Chemistry Reference
In-Depth Information
Table 6.7 Percentages of [Eu
2
L
3
] helicate in aqueous solution at pH 7.4 (Tris-HCl 0.1M)
containing a total ligand concentration of 4.510
4
M.
(L6)
2
(L7)
2
(L8)
2
(L9)
2
(L10)
2
(L11)
2
Ligand
82.0
a
92.0
b
%Eu
2
L
3
97.1
88.7
92.5
94.6
Ref.
[45]
[46]
[49]
[50]
[50]
[50]
a
For total ligand concentration 1
10
4
M.
b
For total ligand concentration 1
10
3
M.
data reported in Table 6.7, the development of lanthanoid bioprobes has focused on heli-
cates with ligands (L7)
2
and, to a lesser extent, (L11)
2
. The thermodynamic stability
and kinetic inertness of the [Eu
2
(L7)
3
] helicate has further been assessed as a function of
pH and by competitive reactions with various chelating agents (edta, dtpa), anions
(citrate,
L
-ascorbate) and cations (Ca
II
,Zn
II
). It has proved to be satisfying: addition of
100 equivalent of exogenous reactants resulted in no, or at most 10%, loss in lumines-
cence properties after 24-96 h. The time for a bioanalysis being usually
1 h, the slight
dissociation observed after 24 h in some cases should not be a problem. In fact the
stability of [Eu
2
(L7)
3
] is intermediate between that of the polyaminocarboxylate with
dtpa and the well known macrocyclic [Eu(dota)]
complex [46]. The helicate is
however sensitive to the addition of one equivalent of 3d metal ions, such as Mn
II
,
Co
II
or Cu
II
,butnotFe
II/III
[76].
A second important point is the cytotoxicity of the helicates, which has been tested by
incubating several cell lines in the presence of various concentrations of helicates and
measuring the cell viability by WST-1 assays. These assays encompassed 5D10 mouse
hybridoma, Jurkat human T leukemia, HeLa cervical adenocarcinoma, MCF-7 human
breast carcinoma and nonmalignant epithelial HaCat (human keratinocytes) cells. They
were conducted for [Eu
2
(L6)
3
] [45], [Eu
2
(L7)
3
] [46], [Eu
2
(L8)
3
] (HeLa only) [49] and
[Eu
2
(L11)
3
](HeLaonly)[50]andevidencednocytotoxicity up to a concentration of
500 mM. Due to its smaller solubility, [Eu
2
(L5)
3
] was tested only up to 0.125 mM but was
also found to have no influence on cell growth and morphology [63]. With these data at
hand, combined with the interesting photophysical properties described above, the heli-
cates have been tested on three aspects: luminescent cell staining, specific targeting of
cancerous cells and tissues and DNA analysis.
<
6.2.5.1 Cell Penetration and Staining
Internalization experiments have involved helicates with (L5)
2
[63] and (L6)
2
(Ln
¼
Eu)
[45], (L7)
2
(Ln
Sm, Eu, Tb, Yb) [46,77] and (L11)
2
(Ln
Eu) [50]. Only a few rele-
vant features are described here. All tested helicates penetrate into the various cell lines
mentioned above without altering their morphology, as shown in Figure 6.7 (top panel, top
row): although time-resolved luminescence microscopy (TRLM) reveals that the cells con-
tain a sizeable amount of the Eu
III
chelate (bottom panel), no swollen nuclei or granules
are visible, contrary to what happens when the cells are incubated in presence of acridine
orange (top panel, bottom row). From the TRLM images (bottom panel), it is obvious that
the helicates are not uptaken in the nucleus of the cells. The first bright images can be seen
for helicate loading concentrations
¼
¼
5 mM and already after 10-15min incubation. The
chelates are first uptaken in isolated vesicles which diffuse into the cytoplasm and
>
Search WWH ::
Custom Search