A “Multi-Heavy-Atom” Approach toward Biphotonic Photosensitizers with Improved Singlet-Oxygen Generation Properties

Article abstract of DOI:10.1002/chem.201901047

Two trispicolinate 1,4,7-triazacyclonane (TACN)-based ligands bearing three picolinate biphotonic antennae were synthetized and their Yb³⁺ and Gd³⁺ complexes isolated. One series differs from the other by the absence (L¹)/

Full text of DOI:10.1002/chem.201901047

A Journal of
Accepted Article
Title: A “Multi-Heavy-Atom” Approach Toward Biphotonic
Photosensitizers With Improved Singlet Oxygen Generation
Properties
Authors: Galland Margaux, Tangui Le Bahers, Akos Banyasz, Noelle
Lascoux, Alain Duperray, Alexei Grichine, Raphael Tripier,
Yannick Guyot, Marie Maynadier, Christophe Nguyen, Magali
Gary-Bobo, Chantal Andraud, Cyrille Monnereau, and Olivier
Maury
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To be cited as: Chem. Eur. J. 10.1002/chem.201901047
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A “Multi-Heavy-Atom” Approach Toward Biphotonic
Photosensitizers With Improved Singlet Oxygen Generation
Properties.
Margaux Galland,[a] Tangui Le Bahers,[a] Akos Banyasz,[a] Noëlle Lascoux,[a] Alain Duperray,[b] Alexei
Grichine,[b] Raphaël Tripier,[c] Yannick Guyot,[d] Marie Maynadier,[e] Christophe Nguyen,[f] Magali Gary-
Bobo,*[f] Chantal Andraud,*[a] Cyrille Monnereau,*[a] and Olivier Maury*[a]
Abstract: Two TACN based ligands bearing three picolinate
biphotonic antennae were synthetized and their Yb3+ and Gd3+
complexes isolated. One series differs from the other by the absence
(L1) / presence (L2) of bromine atoms on the antenna backbone
offering respectively improved optical and singlet oxygen generation
properties. Photophysical properties of the ligands, complexes and
micellar suspension in Pluronic were investigated. Complexes
exhibit high two-photon absorption cross-section combined either
with NIR emission (Yb) or excellent 1O2 generation (Gd). The very
large inter-system crossing efficiency induced by the combination of
bromine atom and heavy rare-earth element was corroborated with
theoretical calculations. The 1O2 generation properties of L2Gd
micellar suspension under two-photon activation leads to tumour
cells death introducing the potential of such structures to offer
theranostic applications.
have become a reality following the groundbreaking works of
Dougherty in the 1970’s.[5] PDT has experienced a major burst
of interest in the last 15 years: on the one hand various recent
proofs of principles have indeed illustrated the relevance and
benefits of two-photon activation in the framework of PDT; on
the other hand, singlet oxygen photosensitizers have often been
a key component in theranostic architectures.[7]
In that framework, a significant number of molecular engineering
works have attempted to substitute porphyrin macrocycle by
better suited photosensitizers. In most of these works, strategies
aimed at improving Inter-System Crossing (ISC), i.e. the
transition from the singlet excited state to a more long-lived and
thus reactive triple state able to achieve energy transfer to the
surrounding triplet oxygen, have relied on the so called “Heavy
Atom Effect”.[8] Briefly, introduction of atoms of high mass
number within the molecule enhance spin-orbit coupling effects,
which partially releases the spin forbidden nature of the ISC
process.[8d] Halogen substituted extended π-conjugated
chromophores[9] or third row (Ru2+, Ir3+) transition metal
complexes[10] have been particularly used in that regard.
[6]
Introduction
Since the discovery of Raab and Von Tappenier in the early
1900 that the activation of organic dyes by light could efficiently
induce cell death[1] followed with the identification by Foote of
the
related
singlet
oxygen
mediated
mechanism,[2]
PhotoDynamic Therapy (PDT) has never ceased to be the
object of many research endeavours.[3] Porphyrins have played
a major role in that field,[4] and their use in clinical application
[a]
Dr. M. Galland, Dr. A. Banyasz, Dr. N. Lascoux, Dr. T. Le Bahers,
Dr. C. Andraud, Dr. C. Monnereau, Dr. O. Maury
Laboratoire de Chimie de l’ENS de Lyon
Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude
Bernard Lyon 1, F-69342 Lyon, France.
E-mail: chantal.andraud@ens-lyon.fr; cyrille.monnereau@ens-
lyon.fr; olivier.maury@ens-lyon.fr
[b]
[c]
Dr. A. Duperray, Dr. A. Grichine
INSERM, U1209, Université Grenoble Alpes, IAB, F-38000,
Grenoble, France
Pr. R. Tripier,
Univ Brest, UMR CNRS-UBO 6521 CEMCA, IBSAM, UFR des
Sciences et Techniques, 6 avenue Victor le Gorgeu, C.S. 93837,
29238, Brest, Cedex 3, France.
[d]
[e]
Dr. Y. Guyot,
Univ. Lyon, Institut Lumière Matière, UMR 5306 CNRS–Université
Claude Bernard Lyon 1, 10 rue Ada Byron, 69622 Villeurbanne
Cedex, France.
Figure 1. Structure of the lanthanide complexes.
C. Nguyen, Dr. M. Gary-Bobo,
Faculté de Pharmacie, Institut de Biomolécules Max Mousseron,
UMR 5247 CNRS-UM, 15 Avenue Charles Flahault, 34093
Montpellier Cedex 05, France.
E-Mail: magali.gary-bobo@univ-montp1.fr
Dr. M. Maynadier,
Conversely, in spite of their large atomic mass which ensures
efficient ISC, the use of lanthanide complexes as potential PDT
[f]
NanoMedSyn, 34093 Montpellier, France.
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
1
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photosensitizers has been relatively less studied to date.[11]
Pioneering works by Sessler and co-workers in in the late 90’s
pointed out the potentialities of texaphyrin lutetium or gadolinium
complexes for PDT, one of this complex lutexaphyrin being a
clinically used one photon sensitizer.[11d,e] Several examples
described the influence of oxygen as quencher of lanthanide
luminescence leading to the design of oxygen sensors[12] and
the competition between lanthanide sensitization and singlet
oxygen generation has recently been exemplified on Yb(III) by
Faulkner and co-workers using a pyrene antenna.[13] Recently,
the particularly strong heavy atom effect of Gd(III), a non-
emissive lanthanide ion has been reported leading to very
efficient singlet oxygen generation efficiency ( over 80%).[14]
This observation led us to consider that lanthanide complexes
may have been overlooked to date particularly in the framework
of two-photon PDT, in spite of little matched potential. Indeed
the association of their sensitivity to oxygen with the intrinsic
features of lanthanide complex, such as long lived luminescence
ranging from the visible to the Near Infra-Red (NIR) (Tb3+, Eu3+,
Yb3+, Nd3+, Sm3+) or magnetic properties (Gd3+, used in
Magnetic Resonance Imaging (IRM) contrast agents) could be of
high interest in view of theranostic applications.[15] In this context,
recent works from the groups of Heitz and Tóth reported
interesting theranostic bimetallic complex combining two-photon
PDT and MRI in which the Gd(III) ion is mainly confined in the
role of contrast agent.[16]
chromatography. The methylester functions were then
hydrolyzed leading to free ligands L1,2 and complexed with the
LnCl3.6H2O salts (Ln = Gd or Yb) using classical reported
procedures.[17b]
In the present article, and in the framework of ongoing
researches dealing with the use of lanthanide complexes for
one- and two-photon imaging application,[12c, 17] we explored the
potential of Yb(III) and Gd(III) complexes based on
functionalized tris-picolinate 1,4,7-Triazacyclonane (TACN)
macrocyclic ligands as photosensitizers. In order to improve
their nonlinear optical properties, and render them compatible
with two-photon activation, the ligand was functionalized with
extended π-conjugated Charge Transfer (CT) antennae
featuring or not additional bromine atoms along their backbone
(Ligand L1 and L2, Figure 1) known to favor ISC and
consequently singlet oxygen generation, when localized at the
right position.[18] We demonstrated a strong synergy for singlet
oxygen generation efficiency when combining
a bromine
Scheme 1. Synthetic procedure for LOMe1 and LOMe2
.
substituted ligand to a Gd(III) cation. As a result, a strong
enhancement in singlet oxygen generation efficiency is
characterized, making our approach introduced herein
particularly effective towards the design of improved two-photon
photosensitizers with potential utility in theranostics. To illustrate
this prospective, incorporation of the Gd3+ complex into pluronic
micelles was explored, affording nano-objects capable of
inducing breast cancer cell death under two-photon excitation.
The photophysical properties of LOMe1,2 and their related LnL1,2
(Ln Yb, Gd) complexes were studied in diluted
=
dichloromethane (DCM) solution (Figures 2-4, Figure S2,3, and
Table 1). The experimental data were systematically compared
with theoretical analysis performed on Y(III) model complexes
using simplified ligands featuring only one -conjugated antenna
YL1’,2’ (Table S1, Figure 5).[19] Computational and photophysics
details are reported in the supplementary information. As
expected, the low energy part of the absorption spectra of the
ligands is in all cases dominated by a broad and structureless
transition presenting the characteristic features of a Charge
Transfer (CT) transition from the strong electro-donating
dialkylamino group to the moderate electron withdrawing
Results and Discussion
The synthesis of the target ligands LOMe1,2 is described in
scheme 1. Details about the materials, methodology, synthetic
protocol and complete chemical characterization of all
intermediates are given in the supporting information. Briefly,
picolinate moieties (with a strong HOMO-LUMO character).[16]
A
both ligands were obtained using
a similar convergent
methodology: the antennae were prepared using a series of
Sonogashira coupling and deprotections of the intermediate
silylated alkynes, terminal OH were mesylated and the resulting
molecules were involved in a triple nucleophilic substitution on
the secondary amines of TACN, to afford the triply functionalized
ligands LOMe1 and LOMe2 after purification by column
second transition is also observed at higher energy that can be
assigned to a LE -* transition corresponding to the HOMO-1
to LUMO (figure 5) localized on the central part of the
conjugated scaffold. Interestingly, whereas complexation has a
little influence on the absorption maxima, the presence of
1
2
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10.1002/chem.201901047
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bromine atoms induces a bathochromic shift of ca. 35 nm of the
CT transition. This can be ascribed to the electron withdrawing
character of the bromine substituent, which increases the CT
character of the transition and is also consistent with
computations.
characterized by a large Stokes shift (ca 7500 cm-1) typical for
charge transfer transitions. Concerning the ligands, a similar
bathochromic effect as for the absorption spectra is observed
upon introduction of bromine substituents (LOMe1 566 nm and
LOMe2 600 nm, em
= 34 nm). As already observed,
complexation by a lanthanide cation (Gd or Yb) results in a
significant quenching of the ligand centred emission; the weak
residual emission band presents
a marginal red-shift as
compared to the free ligand. Ligand LOMe1 features a moderately
high emission quantum efficiency (=24%). In addition, the
introduction of bromine substituents onto the molecule results in
a marked quenching of the luminescence (LOMe2 =9%) which,
can be assessed to the increased ISC rate in the latter (vide
infra), in agreement with what has already been observed on a
related organic quadrupolar chromophore.[18]
Table 1. Photophysical properties of ligands LOMe1 LOMe2 and related LnL1,2
(Ln = Yb, Gd) complexes in DCM solution and NP1,2 suspension in PBS buffer
at room temperature.
[b]
λabs
(nm)
λem
(nm)

Φ [a]
(%)
Φ
(L.mol-1.cm-1)
(%)
LOMe1
LOMe2
GdL1
390
424
400
566
600, 1280
615, 1280
66000
117000
92000
24
9
2
0
24
54
GdL2
YbL1
YbL2
NP1
426
400
430
384
417
624, 1280
580, 980
580, 980
593
79000
87000
85000
-
-
1
< 1
< 1
-
80
0
0
8
1.0
7
-
NP2
636
-
21[c]
6
5
[a] luminescence quantum yield is measured in the visible considering only the
residual emission of the ligand centered transition with coumarine 153 in
MeOH used as a reference (ex = 400 nm, Φref = 45 %); [b] phenalenone in
DCM used as a reference compound (ΦΔ = 95%); [c] mesured in water using
competitive reacation method with DPAA scavenger (see SI).
4
0.5
3
2
1
0.20
0.0
0
400
600
1000
1200
1400
1.0
wavelength (nm)
0.15
0.8
Figure 3. Normalized absorption (red), visible emission (blue) and NIR
emission (black) of GdL2 in DCM at RT (ex = 400 nm).
0.6
0.10
0.4
0.05
0.2
1.0
0.5
0.0
0.0
0.00
1400
300 400 500 600 700
1200
1300
wavelength (nm)
Figure 2. Normalized absorption (red), visible emission (blue) and NIR
emission (black) of LOMe1 (dashed lines) and LOMe2 (straight lines) in DCM at
RT (ex = 400 nm).
400
600
900 1000 1100 1200 1300 1400 1500
wavelength (nm)
To confirm this assignment, the amount of charge transferred in
this transition could be quantified using the theoretical qCT index
based on the integration of the computed electron density
variation. As expected an important qCT of 0.6 and 0.7 was
computed for non-brominated molecules and for brominated
molecules, respectively (Table S1). Qualitatively speaking, the
charge transfer character of the transition is also demonstrated
by the drawing of the molecular orbitals involved in the first
transition, namely the HOMO and the LUMO (Figure 5). The
HOMO is mainly localized on the dialkylaniline group while the
LUMO is delocalized on central phenyl and pyridine groups, in
line with the aforementioned description. The molar extinction
coefficients are within experimental error, similar for all studied
compounds ca 9.104 L.mol-1.cm-1. All compounds also present a
broad, structureless ligand centred emission, which is
Figure 4. Normalized absorption (red), visible emission (blue) and NIR
emission (black) of YbL2 in DCM at RT (ex = 400 nm).
For the ytterbium complexes YbL1,2 the decrease in ligand
centred emission is accompanied by the apparition of an intense
emission in the NIR at 980 nm, characteristic of the F5/2→2F7/2
2
transition associated with Yb(III). This evolution can be attributed
to a very efficient energy transfer between the ligand excited
2
state and the F5/2 level of ytterbium, via the so-called antenna
effect.[20] The lifetimes of the f-f transition, measured in diluted
DCM solution, were found to be 9.3 and 9.7 µs for YbL1 and
3
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YbL2, respectively (Figure S4). The lifetimes are almost identical
indicating that the emission efficiency of these complexes is
rather similar. Since the Yb sensitization can proceed either from
the CT or from the triplet excited state,[20] the influence of the
bromine antenna functionalization (favoring the ISC process,
hence triplet sensitization) is not apparent on this system.
unambiguously characterized, which translates into a much
improved singlet oxygen generation ability of GdL2 of 80%.
This evolution drastically differs from that observed in our
previous works dealing with halogenated derivatives of purely
organic chromophores.[18] In the latter, we had clearly evidenced
that the essential part of the benefits arising from the
introduction of bromine substituents along the -conjugated
backbone of the chromophore on the ISC efficiency was
obtained after the introduction of a single substituent; additional
substitutions only resulted, at best, in a marginal increase of kISC
and , but were mostly inefficient and in some cases even
detrimental
to
the
photosensitizing
ability
of
the
chromophore.[18a] Here, opposite conclusions can be drawn, as
the singlet oxygen generation efficiency of GdL2 corresponds,
within experimental errors, to the additive effect of the two
contributions: bromine substitution and gadolinium complexation.
As a consequence, the singlet oxygen generation ability of GdL2
compares well with that of some of the best photosensitizers
reported to date, including clinically used porphyrin
derivatives.[4b] The absence of any singlet oxygen in the
corresponding Yb(III) complex can be explained by a much
faster intramolecular energy transfer leading to Yb(III)
luminescence in regard with the intermolecular collisional energy
transfer affording singlet oxygen.[13, 14b]
25
20
15
10
5
12
10
8
25
20
15
10
5
12
10
8
L1’
YL1’
HOMO
LUMO
HOMO-1
Figure 5. HOMO(-1), HOMO and LUMO orbitals of the YL2’ molecule, involved
in the first electronic transition (isocontour 0.02 a.u.).
6
6
4
4
2
2
0
0
0
0
On the other hand, for the Gd(III) complexes, although no
transfer to the lanthanide cation is energetically feasible, the
ligand centred emission intensity is however strongly affected by
the complexation. This can be ascribed to increased ISC rate
resulting from the presence of the heavy Gd cations.[14]
Interestingly, it appears that this quenching of the luminescence
is further enhanced upon introduction of halogen substituents.
Indeed, while GdL1 still presents a residual fluorescence
quantum yield of ca 2%, it is reduced to less than 1% for GdL2
(Table 1). Again, this can be put in line with the increased ISC
rate that is expected to result from the heavy atom effect of
bromine substituents. It is worth noting that, the decrease of
fluorescence quantum yield is by itself not a proof of the
increased ISC-rate as numerous other non-radiative processes
may induce the decrease of the fluorescence. In the following,
though, we present the concomitant singlet oxygen generation
experiments that strongly support the above-mentioned
hypothesis (Table 1).
0
30
60
0
30
60
25
20
15
10
5
12
10
8
25
20
15
10
5
12
10
8
L2’
YL2’
T2
6
6
4
4
T1
PES S1
2
2
0
0
0
0
0
30
60
0
30
60
Angle /
Angle /
Figure 6. Variation of the potential energy surface (PES) of the S1 state along
the rotation around a triple bond (blue curve and squares). Spin-Orbit Coupling
between the S1 and Tn states computed for each angle of rotation (green
symbols T2 state, red symbol T1 state).
The DFT and TD-DFT calculations bring an interesting point of
view on the mechanism of the ISC. It is well documented in the
literature that rotation around triple bond spacers is almost
While the non-substituted ligand LOMe1 does not provide any
measurable singlet oxygen generation, introduction of bromine
substituents (LOMe2) results in a significant improvement in the
photosensitizing properties, as singlet oxygen generation
quantum efficiency reaches 24%. Similarly, complexation of Gd
cation with the halogen free ligand is also seen to efficiently
barrierless at the ground state and is more difficult at the excited
state.[18d,
This behaviour is also observed on our system,
21]
based on the energetic scan along the rotation around the triple
bound spacer, computed at the S1 excited state (the excited CT
state). While more difficult than in the ground state, the rotation
is still possible at the excited state, with an energy increase of
only ~5 kJ.mol-1 for a rotation of 30°. The calculation of the spin-
orbit coupling between the S1 state and Tn states have been
performed as a function of the rotation around the triple bound.
Only the triplet states lower in energy than the S1 state were
considered, namely the T1 and T2 states (Figure S1) and based
on the molecular orbitals involved in these states can be
ascribed to 3CT and 3LE states respectively. Results are given in
drive the ISC process: GdL1 presents
a singlet oxygen
generation efficiency (54%) even higher than that of LOMe2 alone.
The most important result of this study, however, is achieved
upon complexation of the halogen substituted ligand with Gd
cation (GdL2): in the latter case, a synergetic effect of the
different heavy atoms on the ISC efficiency can be
4
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the Figure 6. It clearly appears that, the flat structure (0° of
rotation) undergoes almost no coupling between S1 state and Tn
states. A rotation is necessary to increase notably the spin-orbit
coupling between these states. While the increase is modest for
non-brominated molecules, it is notably larger for brominated
ones. Furthermore, for brominated molecules, the S1-T2 coupling
is much larger than the S1-T1 coupling. The rotation around the
triple bound cannot explain the increase of ISC induced by the
rare-earth element, but is necessary to ISC induction by the
bromine substituents.
by a distortion of the molecule (such as the rotation around a
triple bound spacer), the rare earth element contribution could
also be activated by a deformation, not yet found. Another
explanation sometimes proposed in the literature corresponds to
an increase of the S1-T1 mixing due to the paramagnetic nature
of the Gd3+ ion. This phenomenon, first theorized by Tobita et
al.[23] has been assessed on several rare-earth element based
complexes.[24]
wavelength (nm)
600
800
1000
1200
600
500
400
300
200
100
0
1.0
L2’
0.8
0.6
0.4
0.2
0.0
S2 (1LE)
S1 (1CT)
S1-T2
400
600
wavelength (nm)
T2 (3LE)
T1 (3CT)
Δρ=ρT1-ρS1
YL2’
Δρ=ρT2-ρS1
Figure 8. 1P-absorption spectrum of LOMe in DCM at RT (red line, lower
abscissa). Superimposed on this plot is the 2P-absorption measured by TPEF
method in DCM in wavelength doubled scale (■, upper abscissa).
2
S1-T1
Finally, the nonlinear optical properties of the ligand were
estimated using the Two-Photon Excited Fluorescence (TPEF)
method using a tunable fs-Ti-sapphire source (see supporting
information for details). These measurements were only
performed on the ligands LOMe1 and LOMe2 because of a too weak
residual visible emission of the related complexes to ensure
reliable 2P cross-section measurements by this mean. The two-
photon absorption (2PA) spectra of LOMe1 and LOMe2 are reported
in figure S7 and figure 8, respectively. They are found to
superimpose perfectly with the main CT transition in the one–
photon absorption (1PA) spectra, in good agreement with the
non-centrosymmetric character of the molecule. The maximal
two-photon absorption cross-section (2) is estimated to 540 GM
at 810 nm for LOMe2 and 950 GM at 780 nm for LOMe1. This value
is in the range of the best 2PA cross-section already described
for lanthanide ligand and/or complexes.[17, 25]
In conclusion of this spectroscopic study, it appears that the
“multi-heavy atoms” strategy that consists in combining bromine
atoms on the ligand and a complexation with Ln(III) to increase
ISC rate leads to the design of a very straightforward singlet
oxygen photosensitizer GdL2, combining large  value (80%)
with very high one- and two-photon absorption efficiency ( ~
80000 L.mol-1.cm-1 and 2 ~ 540 GM). Keeping in mind that
figure-of-merit of a 1P- or 2P-sensitizer is the product xand
x2, respectively (for GdL2 x = 64000 L.mol-1.cm-1 and
x2 = 430 GM) GdL2 present real potentiality for practical
applications in PDT. In addition, the computational results
highlight the importance to have a dynamical point of view on
the strength of the spin-orbit coupling that depends not only on
the composition of the system (i.e. the presence of heavy atoms)
but also on the possibility to undergo geometrical distortions
able to remove selection rules associated to spin-orbit
coupling.[18d]
S0
Δρ=ρT1-ρS1 Δρ=ρT2-ρS1
Figure 7. Simplified Perrin-Jablonski diagram (left) and (right) variation of the
electron density between the S1 and the Tn (n = 1, 2) states for L2’ (top) and
YL2’. Red and yellow areas correspond to electron increase and depletion
regions respectively (isocontour 0.0002 a.u.).
To understand the origin of the larger coupling with the T2 state,
the variation of the electron density between S1 and Tn states
were computed (Figure 7). Both transitions (S1→T1 and S1→T2)
have a charge transfer character with an increase of the electron
density of the aniline group. It means that the Tn states have a
less charge-separated character than the parent S1 state. It
appears that the bromine atoms are much more involved in the
S1→T2 transition than in the S1→T1 transition. Since the bromine
atoms have an important contribution to the Spin-Orbit Coupling,
it explains why the S1→T2 coupling is larger than the S1→T1 one.
The spin-orbit coupling exaltation induced by deformations has
also been proposed and rationalized in the field of Thermally
Activated Delayed Fluorescence (TADF) by Monkman et al..[22]
In addition to this increase of spin-orbit coupling due to the
molecular vibrations, Monkman et al. also suggest a mechanism
of S1-T1 coupling involving another triplet state. Our
computational results prove that the three-states model can be
used to compute triplet population for other application such as
singlet-oxygen generation. For the rare-earth containing
molecules, a slight contribution of the rare-earth element to the
variation of the electron density is computed. It highlights that
this element is also involved in the transition. We have shown
that bromine contribution to the spin-orbit coupling is activated
As a consequence of the presence of lipophilic alkyl substituents
on their periphery (chosen to ease their spectroscopic study in
organic solvents, by alleviating stacking issues) these GdL1,2
5
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complexes are absolutely not soluble in water nor in any
biological media. We thus decided to use a cargo strategy in
order to stabilize micellar suspensions of the complexes
compatible with such media. To that end, a dispersion using an
amphiphilic polymer (Pluronic F127, n = 100, m =65, figure 9)
has been envisaged, because of the well-known compatibility of
this polymer with in cellulo PDT, drug delivery or bioimaging
applications.[26] The Pluronic/GdL1,2 nanoparticles, noted NP1,2
are prepared through self-assembly, achieved by adding a
phosphate saline buffer (PBS) solution to a THF solution of
GdL1,2 complexes and Pluronic polymers. After slow evaporation
of the THF and filtration on 100 nm pore size filter, a bright
orange colloidal suspension in water is obtained. Both particles
population, measured by dynamic light scattering present a
homogeneous dispersion size centered at 50 nm (Figures 9, S9-
10). The spectroscopic properties of the nanoparticles in PBS
(Figures S5, S6, Table 1) compare well with their respective
parent complexes in DCM indicating that the structure of the
complex is preserved in the NPs. The singlet oxygen generation
quantum yield cannot be measured in water by relative
luminescence measurement because of the very short lifetime of
this reactive species in such protic medium. Consequently, the
singlet oxygen quantum yield was estimated via a competitive
reaction method using dipropionic acid anthracene (DPAA), a
emission of GdL1, live human T24 cancer cells have been
stained with NP1 and successfully imaged with one (ex = 405
nm) or two-photon (ex = 800 nm) microscopy (Figure 10). The
pictures clearly indicate the presence of the emissive species in
the endoplasmic reticulum of the cell, indicating that the NPs
have successfully transported the lipophilic complex inside the
cells. The perinuclear intracellular localization of the compound
overlaps well the typical mitochondria-rich region. This proximity
(and probable co-localization) would further favour PDT
efficiency through apoptosis and other reported anticancer
mechanisms.[29]
fluorescent singlet oxygen scavenger evolving into
a
nonfluorescent endoperoxyde species upon reaction with singlet
oxygen.[27] The comparison of the disappearance kinetic
constant of DPAA in the presence of NP2 suspension or
benchmark phenalenone as photosensitizers enable us to
estimate the (NP2) to ca. 21% in water (see SI for details,
Figure S7, Table S2).[28]
Figure 11. 2P-PDT experiment. MCF-7 cells were incubated with GdL2 (30
µM) for 24 h and or not irradiated with a pulsed laser by 3 scans of 1.57 sec at
800 nm (maximal power laser input 3W). Cell survival measurements were
done by the MTT assay 48 h after irradiation. (Top) pictures show the
difference between non irradiated and irradiated cells after MTT coloration.
(Bottom) bar graphs are the results of the quantification. Data are mean values
± standard deviation from three experiments.
Finally, the photodynamic therapy (PDT) potential of GdL2 was
performed on human breast cancer cells stained with NP2
nanoparticles under 2P-excitation (TPE). MCF-7 breast cancer
cells were treated with NP2 (leading to an estimated
concentration in GdL2 in the medium of 1 and 30 µM,
respectively) and submitted or not to a laser irradiation safe for
cells (Figures 11 and S11). All controls were done to determine
the part of cell death due to the NP2 treatment followed by
irradiation. In fact, irradiated cells without NP2, or cells incubated
with NP2 and not irradiated, exhibit no significant cell death in the
conditions used here (data not shown). At lower concentration (1
µM), the 2P-PDT effect was not clearly detectable which let us
try with a higher concentration (Figure S11). At 30 µM, the
combination of NP2 and 2P-irradiation (3x 1.57 s) clearly induced
a strong modification of the cells aspect: the initially clustered
cells are completely dispersed in the irradiation area.
Furthermore the quantification of cell viability by MTT assay
staining only living cells, demonstrated 66% of cell death. This
strong effect obtained at 30 µM concentration, after only 3 x 1.57
seconds of irradiation using a medium power laser (Carl Zeiss
10×/0.3 EC Plan-Neofluar, laser power input 3W), demonstrates
the robustness of the 2P-PDT potential of GdL2. We attribute
this high efficiency to the combination of the high two-photon
cross-section and the large singlet oxygen generation quantum
yield of this probe, as shown above.
Figure 9. Synthesis of the Pluronic/GdL1,2 nanoparticles and picture of the
suspension in PBS (top). DLS of NP2 and two-photon images of living T24
cells stained with NP1 (ex = 800 nm, bottom).
Figure 10. Intracellular distribution of GdL1 in lived T24 cells after staining with
NP1 nanoparticles. Overlay of transmitted light (DIC) imaging and 1P-(left) and
2P-(right) fluorescence microscopy imaging. 1P- and 2P-excitation
wavelengths are respectively 405 and 800 nm.
Preliminary tests concerning the behaviour of these NPs in
cellulo were undertaken using NP1. In spite of the weak residual
6
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10.1002/chem.201901047
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Author thanks Dr. Y. Bretonnière and Dr. M. Rémond for their
help in the preparation of Pluronic nanoparticles. The authors
are grateful to ANR (SADAM ANR-16-CE07-0015-01) for
financial support. IAB platform was co-funded thanks to grants of
‘‘Association pour la Recherche sur le Cancer’’ (ARC, Villejuif,
France),‘‘Ligue Contre le Cancer’’ (LCC Ise`re/Arde`che) and the
CPER program. The authors thank C. Chamot and the PLATIM
Imaging and Microscopy platform for the access to the
femtosecond laser system. We thank MRI (Montpellier RIO
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Keywords: Gadolinium • Photodynamic therapy • Intersystem
crossing• Two-photon microscopy • Lanthanide
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8
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
The TACN based gadolinium complex
exhibits high two-photon absorption
cross-section combined with near-
unity 1O2 generation. The very large
inter system crossing efficiency
induced by the combination of
bromine atom and heavy rare-earth
element was corroborated with
theoretical calculations. The 1O2
generation properties of micellar
Margaux Galland, Tangui Le Bahers,
Akos Banyasz, Noëlle Lascoux, Alain
Duperray, Alexei Grichine, Raphaël
Tripier, Yannick Guyot] Marie Maynadier]
Christophe Nguyen, Magali Gary-Bobo,*
Chantal Andraud,* Cyrille Monnereau,*
Olivier Maury*
Page No. – Page No.
A “Multi-Heavy-Atom” Approach
Toward Biphotonic Photosensitizers
With Improved Singlet Oxygen
Generation Properties.
suspensions
under
two-photon
activation lead to tumour cells death
introducing the potential of such
structures for theranostic applications.
9
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