5th EuropEan MolEcular IMagIng MEEtIng - ESMI

5th EuropEan MolEcular IMagIng MEEtIng – EMIM2010
Photochemical activation of endosomal escape of MRI-Gd-agents in tumor cells
Gianolio E. (1) , Arena F. (1) , Hogset A. (2) , Aime S. (3) .
(1) Centro di Imaging Molecolare,
(2) PCI Biotech AS Laboratories,
(3) Molecular Imaging Center, Torino Italy.
eliana.gianolio@unito.it
Introduction: The cellular uptake of xenobiotics often
procedes through the entrapment into endosomes. As
recently reported1,2, in the case of cellular labeling with
Gd-based complexes, the confinment into endosomal
vesicles negatively affects the attainable relaxation enhancement
of water protons. In fact it has been shown
that, upon increasing the number of Gd(III) per cell, a
quenching effect on the observed relaxivity takes place.
It is the consequence of the fact that the term |R1end
– R1cyt| is > than the water exhange rate between the vesicular
and cytosolic compartments. In this communication
we report an efficient method for endosomal escape
of paramagnetic Gd(III) chelates that yields to a marked
improvement of the efficiency in cellular labeling.
Methods: The Photosensitizer TPPS2a (LumiTrans®)
and the Lumisource® lamp were provided by PCI Biotech
AS laboratories (Oslo, Norway). For cellular labeling,
ca. 3-4×106 HTC,K562, NEURO2A and C6 cells
were incubated for 18 hours at 37°C with different concentrations
(5-100mM) of Gd-HPDO3A in the absence
and in the presence of the Photosentisizer (2µl/ml). After
this incubation time cells were washed three times
and incubated for additional 4 hours at 37°C. Then the
cells containing the Photosentisizer were exposed to LumiSource
light for 5 minutes. Then cells were detached,
washed and transferred into glass capillaries for registraion
of MR-images on a Bruker Avance300 spectrometer
operating at 7.1T.
K562 cells
B
K562 cells
A
C
D
E
F
G
R1obs (s -1 )
10
9
8
7
6
5
4
3
2
1
0,0 5,0x10 9
1,0x10 10
Number of Gd 3+ / cell
1,5x10 10
Fig.1: A) T 1 -weighted spin echo images of K562 cells labeled with Gd-HPDO3A. Phantoms E, F
and G contain cells treated with the PCI technology while phantoms B, C, and D contain cells
simply labeled by pynocitotic uptake. B) Observed relaxation rates of cells (HTC stars; NEURO-2a
circles; C6 triangles) labelled with Gd-HPDO3A with endosomic (black symbols) distribution as
a function of the number of Gd(III) found in each cell.
Results: The cellular labeling has been pursued
by pynocitosis (18h at 37°C) at different concentration
of Gd-HPDO3A in the presence and in
the absence of PCI treatment. As shown in the
figure 1A, a marked enhancement in the labeling
efficiency has been observed upon application
of photochemical stimulus to cells entrapping
Gd-HPDO3A and TPPS2a. This considerable
gain in signal intensity achieved when cells are
processed with PCI tehnology is due to the release
of Gd-units from endosomes to cytosol. As
shown in Fig. 1B, the “quenching” effect on the
relaxivity of cells processed with PCI technology
is reached when the number of Gd-HPDO3A per
cell is ca. One order of magnitude higher than the
number causing the same effect when the paramagnetic
complexes are confined into endosomes.
Thus, on going from the endosome-entrapped to
cytoplasm-entrapped Gd(III), the paramagnetic
loading can be several times higher thus allowing
a marked improvement in the MRI detection of
labelled cells.
Conclusions: The PCI methodology appears an
excellent route to pursue the endosomal escape of
Gd-HPDO3A molecules entrapped by pynocitosis
in different types of cells. It as been shown that
it allows to exploit high payload of Gd-HPDO3A
before the quenching effect becomes detectable.
Fig. 1: A) T1-weighted spin echo
Neuro
images of K562 cells Neuro_lumi labeled with
Gd-HPDO3A. Phantoms Htc E, F and G
contain cells treated Htc_lumi with the PCI
C6
technology while phantoms C6_Lumi B,C and
D contain cells simply labeled by
pynocitotic uptake. B) Observed
relaxation rates of cells (HTC stars;
NEURO-2a circles; C6 triangles)
labelled with Gd-HPDO3A with
endosomic (black symbols) or
cytoplasmatic (red symbols)
distribution as a function of the
number of Gd(III) found in each cell.
References:
1. Terreno, E et al., Magnetic
Resonance in Medicine,
2006, 55, 491-497.
2. Strijkers G.J. et al. 2009,
61, 1049-58.
EuropEan SocIEty for MolEcular IMagIng – ESMI
day1
Parallel Session 4: PROBES - supported by COST