Water-soluble dendrimeric two-photon tracers for in vivo imaging

Article abstract of DOI:10.1002/anie.200601246

(Figure Presented) In a (nut)shell: Covalently wrapped in a protecting and solubilizing dendrimeric shell (see picture), an initially Hydrophobic two-photon fluorophore becomes a biocompatible contrast agent with strong emission for imaging in vivo by mul

Full text of DOI:10.1002/anie.200601246

Angewandte
Chemie
Contrast Agents
the combination of reduced absorption and scattering).
Molecular engineering of optimized chromophores with
large TPEF cross-sections has been particularly active in the
last decade[3–5] and has led, for example, to bolamphiphilic TP
markers of interest for imaging of biological membranes and
cells or pH sensing.[4–6] However, water-soluble TP fluoro-
phores that maintain both a high fluorescence quantum yield
and a large TPA cross-section in the spectral range of interest
are still rare. In addition, fluorophores with a high TPA cross-
section in toluene (that is, in a lipophilic environment) have
been reported to undergo a significant decrease of their TPA
characteristics in water as a result of molecular aggregation.[7]
Herein, we report a general route for the design of water-
soluble TP markers from lipophilic TP fluorophores that
could be applied to a variety of TP chromophores. To prevent
aggregation while ensuring water solubility, we included a TP-
active lipophilic chromophoric unit in the core of a dendrimer
whose periphery is covered with cationic groups[8] (G3 in
Scheme 1). The dendritic sheaths should, in principle, provide
DOI: 10.1002/anie.200601246
Water-Soluble Dendrimeric Two-Photon Tracers
for In Vivo Imaging**
Thatavarathy R. Krishna, Manuel Parent,
Martinus H. V. Werts, Laurent Moreaux, Said Gmouh,
Serge Charpak, Anne-Marie Caminade, Jean-
Pierre Majoral,* and Mireille Blanchard-Desce*
Novel microscopies based on nonlinear optical (NLO)
phenomena such as two-photon excited fluorescence
(TPEF)[1] are rapidly gaining popularity in biological imaging
owing to the many advantages they provide. These include a
capacity for a highly spatially confined excitation and intrinsic
three-dimensional resolution, as well as the ability to image at
an increased penetration depth in tissue with reduced
photodamage and background fluorescence by operat-
ing with excitation radiation in the near-infrared (NIR)
region. At first, TPEF was developed using conventional
fluorophores whose two-photon absorption (TPA) char-
acteristics were not optimized,[2] but it was soon realized
that molecules specifically engineered for TPEF can
significantly outperform standard fluorophores opti-
mized for one-photon excitation. This realization trig-
gered the search for fluorophores combining a high
fluorescence quantum yield (F) and a high TPA cross-
section (s2) in the spectral region of interest for
bioimaging (typically 700–1000 nm, corresponding to
[*] Dr. T. R. Krishna, Dr. A.-M. Caminade, Dr. J.-P. Majoral
Laboratoire de Chimie de Coordination
CNRS
205 route de Narbonne, 31077 Toulouse Cedex 4 (France)
Fax: (+33)5-6155-3003
E-mail: majoral@lcc-toulouse.fr
M. Parent, Dr. M. H. V. Werts, Dr. S. Gmouh, Dr. M. Blanchard-Desce
Synth›se et ElectroSynth›se Organiques (CNRS, UMR 6510)
Institut de Chimie
UniversitØ de Rennes 1
Campus de Beaulieu, Bât. 10A, 35042 Rennes Cedex (France)
Fax: (+33)2-2323-6277
Scheme 1. Chromophore Q’ and a schematized structure of dendrimer G3.
isolation of the central chromophore from the outer environ-
ment, thus preventing fluorescence quantum yield decrease
by nonradiative processes mediated by water molecules, as
well as aggregation processes that are detrimental to the TPA
response. An alternative (and simpler) route consisting in
grafting water-solubilizing cationic groups directly onto the
TP-active lipophilic chromophore (chromophore Q’ in
Scheme 1) was also tested to evaluate the relevance of the
dendrimer approach.
We will show herein that the strategy to incorporate a
lipophilic chromophore in a protecting dendrimer shell is
indeed successful and leads to nanometric water-soluble
fluorescent TP-active tracers. These molecules were built
from the core reagent C by successive repetition of nucleo-
philic substitution and condensation reactions. The water
solubility is ensured by the ammonium groups that are linked
to the surface of G1–G3 in the last step by reaction with N,N-
diethylethylenediamine (Scheme 2).
E-mail: mireille.blanchard-desce@univ-rennes1.fr
Dr. L. Moreaux, Prof. S. Charpak
Neurophysiology & New Microscopies Laboratory
INSERM U603–CNRS FRE 2500
45 rue des Saints P›res, 75006 Paris (France)
[**] Financial support from the Rennes MØtropole and RØgion Bretagne
(“Renouvellement des CompØtences” Program) is gratefully
acknowledged. J.-P.M. thanks the Minist›re de la Recherche
(France) for a postdoctoral grant to T.R.K. The authors wish to thank
Fayçal Ksar for his experimental contribution.
Supporting Information for this article (synthesis of chromophores
Q and Q’; preparation of dendrimers G1–G3; experimental details
of spectroscopic measurements; comparison of emission spectra
of G1–G3 in water and ethanol (Figure S1); fluorescence anisotropy
results) is available on the WWW under http://www.angewand-
te.org or from the author.
Angew. Chem. Int. Ed. 2006, 45, 4645 –4648
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4645

Communications
Scheme 2. Synthesis of G1 from Q, and structures of G2 and G3. The core reagent C was prepared from the bisphenol quadrupolar chromophore
Q by treatment with N3P3Cl6 under basic conditions.
Table 1: Photophysical properties of compounds Q, Q’, and G1–G3.
Solvent labs,max [nm] emax [104 mÀ1 cmÀ1 lem [nm] F[a] s2 [GM][b]
Dendrimers G1–G3 show strong one-photon absorption
in the near UV and strong emission in the blue-visible region
(Figure 1). Interestingly, their photoluminescence (PL) effi-
]
Q
EtOH 379
7.80
–
435
354
442
444
443
0.79 155[5]
0.22
Q’ water
G1 water
G2 water
G3 water
349
377
381
383
8
7.27
6.24
6.31
0.53 104
0.71 119
0.66 127
[a] Fluorescence quantum yield determined relative to fluorescein in 0.1n
NaOH. [b] At 705 nm; 1GM=10À50 cm4 sphotonÀ1
.
and water tend to disappear with increasing generation
number (see the Supporting Information). As a result, the
emission spectra of the highest-generation dendrimer G3
show similar vibronic intensities in ethanol and water, thereby
indicating that the dendritic branches provide shielding layers
that isolate the core chromophore from interactions with the
external environment.
TPA measurements were conducted by investigating the
TPEF of dendrimers G1–G3 as well as model chromophores
Q and Q’ in solution. TPEF measurements allow for direct
determination of the TPEF action cross-section s2F, the
relevant figure of merit for imaging applications and from
which the TPA cross-sections can be derived.[9,10] In addition,
the TPEF method has been recognized to be more reliable
than nonlinear transmission measurements.[11] The quadratic
dependence of the TPEF signal on the excitation intensity
was checked for each data point and indicated that no
photodegradation or saturation occurs. We should stress that
the reported values, although moderate, are non-one-photon
resonant values, which means that these chromophores could
actually allow for the 3D resolution offered by selective two-
photon excitation in the NIR region. This is not the case when
even a small one-photon absorption is present at the
excitation wavelength, which is the case in a number of
Figure 1. Absorption and emission spectra of G1 (c), G2 (a),
and G3 (b) in water. Inset: rotational correlation time, q, as a
function of molecular weight m for dendrimers G1–G3 in water
(T=298 K), calculated from the steady-state fluorescence anisotropy
and the fluorescence lifetime using the Perrin equation. These aniso-
tropy data indicate that G1–G3 behave as isolated globular objects as
their rotational correlation times increase linearly with the molecular
weight. The hydrodynamic diameters of G1–G3 were estimated from
the rotational correlation times to be approximately 2.4, 3.8, and
5.2 nm, respectively.
ciencies are similar in water and ethanol. This indicates that
the dendrimeric approach is efficient in providing water
solubility while maintaining high PL efficiency. In contrast,
adding water-solubilizing ammonium groups directly to the
aliphatic chain of the quadrupolar chromophore Q (to give
the water-soluble chromophore Q’) does not lead to retention
of the PL efficiency in water (Table 1). This decrease in PL
efficiency is most probably related to molecular aggregation
phenomena. Indeed, the PL is restored upon addition of a
surfactant (sodium dodecylsulfate) to a water solution of Q’.
Interestingly, the differences in relative vibronic intensi-
ties in the emission spectra of dendrimers G1–G3 in ethanol
chromophores with
a
giant resonant TPA reported
recently.[12,13]
Strikingly, the water-soluble quadrupole Q’ undergoes a
marked decrease of its TPA characteristics (Table 1) in water
as compared to Q in ethanol. As a result, although being
water-soluble and based on a quadrupolar chromophore that
4646 www.angewandte.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 4645 –4648

Angewandte
Chemie
shows reasonable TPA efficiency in nonaqueous protic media,
Q’ has a low TPEF cross-section in water. In contrast,
dendrimers G1–G3 maintain a significant TPA cross-section
in water, thereby confirming that the dendritic branches
indeed isolate the chromophoric core, thus preventing
aggregation and PL quenching. Interestingly, the TPA char-
acteristics appear to increase slightly with increasing gener-
ation. This effect could be related to the slight increase of
local polarity at the core with increasing generation due to the
nature of the dendritic branches, which provide a somewhat
polar environment.[14] Recent calculations and experiments[15]
have shown that increasing solvent polarity, in a certain range,
can lead to TPA enhancement.
Our results demonstrate that water-soluble, luminescent
two-photon markers can be obtained by incorporating a
lipophilic two-photon chromophoric unit within shielding
layers built by a dendritic approach. This covalent layer-by-
layer approach allows us to modulate the solubility by varying
the nature of the peripheral groups while isolating the core TP
chromophore from deleterious effects. This route allows us to
overcome the drawbacks encountered when solubilizing
groups are grafted directly onto a TP chromophore and
provides a unique modular approach. In particular, replacing
the core unit by more efficient TP chromophores[3] and taking
advantage of the polar environment provided by the dendritic
environment should lead to more efficient TP tracers, while
further surface functionalization opens up a route for further
functionality (such as recognition or targeting). This strategy
can, in principle, be applied to various lipophilic chromo-
phores that show high TPA cross-sections and large fluores-
cence quantum yields as long as phenol moieties can be
grafted onto them, thus allowing for the building of the
dendritic sheath.
This is of particular interest for biological applications due
to the low toxicity detected for phosphorus-based dendrim-
ers.[16] This is a major advantage—in addition to the accessi-
bility for both inner and surface functionalization using
covalent chemistry—over semiconductor quantum dots
(QD). These inorganic nano-objects have gained a lot of
popularity as photonic imaging agents[17] but their toxicity is
still a main concern. Taking into account potential risks is one
of the most important issues in nanoscience. With this aim in
mind, we have demonstrated that the organic-nanodots route
can provide a promising alternative in terms of two-photon
brilliance,[18] and that it allows us to maintain excellent PL
properties while conveying water solubility, which leads to
water-soluble tracers that can indeed be used for in vivo
imaging (Figure 2). This is an important step that establishes
that organic nanodots definitely merit consideration as an
alternative to semiconductor QDs for two-photon imaging,
although further studies focusing on toxicity and (photo)-
stability are needed. In this respect, it is important to add that
in preliminary experiments we have found that polycationic
phosphorus dendrimers bearing the same ammonium end
groups are stable for months towards hydrolysis and that the
photostability of dendrimers G1 and G2 is significantly higher
than that of chromophore Q’ in water, thus indicating that this
strategy is also of interest in terms of photostability improve-
ment.
Figure 2. Two-photon imaging of the vascular network in the dorsal
part of the rat olfactory bulb. Vessels were labeled after injecting
intravenously a small bolus of 500 mm of dendrimer G2 in water. The
fluorescence was excited at 710 nm, epi-collected, and band-pass
filtered (440/40 nm). The image was taken at a depth of about
200 mm. No obvious toxic effects were observed during the experiment
(for technical details, see reference [19]).
Received: March 30, 2006
Published online: June 21, 2006
Keywords: chromophores · contrast agents · dendrimers ·
.
fluorescence · two-photon absorption
[1] W. Denk, J. H. Strickler, W. W. Webb, Science 1990, 248, 73 – 76.
[2] C. Xu, W. W. Webb, J. Opt. Soc. Am. B 1996, 13, 481– 491.
[3] a) S.-J. Chung, K.-S. Kim, T.-C Lin, G. S. He, J. Swiatkiewicz,
P. N. Prasad, J. Phys. Chem. B 1999, 103, 10741 – 10745; b) L.
Ventelon, L. Moreaux, J. Mertz, M. Blanchard-Desce, Chem.
Commun. 1999, 2055 – 2056; c) M. Rumi, J. E. Ehrlich, A. A.
Heikal, J. W. Perry, S. Barlow, Z.-Y. Hu, D. McCord-Maughon,
T. C. Parker, H. Rꢀckel, S. Thayumanavan, S. R. Marder, D.
Beljonne, J.-L. Brꢁdas, J. Am. Chem. Soc. 2000, 122, 9500 – 9510;
d) B. R. Cho, K. H. Son, S. H. Lee, Y.-S. Song, Y.-K. Lee, S.-J.
Jeon, J. H. Choi, H. Lee, M. Cho, J. Am. Chem. Soc. 2001, 123,
10039 – 10045; e) O. Mongin, L. Porrꢂs, L. Moreaux, J. Mertz, M.
Blanchard-Desce, Org. Lett. 2002, 4, 719 – 722; f) W. J. Yang,
D. Y. Kim, C. H. Kim, M.-Y. Jeong, S. K. Lee, S.-J. Jeon, B. R.
Cho, Org. Lett. 2004, 6, 1389 – 1392; g) L. Porres, O. Mongin, C.
Katan, M. Charlot, T. Pons, J. Mertz, M. Blanchard-Desce, Org.
Lett. 2004, 6, 47 – 50; h) C. Le Droumaguet, O. Mongin, M. H. V.
Werts, M. Blanchard-Desce, Chem. Commun. 2005, 2802 – 2804.
[4] L. Ventelon, S. Charier, L. Moreaux, J. Mertz, M. Blanchard-
Desce, Angew. Chem. 2001, 113, 2156 – 2159; Angew. Chem. Int.
Ed. 2001, 40, 2098 – 2101.
[5] M. H. V. Werts, S. Gmouh, O. Mongin, T. Pons, M. Blanchard-
Desce, J. Am. Chem. Soc. 2004, 126, 16294 – 16295.
[6] M. Blanchard-Desce, C. R. Phys. 2002, 3, 439 – 448.
[7] H. Y. Woo, J. W. Hong, B. Liu, A. Mikhailovsky, D. Korystov,
G. C. Bazan, J. Am. Chem. Soc. 2005, 127, 820 – 821.
[8] J. Leclaire, Y. Coppel, A.-M. Caminade, J.-P. Majoral, J. Am.
Chem. Soc. 2004, 126, 2304 – 2305.
[9] TPEF measurements were performed by excitation with 150-fs
pulses from a Ti:sapphire laser, using the protocol of Xu and
Webb,[2] taking into account refractive index effects.[10]
Angew. Chem. Int. Ed. 2006, 45, 4645 –4648
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 4647

Communications
[10] M. H. V. Werts, N. Nerambourg, D. Pelegry, Y. Le Grand, M.
Blanchard-Desce, Photochem. Photobiol. Sci. 2005, 4, 531– 538.
[11] D. A. Oulianov, I. V. Tomov, A. S. Dvornikov, P. M. Rentzepis,
Opt. Commun. 2001, 191, 235 – 243.
[12] R. Misra, R. Kumar, T. K. Chandrashekar, A. Nag, D. Goswami,
Org. Lett. 2006, 8, 629 – 631..
[13] D. Y. Kim, T. K. Ahn, J. H. Kwon, D. Kim, T. Ikeue, N. Aratani,
A. Osuka, M. Shigeiwa, S. Maeda, J. Phys. Chem. A 2005, 109,
2996 – 2999.
[14] J. Leclaire, R. Dagiral, S. Fery-Forgues, Y. Coppel, B. Donna-
dieu, A.-M. Caminade, J.-P. Majoral, J. Am. Chem. Soc. 2005,
127, 15762 – 15770.
[15] A. Masunov, S. Tretiak, J. W. Hong, G. C. Bazan, Abstracts of
Papers, 229th ACS National Meeting, San Diego, CA, March 13 –
17, 2005, COMP-295.
[16] a) M. Maszewska, J. Leclaire, M. Cieslak, B. Nawrot, A.
Okruszek, A.-M. Caminade, J.-P. Majoral, Oligonucleotides
2003, 13, 193 – 205; b) J. Solassol, C. Crozet, V. Perrier, J.
Leclaire, F. Bꢁranger, A.-M. Caminade, B. Meunier, D. Dor-
mont, J. P. Majoral, S. Lehmann, J. Gen. Virol. 2004, 85, 1791.
[17] D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P.
Bruchez, F. W. Wise, W. W. Webb, Science 2003, 300, 1434 – 1437.
[18] O. Mongin, T. R. Krishna, M. H. V. Werts, A.-M. Caminade, J.-P.
Majoral, M. Blanchard-Desce, Chem. Commun. 2006, 915 – 917.
[19] E. Chaigneau, M. Oheim, E. Audinat, S. Charpak, Proc. Natl.
Acad. Sci. USA 2003, 100, 13081 – 13086.
4648 www.angewandte.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 4645 –4648