiles* at 7 T
Dw = 50 ppm
80
60
k ex = 3300 s -1
B 1 = 11.75 mT
40
20
0
0 50 100 150 200 250
[mobile protons] - mM
A ST of 10% requires a total concentration of mobile protons of few mM
[CEST] agent =
[mobile protons]
number of mobile protons per molecule
3

How to increase the number of mobile protons
Use of nanoparticles
Liposomes
- well-known biocompatible systems
- a liposome with 200 nm of diameter contains
k ex
about 2.410 8 mobile water protons!
- k ex values cover a wide range (10-10 6 s -1 )
depending on the chemical composition of
the liposome membrane
In order to act as CEST agent, the resonance frequency of
the water protons inside the liposome must be shifted
LIPOCEST agents: a new entry for highly-sensitive CEST probes
The encapsulation of a paramagnetic shift reagent (SR) in liposomes
affects the resonance frequency of the water protons inside the vesicle
bulk
water
k ex
SR
Ln
d wat-lip
water
inside lipos.
40 30 20 10 0 -10 -20 -30 -40
KHz
d wat-lip depends on the type and concentration of SR
k ex depends on the membrane composition and liposome size
4

ST%
SR
LIPOCEST agents
Hydration
Vortexing
MLV
Extrusion
SUV
Dialysis
55 °C
55 °C
Lipidic film
SR unit
Tm
Water protons
inside liposomes
Bulk water
1
H-NMR spectrum (7 T, 298 K)
[TmDOTMA] -
0.12 M inside liposomes
DPPC/DPPG 95/5 (w/w) liposomes
LIPOCEST agents: sensitivity
POPC/DPPG/Chol (55/5/40 w/w) liposomes
312 K - [TmDOTMA] - 0.1 M – d WAT-LIP 3.2 ppm
MR-CEST images
(7 T - 312K - B 1 field 12 mT)
60
on-image
off-image
50
40
30
20
10
1 8 0 3 6 0
7 2 0
90
2 2 . 5
1 4 4 0
45
0
0 1000 2000 3000
[liposome] - pM
2 8 8 0
on-off
difference image
The sensitivity is augmented to ca. 100 pM !
5

LIPOCEST agents: passive tumor targeting in mice
Liposomes can passively target tumors by exploiting the increased
vascular permeability and the lack of an efficient lymphatic drainage
But in order to prevent opsonisation processes and prolong blood lifetimes
liposomes must be protected
use of STEALTH Liposomes
STEALTH Liposomes
Liposome coated by PEG chains
(t 1/2 in blood up to 3 days)
Experimental set-up
Animal model: Neuro 2A cells (murine neuroblastoma) inoculated
subcutaneously to mice
STEALTH LIPOCEST: POPC/Chol/DSPE-PEG (55/40/5) liposomes
SR = [TmDOTMA] - - d WAT-LIP = 2.6 ppm
diameter 110 nm
LIPOCEST agents: passive tumor targeting in mice
i.v administration of STEALTH LIPOCEST (dose: 0.04 mmol SR/kg)
MR images at 7 T overlaid with ST-effects
Pre-administration
Tumoral region
3 min post 30 min post
24 h post
6

Innovative Imaging Probes
CEST agents
Hyperpolarized molecules
What does “hyperpolarization” mean
Normal Polarization:
b
a
DN/N
10 -5
Low
NMR
sensitivity
No field B 0
Hyperpolarization:
b
a
10 5
enhancement
in sensitivity
No field B 0
7

Routes to hyperpolarization
Laser excitation ( 3 He or 129 Xe)
“Brute Force”
Dynamic Nuclear Polarization (DNP)
Para-hydrogen Induced Polarization (PHIP)
Laser-Polarized 3 He and 129 Xe
h n
Rb
Rb
Rb
Rb
Rb
Rb
Hyperpolarization
of Rb atoms
The radiation is circularly polarized;
the Rb D1 line is used (corresponding
to the 5s 1 -/ 25p transition)
1 / 2
Collisions between
Rb and Xe
Rb
Xe
(Van der Waals molecule)
Polarization transfer to Xe atoms
via “spin exchange”
Xe
Xe
Xe
8

Dynamic Nuclear Polarization (DNP)
Solid material doped with unpaired
electrons in a ratio of ~1:1000
13
C
13
13
13
C
C
C
13 13
C
13
13
13
C C
C
C
13
C e -
13
C
13
C
13 13
13
13
C
C
C C
13
13 C
13
C
C
13 13
C
13
13 C
C
13
C
C
13
13
C
13 13
C
C
C
e -
13
C
e -
13
C
13
C
13
13 13
C
C
13
C
13 C
13 C
13
C
C
13
13
C
13 13 C
C C
13
13 C
13
13 C
C
C
13
C
13
C
P e = 94% and P C = 0.086%
3.35 T and ~1.2K Microwaves transfer polarization
from electrons to nuclei
NMR and its Holy Grail
PNAS 100, 10158-10163 (September 2003)
< 1s
65 h
9

NMR and its Holy Grail
Imaging 13 C-labelled urea in a rat
Metabolic imaging of a rat tumor
with hyperpolarized 13 C-pyruvate
R3230AC,
mammary
adenocarcinoma
1
H-reference
pyruvate
alanine
lactate
10

Equilibrium p-H 2 percentage
Para-hydrogen Induced Polarization
(PHIP)
What Para-Hydrogen is
bb
c(ab+ba)
Triplet (ortho)
aa
c(ab-ba)
Singlet (para)
100
80
60
Normal Hydrogen =
75% ortho + 25% para
40
20
0 100 200 300 400 500
Temperature (K)
Para-Hydrogen Induced Polarization
(PHIP)
D
D
H H
D D
d 5
D
+ p-H 2
D
d 5
Resulting 1 H NMR spectrum:
Ortho-H 2
I = 0
ab
bb
Para-H 2
I = 0
Para-H 2
aa
AX spin system
ba
Signal enhancement
up to 10 5
11

Para-H2 containing molecules
as hyperpolarized contrast agents
In order to obtain a 13 C image by
using the 13 C polarized signal, it
must be transformed from
anthiphase to in-phase
bbb
bba
FIELD CYCLING (ab+ba)b
(ab+ba)b
Earth magnetic field (50 mT)
Non adiabatic (fast)
Zero magnetic field
(ab+ba)a
(ab+ba)a
aab
aaa
fast
m-Metal

The Nycomed approach: Para-H 2 containing
molecules as hyperpolarized CA
100% 13 C enriched
Sub-second 13 C-angiography
O
O
C C C C
D 3 C O O C D 3
p - H 2
c a t .
O
O
D 3 C O
C
C
H
C
C *
H
O C D 3
T 1 ( 13 C) = 75 s (7.05 T)
normal spin echo (SE) image
( 1 H) ( 13 C)
single shot RARE sequence
Magn. Res. In Med., 2001, 46, 1
Targets for Molecular Imaging
Apps.gemedicalsystems.com
13

Medical Imaging and range of detection
CT/x-ray
US
MRI
PET/NM
Optical
Anatomy Physiology Metabolism Molecular
Gd-chelates
Iron oxide
particles
Hyperpolarized
13
C-molecule
CEST
…a look at the market
S truc tura l Im a ging 1 )
22000
20000 MID CAGR 4.8%
18000 IT
16000 MIA
3200
$ 21.9 bn
3990
1742
Func tiona l/M ole c ula r Im a ging2 )
24000
22000
20000
18000
16000 MID
CAGR 11.5%
$ 21.8 bn
8823
14000
12000
10000
2451
707
1486
16203
14000
12000
10000
IT
MIA
4444
4065
8000
6000
9926
13350
8000
6000
2222
2229
8917
4000
4000
707
5960
2000
2000
3673
0
2000 2002 2004 2006 2008 2010
2001
2007
2012
0
2000 2002 2004 2006 2008 2010
2001
2007
2012
Main drivers
for growth
• Ageing population
(e.g.U.S., Europe, Japan)
• Further penetration of
unsatured markets
(e.g.China, India)
• Ageing population (e.g. U.S., Europe,
Japan)
• Broadening of indications for existing
MIAs
• Market entry of new MIA
generations/categories
• Technological improvement of MID
14