Dendrimeric Nanoparticles for Two-Photon Photodynamic Therapy and Imaging: Synthesis, Photophysical Properties, Innocuousness in Daylight and Cytotoxicity under Two-Photon Irradiation in the NIR

Article abstract of DOI:10.1002/chem.201805617

The synthesis and the photophysical properties of a new class of fully organic monodisperse nanoparticles for combined two-photon imaging and photodynamic therapy are described. The design of such nanoparticles is based on the covalent immobilization of a dedicated quadrupolar dye that combines excellent two-photon absorbing (2PA) properties, fluorescence and singlet oxygen generation ability, in a phosphorous-based dendrimeric architecture. First, a bifunctional quadrupolar dye bearing two different grafting moieties, a phenol function and an aldehyde function, was synthesized. It was then covalently grafted through its phenol function to a phosphorus-based dendrimer scaffold of generation 1. The remaining aldehyde functions were then used to continue the dendrimer synthesis up to generation 2, introducing finally 24 water-solubilizing triethyleneglycol chains at its periphery. A dendrimer confining 12 photoactive quadrupolar units in its inner scaffold and showing water solubility was thus obtained. Interestingly, the G1 and G2 dendrimers retain some fluorescence as well as significant singlet oxygen production efficiencies while they were found to show very high 2PA cross-sections in a broad range of the NIR biological spectral window. Hydrophilic dendrimer G2 was tested in vitro on breast cancer cells, first in one- and two-photon microscopy, which allowed for visualization of their cell internalization, then in two-photon photodynamic therapy. While being nontoxic in the dark and, more importantly, under exposure to daylight, dendrimer G2 proved to be a very efficient cell-death inducer only under two-photon irradiation in the NIR.

Full text of DOI:10.1002/chem.201805617

A Journal of
Accepted Article
Title: Dendrimeric nanoparticles for two-photon photodynamic therapy
and imaging: synthesis, photophysical properties and cytotoxicity
under two-photon irradiation in the NIR
Authors: Aude Sourdon, Magali Gary-Bobo, Marie Maynadier, Marcel
Garcia, Jean-Pierre Majoral, Anne-Marie Caminade, Olivier
Mongin, and Mireille Blanchard-Desce
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Chem. Eur. J. 10.1002/chem.201805617
Link to VoR: http://dx.doi.org/10.1002/chem.201805617
Supported by

Chemistry - A European Journal
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FULL PAPER
Dendrimeric nanoparticles for two-photon photodynamic therapy
and imaging: synthesis, photophysical properties,
innocuousness in daylight and cytotoxicity under two-photon
irradiation in the NIR
[
a]
[c]
[c]
[c]
[d,e]
Aude Sourdon, Magali Gary-Bobo, Marie Maynadier, Marcel Garcia, Jean-Pierre Majoral,
Anne-Marie Caminade,[d,e] Olivier Mongin* and Mireille Blanchard-Desce*
[a]
[b]
Dedication ((optional))
Abstract: The synthesis and the photophysical properties of a new
class of fully organic monodisperse nanoparticles for combined two-
photon imaging and photodynamic therapy are described. The
Introduction
design of such nanoparticle is based on the covalent immobilization
of a dedicated quadrupolar dye - which combine large two-photon
absorbing (2PA) properties, fluorescence and singlet oxygen
generation ability - in a phosphorous-based dendrimeric architecture.
Firstly, a bifunctional quadrupolar dye bearing two different grafting
Photodynamic therapy (PDT) is a clinical treatment used in
oncology for the treatment of certain types of tumors including
esophagus, skin, bladder and non-small cell lung carcinomas, as
well as in ophthalmology for the treatment of age-related
moieties,
a
phenol function and an aldehyde function, was
[1]
macular degeneration (ARMD). PDT is based on the activation
synthesized. It was then covalently grafted through its phenol
function to a phosphorus-based dendrimer scaffold of generation 1.
The remaining aldehyde functions were then used to carry on the
dendrimer synthesis up to generation 2 introducing finally at its
periphery 24 water-solubilizing triethyleneglycol chains. A dendrimer
confining 12 photoactive quadrupolar units in its inner scaffold and
showing water solubility was thus obtained. Interestingly, the G1 and
G2 dendrimers retain some fluorescence as well as significant
singlet oxygen production efficiencies while they were found to show
very high 2PA cross-sections in a broad range of the NIR biological
spectral window. Hydrophilic dendrimer G2 was tested in vitro on
breast cancer cells, first in one- and two-photon microscopy, which
allowed for visualization of their cell internalization, then in two-
photon photodynamic therapy. While being non-toxic in the dark and,
more importantly, under daylight exposition, dendrimer G2 proved to
be very efficient cell death inducer only under two-photon irradiation
in the NIR.
by light of photosensitizers; non-toxic in the absence of light
excitation, a photosensitizer is able upon excitation by a laser
source, to generate singlet oxygen and/or other reactive oxygen
species (ROS), which leads to the destruction of the targeted
tissues.[2]
The use of
a two-photon excitation (2PE) offers several
advantages for PDT, especially for the treatment of small areas,
[3]
such as small solid tumors. These include the intrinsic three-
dimensional resolution allowed by 2PE, offering the ability for
highly spatially resolved excitation as well as increasing
penetration depth in tissues thanks to the use of near infra-red
[4]
(
NIR) light. Yet, in order to achieve efficient treatments,
optimized 2P photosensitizers (2PP) - i.e., combining very large
PA cross-sections in the biological spectral window (700-1000
2
nm) and high singlet oxygen (or ROS production) quantum
yields - are required. In addition, if the 2PP remains fluorescent,
in vitro monitoring is allowed, paving the way to theranostics
applications.[3] Most of the current clinical or preclinical
photosensitizers are porphyrin derivatives, such as porfimer
sodium (Photofrin) and verteporfin (Visudyne). As model
[
a]
Dr A. Sourdon, Dr O. Mongin
Univ Rennes, CNRS, ISCR (Institut des Sciences Chimiques de
Rennes), UMR 6226, F-35000 Rennes, France.
E-mail: olivier.mongin@univ-rennes1.fr
[5]
tetraphenylporphyrin (TPP, 12 GM), these compounds exhibit
high singlet oxygen quantum yields, significant fluorescence but
low 2PA cross sections (10 GM and 50 GM, respectively) in the
[
[
b]
c]
Dr M. Blanchard-Desce
near infra-red (NIR).[ Giant 2PA cross-sections have been
6]
Univ. Bordeaux, ISM (CNRS-UMR 5255), 33405 Talence, France.
E-mail: mireille.blanchard-desce@u-bordeaux.fr
Dr M. Gary-Bobo, Dr M. Maynadier, Dr M. Garcia
Institut des Biomolécules Max Mousseron, UMR CNRS-UM-ENSCM
obtained with expanded porphyrins,[7] conjugated porphyrin
dimers, trimers and oligomers,[8] planarized fused or bridged
porphyrins[9] and supramolecular assemblies,[8a, 10] however at
the expense of a strong modification of the other photophysical
properties. In particular, it often leads to decrease or
suppression of fluorescence and onset of residual one-photon
absorption (1PA) overlapping with the 2PA band located in the
NIR region. The huge gain in 2PA response therefore results in
the loss of some of the intrinsic advantages of selective 2PA, in
particular of the 3D confinement of excitation and therefore
spatial resolution in laser-assisted tumor resection.
5247, UFR des Sciences Pharmaceutiques et Biologiques,
15 Avenue Charles Flahault, 34093 Montpellier Cedex 5, France.
[
d]
e]
Dr J.-P. Majoral, Dr A.-M. Caminade
Laboratoire de Chimie de Coordination du CNRS,
205 route de Narbonne, BP 44099, F-31077 Toulouse Cedex 4,
France
[
Dr J.-P. Majoral, Dr A.-M. Caminade
LCC-CNRS, Université de Toulouse, CNRS,
31013 Toulouse, France
Supporting information for this article is given via a link at the end of
the document. It contains 2PA and 1PA spectra of dendrimers and
corresponding dye.
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To increase the 2PA cross-sections (while avoiding one-photon
absorption at the same wavelength) and still retain the excellent
photosensitization and luminescence properties of the
porphyrins, different systems have been designed, such as
symmetrically or dissymmetrically -extended phosphorescent
metalloporphyrins,[
11]
as well as dendritic antenna systems
based on resonant energy transfer (FRET) from peripheral 2P
single porphyrin core,[
12]
and “semi-
absorbers towards
a
disconnected” assemblies with a weak conjugation between 2P
absorbing dendrons and a porphyrin core,[13] or between a
central 2PA unit and peripheral porphyrins.[14] FRET between 2P
absorbers and porphyrinic photosensitizers could also be
achieved without covalent architectures by coencapsulating
them within silica nanoparticles.[15] Other types of nanoparticles
based on energy transfer have been designed, including
quantum dot-porphyrin[16] and quantum dot-phthalocyanine[17]
conjugates and porphyrin-doped conjugated polymers.[18]
Non-porphyrinic 2P absorbing photosensitizers have also been
developed,[19] among which should be mentioned quadrupolar
chromophores substituted with heavy atoms such as bromine or
iodine,[20] which leads to efficient intersystem crossing and
singlet oxygen generation quantum yields but concomitantly to
vanishing fluorescence. We have previously developed
alternative 2P fluorescent photosensitizers based on banana-
shaped quadrupolar systems, that combine large 2PA cross-
sections and significant singlet oxygen quantum yields, while
maintaining fair fluorescence quantum yields.[21] These
symmetric photosensitizers were covalently incorporated within
mesoporous silica nanoparticles for efficient two-photon
photodynamic therapy of solid tumors.[4e, 4f]
Figure 1. Molecular design.
The Mitsunobu condensation of N-ethyl-N-hydroxyethylaniline
1) with hydroquinone afforded 2a. Phosphonium salts 2b and 6
(
[
26]
were synthesized according to the procedures reported. Wittig
condensations of 5-iodo-2-thiophenecarboxaldehyde (3)[ with
phosphonium salts 2b and 6 led to mixtures of stereoisomers.
Their isomerization with iodine afforded 4a and 7a, respectively,
as pure trans isomers (Scheme 1).
27]
As an alternative to these hard nanoparticles, our aim was to
develop soft and monodisperse fully organic nanodots by
gathering quadrupolar 2P photosensitizers in phosphorus-based
dendrimer architectures, and to graft at their surface water-
solubilizing groups. Among the major advantages of
phosphorus-based dendrimers[22] are their low toxicity[23] and
their accessibility for both inner and surface functionalization
using covalent chemistry.
This highly modular “organic nanodots” strategy was previously
used successfully to obtain covalent assemblies of fluorophores,
leading to luminescent nanoparticles that can outperform
semiconductor quantum dots in terms of one- and two-photon
brightness.[24] These fluorescent nanodots have been proved to
be of major interest for in vivo imaging.[25]
We report here the first two-photon photosensitizing nanodots
and their use in photodynamic therapy.
Results and Discussion
Scheme 1. Reagents and conditions. a) hydroquinone, PPh
3
, DIAD, THF, RT,
O, acetic acid, 66h,
1°C, (83% for 6 and 84% for 2b); c) 3, tBuOK, anhyd CH Cl , RT, 16h (50%
N/CH Cl , RT, 16h (97%);
N/toluene, 40°C, 16h (87% for
8 and 94% for 5); f) 4 hydroxybenzaldehyde, PPh , DIAD, THF, RT, 16h
3 2
16h (36%); b) PPh , paraformaldehyde, toluene, NaI, H
6
2
2
Synthesis of the graftable two-photon photosensitizer
for 7a and 54% for 4a); d) acetyl chloride, DMAP, Et
e) Pd(PPh Cl , CuI, ethynyltrimethylsilane, Et
3
2
2
)
3 2
2
3
2
The photosensitizer needed for the synthesis of 2-G (Fig 1)
3
should exhibit two different orthogonal grafting moieties, a
phenol function to attach it to the dendrimeric scaffold, and an
aldehyde function to continue the dendrimer synthesis and
introduce water-solubilizing peripheral groups. The synthesis of
this dissymmetric photosensitizer is described in Scheme 1.
(81%); g) 8, 9, Pd(PPh
Pd(PPh Cl , CuI, TBAF/THF, 40°C, 16h (70%); i) NaOH, EtOH/THF, RT, 20
min (84%).
3 2 2
) Cl , CuI, TBAF/THF, 40°C, 16h (77%); h) 5,
)
3 2
2
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1
Scheme 2. Synthesis of spherical dendrimer 2-G .
On one hand, the phenol function of 4a was esterified with acetyl
chloride, giving 4b, which was reacted with
dendrimer 1-G
1
,[22a, 29] built from a cyclotriphosphazene core[30]
(Scheme 2). The substitution of the twelve chlorine atoms of 1-
ethynyltrimethylsilane in a Sonogashira coupling to afford 5, and
on the other hand, Mitsunobu condensation of 7a with 4-
hydroxybenzaldehyde gave 7b, which was also coupled with
ethynyltrimethylsilane, leading to 8. Finally, graftable
chromophore 10 was obtained as a single E,E-stereoisomer,
from 9,9-dibutyl-2,7-diiodo-9H-fluorene (9)[28] by means of two
successive Sonogashira couplings with 8 and 5, followed by
saponification of the acetate protective group (Scheme 1).
1
G with such a bulky substituent was slow and needed 7 days to
go to completion. This reaction was monitored by 31P NMR
which shows a total disappearance of the singlet at δ = 62.3
ppm corresponding to the phosphorus P
new singlet at δ = 64.3 ppm due to the phosphorus P
An intermediate signal observed at 69.3 ppm and
1
in 1-G
1
on behalf of a
1
in 2-G .
1
δ
=
corresponding to the monosubstitution (i.e. P(S)ClOAr) totally
disappears when the reaction is over. The second step
consisted in the condensation of 12 equivalents of the
The synthesis of the spherical first generation dendrimer 2-G
1
was carried out by grafting 12 equivalents of the 2PA
chromophore 10 in the presence of caesium carbonate, to the
phosphorhydrazide 11 with the aldehyde functions of 2-G
chloroform at room temperature, and afforded the second
generation of the dendrimer 1-G (Scheme 3).
1
in
P(S)Cl
2
end groups of the first generation phosphorous
2
2
Scheme 3. Synthesis of spherical dendrimer 2-G .
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1 2
Table 1. Photophysical properties of compound 10, 2-G and 2-G in THF.
[
a]
max/n[b]
λ
abs
ε
max
ε
λ
/nm
em
Stokes shift
/10 cm
[c]
/ns [d]
[e]
Cpd

F


-
1
-1
-1
-1
3
-1
/nm
/M .cm
/M .cm
1
0
434
1.2 105
1.2 105
544
4.66
0.51
0.50
1.3
0.16
0.10


1= 0.56 ;n1=63 %
2=1.5; n2=37 %
2
2
-G
-G
1
2
433
1.2 106
1.0 105
8.3 104
547
560
4.81

1= 0.52 ;n1=66 %
2=1.4; n2=34 %
434
1.0 106
5.18
0.21
0.07

[
a] Molar extinction coefficient
.
[b] Molar extinction coefficient per chromophoric subunit [c]
F
= fluorescence quantum yield, using as a standard
quinine bisulfate ( = 0.546) in 0.5M aq H
F
2
SO . [d] Fluorescence lifetime. [e] = singlet oxygen formation quantum yield determined relative to
4
tetraphenylporphyrin in methylene chloride ([TPP] = 0.60 in methylene chloride).
The reaction was monitored by 1H NMR, its completion being
characterized by the total disappearance of the aldehyde singlet.
The 31P NMR spectrum of 2- G
consists of three singlets δ = 8.6
ppm for the core, δ = 63.3 ppm for P1, δ = 64.4 ppm for the newly
grafted phosphorus P
The next step involved the grafting of 24 equivalents of the
monodisperse triethyleneglycol chain 12 on 1-G to improve
solubility in water, affording dendrimer 2-G (Scheme 3). Here
1
2
.
2
2
again, the reaction was monitored by 31P NMR. Almost no
change occurs for the signal corresponding to the core, but we
observed the total disappearance of the singlet at δ = 63.4 ppm
corresponding to the phosphorus P
singlet, slightly deshielded. An overlap occurs for the signals of
phosphorus P and P at δ = 64.6 ppm. To ascertain the full
2
1
in 2-G on behalf of a new
1
2
substitution or condensation, we used in all cases a slight
excess of reagents (10%), which was relatively easy to eliminate
by washings.
Figure 2. Normalized absorption and emission spectra of compounds 10, 2-G
1
and 2-G , in THF.
2
One photon absorption (1PA) and fluorescence properties
Dye 10 exhibits a significant singlet oxygen quantum yield as
well as a reasonable fluorescence quantum yield (Table 1), as
was initially required for allowing both therapy (by
photosensitized production of singlet oxygen) and monitoring (by
fluorescence imaging).
1 2
The compared photophysical properties of 10, 2-G , and 2-G in
THF are shown in Table 1 and their absorption and emission
spectra are represented in Fig 2. THF was chosen as a common
solvent as all three compounds are soluble in this solvent. All
compounds exhibit an intense absorption band from the near UV
to the blue visible range and an emission band from the green
visible range to the yellow visible range (Fig 2). Dendrimers also
show strong one-photon absorption in the UV.
The grafting of photosensitizers on the dendrimeric scaffold
(
1
from 10 to 2-G ) leads to a slight red shift and broadening of the
emission (Fig 2), in relation with an increase of the polarity
produced by polarizable environment produced by the
dendrimeric scaffold and the closeness of adjacent quadrupolar
dyes.[32] Interestingly, in spite of the confinement of the dyes
The low-energy absorption band located at 434 nm can be
ascribed to a periphery-to-core intramolecular charge transfer
1
chromophores in a reduced volume, 2-G maintains the same
transition, characteristic of bis-donor quadrupolar dyes built from
a fluorene core.[31] We note a broadening on the high energy
side for organic nanodots 2-G and even more for 2-G as
1 2
fluorescence quantum yield than the isolated dye 10. This
indicates that no significant through-space interchromophoric
interactions which would lead to fluorescence quenching occurs
compared to the isolated dye 10 in solution (Fig.2). This can be
related to the residual absorption of the dendrimeric backbone.
In addition, a hypochromic effect is observed: the extinction
coefficients of the chromophoric building blocks are reduced by
1
within 2-G . At opposite, fluorescence quenching of fluorescence
is often observed when widespread fluorescent dyes (such as
Nile red, dioxaborine…) are grafted in close proximity onto a
similar dendritic architecture.[33] In contrast, rigid extended
dendrimers where quadrupolar subunits are directly connected
by triphenylamine nodes were reported to show strong through-
bond coupling leading to cooperative enhancement of 2PA and
preservation of high fluorescence quantum yield.[34] In
1 2
about 17 % for 2-G and 30% for 2-G (Table 1).
This suggests that different conformations or relative positioning
of the dyes within the dendrimeric architecture might occur, in
particular in the case of 2-G2, suggesting that partial interactions
and more compact arrangement might occur in that case.
1 2
comparison, the design of dendrimers 2-G and 2-G does not
allow through-bond coupling between dye subunits due to the
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Chemistry - A European Journal
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FULL PAPER
presence of alkyl linkers (CH
moieties.
2 2
-CH ) between conjugated
We also note that the fluorescence decay is biexponential most
probably in relation with conformational flexibility of the dye.
Such phenomenon was not observed in the case of ultra-bright
organic nanodots confining more rigid and shorter quadrupolar
dyes.[24a]
A more pronounced red shift of the emission band is observed
with 2-G , indicating that the addition of a water-solubilizing layer
2
increases the polarity of the environment of the buried
chromophores. The most prominent feature is a significant
decrease of the fluorescence quantum yield. This behavior
indicates that competing deactivation process takes place and/or
2
Figure 4. Normalized absorption and emission of dendrimer 2-G in solvents
of different polarities.
interaction between the dyes occurs within dendrimer 2-G
2
compared to 2-G Most probably, the presence of the
1
.
hydrophilic layer created by the TEG surface moieties produces
a different external environment which may induce different
organization of the dendritic backbone, as discussed earlier, and
favor interchromophoric ground-state interactions that are
deleterious to fluorescence.
2
However, it appears clearly that the emission of nanodot 2-G in
a low polarity solvent such as toluene is strongly red-shifted in
comparison with the emission of 10 in the same solvent, which
confirms that the dendrimeric scaffold and the triethyleneglycol
chains do influence the polarity of the environment of the
chromophores by creating a cybotactic region reminiscent of that
of ethyl acetate.
The singlet oxygen production (

) is also reduced in
dendrimers 2-G (by 37 %) and 2-G (by 56%) as compared to
1
2
isolated dye 10. We note that this progression is similar to the
decrease in extinction coefficient and could be related to the
different conformations and possible interactions of the dyes
within the dendrimeric backbone. This may also originate from
the hindered access of dissolved dioxygen molecules to the
Two-photon absorption
2PA measurements were conducted by investigating the two-
buried dyes subunits in dendrimer 2-G
moieties creating a barrier to the diffusion of O
2
, the TEG surface
molecules.
photon excited fluorescence (2PEF) of dendrimers 2-G
in THF. The 2PA spectrum of monomeric dye 10 was also
determined for comparison. The quadratic dependence of the
PEF signal on the excitation intensity was checked for each
1
and 2-
2
G
2
2
Solvatochromism
data point, indicating that no photodegradation or saturation
occurs.
Fig 3 and 4 illustrate the dependence of the absorption and
emission properties of dye 10 and soft organic nanodot 2-G on
solvent polarity. Increasing the solvent polarity induces a slight
red shift of the absorption band and a marked bathochromic shift
of the emission band as clearly illustrated in Fig 3 for 10 and Fig
As expected from its symmetric quadrupolar type structure, dye 10
shows an intense 2PA band located around 720 nm (with
maximum 2PA cross-section of about 1900 GM) which can be
ascribed to a higher energy excited state which is strongly two-
photon allowed (see figure S2). Yet the lowest-energy excited
state also leads to significant 2PA response at 870 nm (Table 2),
indicating that this state is both one-photon and two-photon
2
4
for 2-G
2
. This is consistent with symmetry breaking occurring
[
35]
in the excited state prior to emission.
allowed. This effect can be attributed to
a breaking of
centrosymmetry induced by the dissymmetrical environment of the
dye. Indeed, dye 10 bears an alkoxybenzaldehyde moiety only on
one side. The presence of this dipolar unit (> 5 D)[ generates a
local electric field which in turns affects the polarization of the
quadrupole thus breaking its centrosymmetry. This effect is clearly
noticeable, in agreement with the reported strong effect of local
electric field on the 2PA of dyes.[ Along this line, we also note
that the two-photon allowed excited state is also slightly one-
photon allowed (see Figure S2). Hence the breaking of
centrosymmetry results in multiple absorption bands (and thus
broader response) as compared to purely symmetrical
quadrupolar chromophores.
36]
37]
Figure 3. Normalized absorption and emission spectra of quadrupolar dye 10
in solvents of different polarities.
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Table 2. Two-photon absorption data of compounds 10, 2-G
1
, 2-G
2
in THF.
max
1PA
max1
2PA
max1
max1/n
max2
2PA
max2
max2/n
2

/GM
680
6050
4700
2

2
/n


2

2


2

2

2

F
[d]
 

/GM 
300
1920
920
2
Cpd
[
a]
/nm [b]
820
820
[c]
/nm [b]
720
730
[c]
[e]
/nm
/GM
680
504
392
/GM
/GM
1240
933
/GM
/GM
1890
1517
1100
/GM 
960
9600
2770
1
0
868
866
868
1240
11200
8160
1890
19200
13200
2
2
-G
-G
1
2
820
680
730
max
[
a] 2PA cross section at 21PA . [b] Maximum 2PA wavelength. [c] 2PA cross section at 2PAmax. [d] 2P-brightness at 2PAmax2. [e] Figure of merit of the 2P-
max2
-50
4
-1
induced singlet oxygen generation at2PA
.1 GM = 10
cm .s.photon .
groups leads to a further decrease of about 30% of the 2PA cross-
sections for dendrimer 2-G as compared to 2-G
2
1
.
This decrease is reminiscent of the decrease noted in one-photon
absorption, although the trend is even stronger. This once again
confirms that different positioning and possibly partial ground-state
interactions between dye subunits occur in the dendrimeric
architecture, leading to both decrease of 1PA and 2PA.
1 2
Yet we note that dendrimers 2-G and 2-G retain very large 2PA
cross-sections at 730 nm (19200 and 13200 GM respectively) and
maintain high 2PA response over 5000 GM down to 900 nm. This
effect can be ascribed to symmetry breaking (also noted in
1 2
dendrimers 2-G and 2-G , see Figure S3 and S4) promoted by
the presence of dipolar moieties in close proximity. This results in
a broadening of the 2PA spectrum, which is of interest for
biological applications and use in two-photon bioimaging.
1 2
Figure 5. 2PA spectra of 10, 2-G , 2-G in THF (with solid curves as guides for
the eyes only).
Investigation of cell internalization by fluorescence imaging
1 2
Dendrimers 2-G and 2-G also show the same intense 2PA
bands located at 820 and 730 nm as clearly seen from Fig. 5,
leading to very large 2PA responses at 730 nm (up to 19200 GM
for 2-G and 13200 GM for 2-G2,). Yet, we note that the molecular
1
response of each individual dye is affected by the confinement
and the modification of the environment induced by the
dendrimeric scaffold (see Figure S1). As seen from Table 2, a
reduction of the 2PA cross-sections per chromophoric subunit of
about 20-25 % (resp. 40-45 %) is observed at 870 nm, 820 nm
Based on its properties (1PA and 2PA responses, fluorescence
properties and presence of water-solubilizing surface groups),
dendrimer 2-G
fluorescence (2PEF) cell imaging. In order to analyze its
penetration in cancer cells, dendrimer 2-G was incubated for
4 h with MCF-7 human breast cancer cells, whose nuclei were
2
was tested in one- and two-photon excited
2
2
stained with Hoechst 33342 as described in the experimental
section. As shown in Figure 6, confocal fluorescence microscopy
showed that 2-G is efficiently internalized.
2
1 2
and 730 nm for 2-G (resp. 2-G ). Addition of the water-solubilizing
Figure 6. Nuclear marker (left, excited at 405nm), 2-G
2
(middle, excited at 458 nm) and merge (right) confocal microscopy images of living MCF-7 human breast
cancer cells, incubated for 24 h with 50 µg.mL-1 2-G
2
.
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Chemistry - A European Journal
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Two-photon excited photodynamic therapy (2PE-PDT)
experiments
2
PE-PDT experiments were performed on MCF-7 breast cancer
-1
cells incubated with or without 2-G (50 g. mL ) for 20 hours.
2
After incubation, cells were washed, maintained in fresh culture
medium and then submitted or not to laser irradiation. Two-
photon irradiation was performed on a confocal microscope
equipped with a femtosecond pulsed Ti:sapphire laser. The laser
beam was focused using a microscope objective lens (10X, NA
0.4). Cells were irradiated at 760 nm by 3 scans of 1.57 sec
each at an average power of 80 mW. The surface of the
2
-2
scanned areas was 1.5 x 1.5 mm (mean energy of 16.6 J cm ).
Two days after irradiation, the percentage of living cells was
determined by MTS enzymatic assay. Results are reported in
Figure 7. Merged multiphoton confocal microscopy images of living MCF-7
breast cancer cells, incubated for 3 h at 37°C with 50 µg.mL-1 2-G
excited at
2
Figure 8, and 2-G
It was also shown that irradiation alone did not damage the cells.
Hence, the photodynamic therapeutic potential of 2-G is
demonstrated, with 78% cell death under two-photon irradiation
of MCF-7 cells incubated with 2-G . The in vitro efficiency of this
compound is thus comparable to that of mesoporous silica
nanoparticles (MSNs) incorporating closely related
2
was found to be non-toxic without irradiation.
750 nm (green) and membrane marker (yellow).
2
The cell distribution of dendrimer 2-G
co-staining with a membrane marker. MCF-7 breast cancer cells
were incubated for 3 h with 2-G , then plasma membranes were
stained with CellMask as described in the experimental section
Fig 7). Merged images of membranes and 2-G showed that
NDs were efficiently internalized after 3 h of incubation. The
2
was also investigated by
2
2
a
chromophore towards the same MCF-7 cell line, these MSNs
leading to 56% cell death after 4 h incubation time and 100%
cell death after 24 h incubation time, followed by irradiation (3 x
(
2
semi-circular shape of NDs localization in cell suggests that 2-G
2
1
s) at the same wavelength and power.[4e] As these MSNs had
molecules are located in lysosomes.
Hence, 2-G is internalized by cancer cells. The amount of
2
internalized dendrimers depends on the incubation time. We
found that more dendrimers were present in cells after 24 h
incubation (Fig 6) than after 3 h (Fig 7).
also been demonstrated to induce tumor regression (70%
decrease in tumor size) on nude mice bearing tumor xenografts
upon 9 min of irradiation at the same wavelength (i.e. 760nm)
and power, a required irradiation time of 10-20 min can be
2
anticipated with organic dendrimer 2-G for in vivo 2PE-PDT. It
2
In all cases, internalization occurs and 2-G was found to be
should be added that 2PE-PDT drugs bearing peptides specific
of tumor vasculature targeting, already led to even higher tumor
regressions for deep-tissue cancer treatment on mice, with
comparable irradiation times.[4c, 4j] This indicates that further
nontoxic at 50 µg.mL-1 without irradiation (not shown). An
incubation time of 20 h was thus selected for PDT experiments.
2
surface functionalization of dendrimer 2-G with similar targeting
peptides would be of major interest for PDT.
Figure 8. 2PE-PDT efficiency on MCF-7 breast cancer cells incubated or not
Figure 9. Absence of toxicity on MCF-7 breast cancer cells of 2-G
2
in the dark
control) for 20 h with 50 g. mL- of dendrimer 2-G
1
and then irradiated or not
control) at 760 nm for 3×1.57 s. Data are mean ± SD of 3 experiments.
(
(
2
(right) or under daylight exposition (left).
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Chemistry - A European Journal
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The two-photon induced toxicity of 2-G
different processes including singlet oxygen production. Indeed
2
can originate from Experimental Section
2-G
2
displays a two-photon induced singlet oxygen generation
Synthetic procedures
(  ), which is 120 times larger than that of TPP (see Table 2).
2

Yet formation of reactive oxygen species via different photo-
induced processes (in particular electron transfer sensitized by
quadrupolar subunits, which become strong electron donor in
the excited state) may also contribute to the two-photon induced
cell death.
General methods. All air or water-sensitive reactions were carried out
under dry argon. Solvents were generally dried and distilled prior to use.
Reactions were monitored by thin layer chromatography on Merck silica
gel 60
chromatography: Merck silica gel Si 60 (40-63 m, 230-400 mesh or 63-
00 m, 70-230 mesh). Melting points were determined on a system
F254 precoated aluminum sheets, or by NMR. Column
2
Interestingly, no cell death was observed when the cells were
1
13
Kofler type WME. NMR: Bruker Avance AV 300 ( H: 300.13 MHz, C:
75.48 MHz, 31P: 121.58 MHz), in CDCl , CD Cl or DMSO-d6 solutions;
exposed to daylight for 4 hours, indicating that dendrimer 2-G
nontoxic under daylight illumination conditions (Figure 9).
Dendrimer 2-G is thus nontoxic in the dark and even under
2
is
3
2
2
1
H chemical shifts () are given in ppm relative to TMS as internal
13
standard, J values in Hz and C chemical shifts relative to the central
at 77.0 ppm, 31P chemical shifts relative to 85% H
peak of CDCl PO .
4
2
3
3
daylight exposition. This represents an important progress in
view of therapeutics as it allows circumventing side effects in
current daylight. The daylight toxicity represents a common
drawback of photodynamic therapy medical treatment as it
requires patients to avoid direct light for a certain time. This
effect can be related to the large number of UV absorbers
present on the outskirt of the phosphorous dendritic backbone of
High and low resolution mass spectra measurements were performed at
the Centre Regional de Mesures Physiques de l'Ouest (C.R.M.P.O,
Rennes) using a Micromass MS/MS ZABSpec TOF instrument with EBE
TOF geometry; LSIMS (Liquid Secondary on Ion Mass Spectrometry) at
kV with Cs+ in m-nitrobenzyl alcohol (mNBA); ES+ (electrospray
8
ionization, positive mode) at 4 kV; EI (electron ionization) at 70 eV.
Elemental analyses were performed at C.R.M.P.O.
2
2-G , which form an effective UV-screen, avoiding excitation of
the inner quadrupolar photosensitizers by UV from sunlight. At
opposite, these constitutive UV absorbers are very poor 2P
absorbers in the 700-950 nm spectral range, thus permitting 2P
excitation of the inner quadrupolar subunits. This leads to a two-
photon versus one-photon excitation on-off process (i.e. the
dendritic platform is transparent to two-photon excitation in the
NIR region but absorbing upon UV excitation).
5-Iodo-2-thiophenecarboxaldehyde
(3).
A
mixture
of
2-
thiophenecarboxaldehyde (1.79 g, 15.92 mmol), iodine (2.00 g, 7.96
mmol) in carbon tetrachloride (4 mL), iodic acid (0.70 g, 3.98 mmol),
distilled water (3 mL), acetic acid (8 mL) and concentrated sulfuric acid
(
0.12 mL) were stirred under reflux for 1h and at room temperature for
another 12h. After washing with 2% NaHCO and Na the organic
layer was dried over anhydrous MgSO and the solvent was removed in
3
2 2 3
S O
4
vacuum. The crude product was purified by column chromatography
eluting with ethyl acetate/ heptane (1:9) to yield 2.95 g (78%) of 3 as
yellowish crystals, mp 53 °C; 1H NMR (CDCl
3
): 9.8 (s, 1H), 7.4 (s, 2H).
Conclusions
13C NMR (CDCl
3
): 181.1, 149.6, 138.2, 137.0, 87.8.
In summary we have described a new class of dendrimeric
nanoparticles for theranostics, which combine unique properties
for bioimaging and anticancer therapy. These fully organic, soft,
yet monodisperse nanoparticles show:
2
-(Ethyl-[4-[2-(5-iodothiophen-2-yl)vinyl]phenyl]amino)ethanol (7a).
Air was removed from a mixture of 3 (1.95 g, 8.19 mmol), phosphonium
salt 6 (6.32 g, 12.29 mmol) and tBuOK (1.84 g, 16.38 mmol) by bubbling
with argon for 20 min. Fresh distilled CH Cl (50 mL) was added. The
2 2
solution was stirred at room temperature for 16 h. It was then filtered
through a short pad of silica gel. The solvent was removed under
reduced pressure, and the crude product was purified by column
(
i) high two-photon absorption in the biological spectral window,
which allows highly confined and three-dimensional photo-
addressing in tissues, thus limiting side damages,
2 2
chromatography eluting with CH Cl / AcOEt (98:2 to 92:8) to yield 2.16 g
(
ii) large two-photon brightness, which allows fluorescent
tracking in cells and investigation of cell internalization,
iii) efficient two-photon photosensitizing properties in the NIR
of a mixture of Z/E stereoisomers. To 1.34 g of this mixture of isomers
O (1 g.L-1,
dissolved in Et O (50 mL) was added a solution of iodine in Et
2 2
(
3.4 mL). The mixture was then stirred 21h at room temperature under
region leading to highly spatially controlled cell death.
light. After washing with sat Na , the organic layer was dried over
2 2 3
S O
In vitro experiments conducted on human breast cancer cells
demonstrated these dendrimeric nanoparticles to be efficient
nanomedicine tools as their distribution and penetration within
cells can be efficiently monitored by fluorescence imaging, while
cancer cell death can be induced efficiently and selectively upon
two-photon excitation in the NIR.
anhydrous MgSO
g (50%) of 7a as yellow crystals. M.p: 93 °C. H NMR (CDCl
4
and the solvent was removed in vacuum to yield 1.02
1
3
): 7.3 (d, J=
8
.8 Hz, 2H), 7.1 (d, J=3.7 Hz, 1H), 6.9 (d, J= 16 Hz, 1H), 6.78 (d, J=16
Hz, 1H), 6.74 (d, J=8.8 Hz, 2H), 6.6 (d,J=3.7 Hz, 1H), 3.8 (q, 2H), 3.5 (m,
4
1
4
H), 1.6 (t, J= 5.8 Hz, 1H), 1.2 (t, J= 7 Hz, 3H). 13C NMR (CDCl
): 149.7,
3
47.7, 137.2, 129.1, 127.5, 125.6, 124.7, 116.5, 112.3, 70.2, 60.0, 52.2,
5.4, 11.8.
Moreover they were found to be non-toxic in the dark and, more
importantly, under daylight exposition, which is
a major
4-[2-(Ethyl-[4-[2-(5-iodothiophen-2-yl)vinyl]phenyl]amino)ethoxy]-
advantage in comparison with other photodynamic therapy
medical treatments. The grafting of targeting functions could
further improve the therapeutic promise of these nano-objects by
adding to the spatial selectivity of the two-photon activation a
high affinity for cancer cells. We are currently exploring this way.
benzaldehyde (7b). Under argon, a mixture of 7a (0.5 g, 1.25 mmol), 4-
hydroxybenzaldehyde (0.31 g, 2.51 mmol) and triphenylphosphine (0.66
g, 2.51 mmol) was dissolved in THF (15 mL) and bubbled with argon for
20 min. A solution of DIAD (0.5 mL, 2.51 mmol) in THF (3 mL) was then
added dropwise and the mixture was stirred overnight at room
temperature. The solvent was removed in reduce pressure and the crude
2 2
product was purified on column chromatography with CH Cl to yield 0.51
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Chemistry - A European Journal
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FULL PAPER
1
g (81%) of yellow crystals. M.p: 108 °C. H NMR (CDCl
d, J= 8.8 Hz, 2H), 7.3 (d, J= 8.8 Hz, 2H), 7.1 (d, J= 3.7 Hz, 1H), 7.0 (d,
J= 8.7, 2H), 6.9 (d, J= 15,5 Hz, 1H), 6.78 (d, J= 16 Hz, 1H), 6.72 (d, J=
3
) 9.9 (s, 1H), 7.8
stereoisomers (45/55). The mixture of isomers was then mixed with I
Et
O under beam of light to obtain 4a (54%). M.p: 114 °C. 1H NMR
(CDCl ): 7.34 (d, J= 8.8 Hz, 2H), 7.11 (d, J= 3.8 Hz, 1H), 6.98 (d, J= 16
2
in
(
2
3
8
2
1
1
.9 Hz, 2H), 6.65 (d, J=3.7 Hz, 1H), 4.2 (t, J= 6 Hz, 2H), 3.8 (t, J= 5.9 Hz,
Hz, 1H), 6.78 (d, J=16 Hz, 1H), 6.77 (d, J= 2.5 Hz, 4H), 6.71 (d, J= 9 Hz,
2H), 6.64 (d, J= 3.8 Hz, 1H), 4.53 (s, 1H), 4.09 (t, J=6.3 Hz, 2H), 3.74 (t,
H), 3.5 (q, 2H), 1.3 (t, J= 7 Hz, 3H). 13C NMR (CDCl
3
): 190.7, 163.6,
47.2, 145.6, 133.4, 131.9, 130.19, 130.16, 129.5, 127.9, 124.3, 120.2,
17.1, 114.7, 111.9, 99.4, 98.2, 65.8, 49.5, 45.8, 12.3.
J=6 Hz, 2H) 3.53 (q, 2H), 1.24 (t, J= 6.9 Hz, 3H). 13C NMR (CDCl
149.9, 149.7, 147.4, 137.4, 129.4, 127.8, 125.8, 124.4, 116.5, 116.1,
15.5, 111.8, 70.3, 66.0, 49.6, 45.6, 12.3.
3
): 152.7,
1
4-[2-(Ethyl-[4-[2-(5-trimethylsilanylethynylthiophen-2-yl)vinyl]
-
phenyl]amino)ethoxy]benzaldehyde (8): Air was removed from the
solution of 7b (0.77 g, 1.54 mmol) dissolved in toluene (15 mL) and Et
3 mL) by bubbling with argon for 20 min. After heating at 40 °C, CuI (6
mg, 0.03 mmol), Pd(PPh Cl (22 mg, 0.03 mmol) and
Acetic
amino)ethoxy]phenyl ester (4b): Under argon, to a mixture of 4a (0.62
g, 1.26 mmol) in CH Cl (36 mL), DMAP (22 mg, 0.18 mmol) was added
and stirred 5 min at 0°C. At 0°C, Et N (0.39 mL) was then added and
acid
4-[2-(ethyl-[4-[2-(5-iodothiophen-2-yl)vinyl]phenyl]-
3
N
(
2
2
3
)
2
2
3
ethynyltrimethylsilane (0.33 mL, 2.31 mmol) were added. The mixture
was stirred at 40 °C overnight. Solvent was removed by reduce pressure
acetyl chloride (0.19 mL, 2.73 mmol) was added dropwise. The mixture
was stirred overnight at room temperature. Ice was added in solution
followed by NaHCO3sat (20 mL) and stirred 5 min. The organic layer was
then extracted (3 × 15 mL), dried and concentrated under vacuum. The
and the crude product was purified by column chromatography with
1
2
CH Cl
2
/ Heptane (6:4 to 7:3) to yield 0.63 g (87%) of 8. H NMR (CDCl
3
):
9
1
1
3
.9 (s, 1H), 7.9 (d, J= 9 Hz, 2H), 7.4 (d, J= 9 Hz, 2H), 7.1 (d, J=3.7 Hz,
H), 7.01 (d, J= 8.7 Hz, 2H), 6.97 (d, J= 16 Hz, 1H), 6.85 (d, J= 16 Hz,
H), 6.82 (d, J= 3.8 Hz, 1H), 6.7 (d, J= 8.9 Hz, 2H), 4.2 (t, J= 6 Hz, 2H),
.8 (t, J= 5.9 Hz, 2H), 3.5 (q, 2H), 1.3 (t, J= 7 Hz, 3H), 0.3 (s, 9H). 13C
crude product was purified on column chromatography with CH
2 2
Cl to
yield 0.61 g (97%) of 4b. 1H NMR (CDCl
3
): 7.34 (d, J= 8.7 Hz, 2H), 7.11
(d, 3.7 Hz 1H), 7.01 (d, 9 Hz, 2H), 6.98 (d, J= 16 Hz, 1H), 6.89 (d, J= 9.3
Hz, 2H), 6.78 (d, J= 16 Hz, 1H), 6.71 (d, J= 9 Hz, 2H), 6.64 (d, J= 4 Hz,
NMR (CDCl
3
): 190.7, 163.6, 147.2, 145.6, 133.4, 131.9, 130.19, 130.16,
1H). 13C NMR (CDCl
3
): 169.8, 156.3, 150.0, 147.3, 144.4, 137.4, 129.5,
1
4
29.5, 127.9, 124.3, 120.2, 117.1, 114.7, 111.9, 99.4, 98.2, 65.8, 49.5,
5.8, 12.3.
127.8, 125.8, 124.6, 122.4, 116.6, 115.0, 111.82, 111.8, 70.3, 65.8, 49.6,
45.7, 12.3.
4
-[2-(Ethylphenyl-mino)ethoxy]phenol
(2a):
2-[ethyl-
Acetic acid 4-[2-(ethyl-[4-[2-(5-trimethylsilanylethynylthiophen-2-
yl)vinyl]phenyl]amino)ethoxy]phenyl ester (5): Air was removed from
the solution of 4b (0.37 g, 0.70 mmol) dissolved in toluene (5 mL) and
(phenyl)amino]ethanol (1) (3.00 g, 18.07 mmol), hydroquinone (3.98 g,
36.14 mmol) and PPh (9.51 g, 36.14 mmol) were dissolved in THF (55
3
mL). Air was removed by bubbling with argon for 20 min. A solution of
DIAD (7.16 mL, 36.14 mmol) in THF (35 mL) was then added dropwise
and the mixture was stirred overnight at room temperature. The solvent
was removed in reduce pressure and the crude product was purified on
Et
3
N (1 mL) by bubbling with argon for 20 min. After heating at 40 °C, CuI
(3 mg, 0.02 mmol), Pd(PPh
ethynyltrimethylsilane (0.19 mL, 1.31 mmol) were added. The mixture
was stirred at 40 °C overnight. Solvent was removed by reduce pressure
and the crude product was purified by column chromatography with
CH
3
)
2
Cl (12 mg, 0.02 mmol) and
2
column chromatography with CH
2
Cl
): 7.38 (q, 2H), 6.87 (m, 7H), 6.58 (s, 1H), 4.17 (t, J= 6.3 Hz, 2H),
.80 (t, J= 6.3 Hz, 2H), 3.59 (q, 2H), 1.31 (t, J= 6.9 Hz, 3H). 13C NMR
): 152.6, 149.6, 147.4, 129.3, 116.1, 115.5, 111.9, 66.0, 49.6, 45.5,
2
to yield 1.68 g (36%). 1H NMR
1
(CDCl
3
2
Cl
2
/ Heptane (6:4 ) to yield 0.33 g (94%) of 5. H NMR (CDCl
3
): 7.37
3
(d, J= 9 Hz, 2H), 7.12 (d, J= 3.9 Hz, 1H), 7.03 (d, J=9 Hz, 2H), 6.98 (d,
J= 16 Hz, 1H), 6.90 (d, J= 9 Hz, 2H), 6.87 (d, J= 16 Hz, 1H), 6.83 (d, J=
3.9 Hz, 1H), 6.72 (d, J= 9 Hz, 2H), 4.13 (t, J= 6 Hz, 2H), 3.77 (t, J= 5.9
Hz, 2H), 3.54 (q, 2H), 2.29 (s, 3H), 1.26 (t, J= 6.9 Hz, 3H), 0.30 (s, 9 Hz).
(CDCl
3
12.1.
13C NMR (CDCl
3
): 169.7, 156.11, 156.09, 147.2, 145.5, 144.14, 144.11,
[
[4-[Ethyl[2-(4-hydroxyphenoxy)ethyl]amino]phenyl]methyl]-
triphenylphosphonium iodide (2b): Compound 7 (0.600 g, 2.34 mmol),
PPh (0.552 g, 2.34 mmol) and paraformaldehyde (0.063 g, 0.70 mmol)
1
1
4
2
42.2, 133.2, 132.2, 130.6, 129.9, 129.5, 127.7, 127.2, 124.0, 122.17,
22.15, 119.9, 119.7, 116.7, 114.8, 111.6, 111.2, 99.1, 98.0, 65.6, 49.42,
9.39, 45.5, 45.4, 31.7, 31.28, 31.22, 29.97, 29.91, 29.48, 29.45, 29.41,
9.29, 29.15, 28.9, 28.7, 22.5, 20.8, 13.9, 12.1.
3
were dissolved in toluene (8 mL). NaI (0.434 g, 2.34 mmol), water (0.4
mL) and acetic acid (1.2 mL) were then added. The solution was stirred
for 66 h at 61 °C. Water (30 mL) was added and the mixture was stirred
for 10 min. The aqueous layer was extracted with CH
combined organic layers were washed with NaHCO (20 mL) and water
20 mL), dried and evaporated under reduced pressure. The crude
2
Cl
2
and the
4-[2-[[4-[(1E)-2-[5-[(9,9-dibutyl-7-iodo-9H-fluoren-2-yl)ethynyl]2-
thienyl]ethenyl]phenyl]ethylamino]ethoxy]benzaldehyde (9a): Air
was removed from a mixture of 8 (0.63 g, 1.34 mmol), 9,9-dibutyl-2,7-
3
(
product was washed with Et
2
O to afford 1.362 g of 2b (88%). 1H NMR
diiodo-9H-fluorene 9 (2.13 g, 4.01 mmol) and Et
toluene (12 mL) by bubbling with argon for 20 min. After heating at 40°C,
CuI (5.7 mg, 0.03 mmol) and Pd(PPh Cl (21 mg, 0.03 mmol) were
3
N (2.4 mL) dissolved in
(
CDCl
J=8.7 Hz, 2H), 4.88 (d, J= 12.9 Hz, 2H), 4.05 (t, J=6.3 Hz, 2H), 3.77
s,1H), 3.71 (t, J=6.3 Hz, 2H), 3.48 (q, 2H), 1.22 (t, J=6.9 Hz, 3H). 13C
NMR (DMSO-d ): 151.2, 151.0, 147.2 (d, J = 2.7 Hz), 134.8 (d, J = 2.9
Hz), 133.9 (d, J = 9.7 Hz), 131.0 (d, J = 9.7 Hz), 129.9 (d, J = 12.2 Hz),
3
): 7.64 (m, 15H), 7.05 (d, J= 8.7 Hz, 2H), 6.76 (m, 4H), 6.44 (d,
3
)
2
2
(
added. Air was removed from the solution of TBAF in THF (1 M, 0.80
mmol) by blowing argon for 5 min and then added to the mixture. The
solution was stirred overnight at 40 °C. Solvent was removed by reduce
pressure and the crude product was purified by column chromatography
6
1
4
18.1 (d, J = 84.7 Hz), 115.6, 115.2, 112.4 (d, J = 8.7 Hz), 111.5, 65.7,
8.8, 44.5, 27.5 (d, J = 45.0 Hz), 11.6. 31P NMR (DMSO-d
): 21.1.
6
with mixed solvent CH
2
Cl
2
/Heptane (6:4) to yield 0.83 g (77%) of orange
): 9.89 (s, 1H),
.85 (d, J= 8.9 Hz, 2H), 7.68 (dd, J= 1.6 Hz, 1H), 7.66 (s, 1H), 7.63 (d, J=
crystals of compound 9a. M.p: 146 °C. 1H NMR (CDCl
3
7
8
1
2
1
3
4-[2-(Ethyl-[4-[2-(5-iodothiophen-2-yl)vinyl]phenyl]amino)ethoxy]-
.6 Hz, 1H), 7.51 (dd, J= 1.4 Hz, 1H), 7.46 (s, 1H), 7.42 (d, J= 8.4 Hz,
H), 7.39 (d, J= 9 Hz, 2H), 7.18 (d, J= 3.6 Hz, 1H), 7.01 (d, J= 8.7 Hz,
H), 6.96 (d, J= 16 Hz, 1H), 6.90 (d, J= 3.3 Hz, 1H), 6.89 (d, J= 15.3 Hz,
H), 6.73 (d, J= 9 Hz, 2H), 4.25 (t, J= 6.3 Hz, 2H), 3.83 (t, J= 5.7 Hz, 2H),
.56 (q, 2H), 1.96 (m, 4H), 1.26 (t, J= 7.2 Hz, 3H), 1.14 (q, 4H), 0.72 (t,
phenol (4a): Air was removed from a mixture of 2b (2.09 g, 3.18 mmol),
(0.556 g, 2.34 mmol) and tBuOK (0.53 g, 4.67 mmol) by bubbling with
argon for 20 min. Fresh distilled CH Cl (25 mL) was added. The solution
3
2
2
was stirred at room temperature overnight, then filtered through a short
pad of mixed celite and silica gel. The solvent was removed under
reduce pressure, and the crude product was purified on column
J= 7.2 Hz, 6H), 0.56 (m, 4H). 13C NMR (CDCl
50.0, 147.0, 145.5, 140.2, 139.8, 135.8, 132.5, 131.87, 131.82, 130.3,
3
): 190.6, 163.4, 153.2,
1
2 2
chromatography with mixed solvent CH Cl to yield 0.62 g of Z/E
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Chemistry - A European Journal
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1
1
2
29.9, 129.3, 127.8, 125.4, 124.7, 124.6, 121.7, 121.5, 120.2, 119.6,
16.9, 114.5, 111.7, 94.6, 92.9, 83.7, 65.6, 55.1, 49.3, 45.6, 39.9, 29.5,
5.7, 22.8, 13.6, 12.2. HRMS (ESI) calcd for C46H46NO
2
INaS [(M+Na)+]
In a solution of this oil (8.46 g, 34.97 mmol) in 25 mL of DMSO, was
added hydroquinone (3.86 g, 34.97 mmol), and KOH (5.89 g, 104.91
mmol). The mixture was stirred overnight at room temperature (water
bath) and 20 mL of water was then added. The solution was extracted (3
m/z 826.21862, found 826.2185.
×
30 mL) with Et
byproduct. The solution was then acidified with concentrated HCl and
extracted with CHCl (3 × 20 mL). The combined organic layers were
washed with water until neutrality, dried over Na SO , and concentrated
in vacuo to yield 3.69 g (41%) of brown oil of 12. 1H NMR (CDCl
): 6.82
1H), 6.73 (d, J = 9 Hz, 2H), 6.69 (d, J = 9 Hz, 2H), 3.98 (t, J = 4.8 Hz,
H), 3.79 (t, J = 4.8 Hz, 2H), 3.70 (m, 6H), 3.55 (m, 2H), 3.34 (s, 3H). 13C
): 152.3, 150.1, 115.9, 115.5, 71.7, 70.5, 70.4, 70.3, 69.7,
7.8, 58.8. HRMS (ESI) calcd for
Na [(M+Na)+] m/z
2
O to remove both unreacted tosylate and disubstituted
4-[2-[[4-[(1E)-2-[5-[2-[9,9-dibutyl-7-[2-[5-[(1E)-2-[4-[ethyl-[2-[4-
3
(formyl)phenoxy]ethyl]amino]phenyl]ethenyl]-2-thienyl]ethynyl]-9H-
2
4
fluoren-2-yl]ethynyl]-2-thienyl]ethenyl]phenyl]ethylamino]ethoxy]-
phenyl acetate (9b): Air was removed from 5 (0.33 g, 0.66 mmol), the
3
(
s
,
previous compound 9a (0.48 g, 0.60 mmol) and Et
in toluene (14 mL) by bubbling with argon for 20 min. After heating at
0°C, CuI (2.3 mg, 12 µmol) and Pd(PPh Cl (8.4 mg, 12 µmol) were
3
N (2.8 mL) dissolved
2
NMR (CDCl
3
4
3
)
2
2
6
2
1
13 20 2 5
C H N O
added. Air was removed from the solution of TBAF in THF (1 M, 0.42
mmol) by blowing argon for 5 min and then added to the mixture. The
solution was stirred overnight at 40 °C. Solvent was removed by reduce
pressure and the crude product was purified by column chromatography
-
1
79.12029, found 279.1203. ATR-FTIR (cm ): 3333.3, 2873.0, 1508.4,
448.9, 1355.7, 1214.9, 1096.0, 1064.3, 824.4, 753.1.
with mixed solvent CH
crystals of compound 9b. M.p: 160 °C. 1H NMR (CDCl
.85 (d, J= 9 Hz, 2H), 7.68 (d, J=8.6 Hz, 2H), 7.52 (m, 4H), 7.39 (dd, J=3
Hz, 4H), 7.19 (d, J=4 Hz, 2H), 7.02 (d, J= 16 Hz, 2H), 7.01 (d, J= 9 Hz,
2
Cl
2
/Heptane (7:3) to yield 0.46 g (70%) of orange
Dendrimer 2-G1. The reaction was shielded from the light
of photosensitizer 10 (152 mg, 142.72 mol) in distilled THF (0.75 mL)
was added Cs CO (93 mg, 285.43 mol). The solution was stirred 2h at
35°C. A solution of dendrimers bearing P(S)Cl 1-G (20 mg, 10.95 mol)
in 0.75 mL of distilled THF was then added and stirred 7days at 35°C.
The reaction was monitored by 31P using a C
capillary. The resulting
mixture was filtered and dendrimer was precipitated in pentane (200 mL).
The orange precipitate was purified on column chromatography
(pentane: THF: 1:1 until THF 100%) and washed several times with
.
To a solution
3
): 9.89 (s, 1H),
7
2
3
2
1
2
4
4
0
1
1
1
4
H), 6.90 (s, 4H), 6.89 (s, 2H), 6.85 (d, J=16 Hz, 2H), 6.74 (dd, J=3 Hz,
H), 4.24 (t, J=6 Hz, 2H), 4.14 (t, J=6 Hz, 2H), 3.82 (m, 4H), 3.53 (qd,
H), 2.29 (s, 3H), 2.03 (m, 4H), 1.28 (td, J= 3.9 Hz, 6H), 1.16 (m, 4H),
6 6
D
.73 (t, J= 7.2 Hz, 6H), 0.63 (m, 4H). 13C NMR (CDCl
3
): 190.9, 170.0,
63.8, 156.4, 151.2, 145.8, 144.5, 132.8, 132.2, 130.6, 130.3, 129.8,
28.1, 125.83, 125.76, 125.0, 124.0, 124.81, 124.77, 122.5, 120.6, 120.1,
17.3, 115.2, 114.9, 111.9, 65.94, 65.92, 55.3, 49.8, 49.62, 49.60, 45.96,
AcOEt, to eliminate the excess of 10, and dried under reduce pressure to
1
yield 106 mg (69%) of orange powder. Mp: 178 °C. H NMR (CD
2
Cl
2
) 9.86
5.86, 26.0, 23.2, 21.1, 13.9, 12.5. HRMS (ESI) calcd for
(s, 12H), 7.82 (d, J = 8.7 Hz, 24H), 7.67 (dd, J = 7.8 Hz, 24H), 7.59 (m,
66H), 7.36 (d, J = 9 Hz, 24H), 7.30 (d, J = 8.4 Hz, 24H), 7.16 (dd, J = 3.9
Hz, 24H), 7.05 (m, 132 H), 6.73 (d, J = 8.7 Hz, 48H), 6.64 (d, J = 7.5 Hz,
+
C
72
H
70
N
2
O
5
NaS
2
[(M+Na) ] m/z 1129.46184, found 1129.4620.
24H), 4.24 (t, J = 6 Hz, 24H), 3.98 (t, J = 6 Hz, 24H), 3.80 (t, J = 6 Hz,
24H), 3.62 (s br, 24H), 3.54 (q, 24H), 3.40 (s br, 24H), 3.21 (d, J = 12 Hz,
1
8H), 2.00 (m, 48H), 1.21-1,02 (m, 120H), 0.77-0.48 (m, 120H). 13C NMR
Photosensitizer 10: The compound 9b (0.46 g, 0.42 mmol) was
dissolved in a mixture of EtOH (10 mL) and THF (28 mL). NaOH (4 mL)
was then added and the solution was stirred 20 min at room temperature,
(
2 2
CD Cl ) 191.42, 164.63, 157.13, 152.18, 148.45, 146.77, 145.22, 141.67,
and HCl (15 mL) was added. Aqueous layer was extracted with CH
10 mL), and organic layer was then washed with water (5 mL). The
organic layer was dried and concentrated under vacuum. The crude
product was purified on column chromatography with CH Cl to yield 0.37
g (84%) of orange crystals of 10. M.p: 94 °C. 1H NMR (CDCl
): 9.89 (s,
H), 7.85 (d, J= 9 Hz, 2H), 7.68 (d, J=8.6 Hz, 2H), 7.52 (m, 4H), 7.39 (dd,
J=3 Hz, 4H), 7.19 (d, J=4 Hz, 2H), 7.01 (d, J= 9 Hz, 2H), 6.99 (2×d, J= 16
Hz, 2H), 6.89 (m, 2H), 6.87 (d, J=16 Hz, 2H), 6.77 (s, 4H), 6.74 (dd, J=3
Hz, 4H), 5.19 (s, 1H), 4.24 (t, J=6 Hz, 2H), 4.10 (t, J=6 Hz, 2H), 3.82 (t,
J= 5.7 Hz, 2H), 3.73 (t, J=5.7 Hz, 2H), 3.53 (m, 4H), 2.01 (m, 4H), 1.28
2 2
Cl
1
1
8
33.73, 132.72, 131.31, 131.16, 130.59, 128.82, 126.64, 125.77, 123.22,
22.68, 122.25, 121.19, 121.00, 117.70, 115.97, 115.65, 112.84, 95.75,
4.76, 84.67, 66.95, 61.16, 56.14, 55.08, 54.72, 54.36, 50.39, 46.68,
(
2
2
41.03, 34.00, 31.01, 26.92, 23.99, 14.93, 14.57, 13.01.
6
31P (CDCl
3
),
3
4.40, 8.63. LC/MS: calcd avg mass for
C
H N O54NaP S
888 852 39 9 30
1
+
[
(M+Na) ] m/z 14198.2 found: 14197.1.
2 1
Dendrimer 1-G . The reaction was shielded from the light. 2-G (86.4 mg,
6.10 mol) was cooled at 0 °C and 11 (508.3 L, 91.5 mol) in solution in
(
2×t, J= 3.9 Hz, 6H), 1.16 (m, 4H), 0.73 (t, J= 7.2 Hz, 6H), 0.63 (m, 4H).
CHCl
3
was added
.
The solution was stirred 5 days at room temperature
1
3C NMR (CDCl
1
3
): 191.1, 163.8, 152.8, 151.2, 149.9, 147.6, 147.3, 145.9,
(monitored by H NMR). Dendrimer was precipitated in pentane (200 mL),
1
1
1
1
2
45.8, 140.76, 140.71, 132.77, 132.16, 130.6, 130.1, 129.8, 129.6,
to remove excess of 11, filtered under argon then dried under reduce
pressure to yield 85 mg (86%) of orange powder. M.p: 184 °C. H NMR
(CDCl ) 7.68 (m, 120H), 7.38 (d, J = 8.4 Hz, 24H), 7.31 (d, J = 8.4 Hz,
3
24H), 7.16 (dd, J = 3.7 Hz, 24H), 7.06- 6.63 (m, 192 H), 4.19 (t, J = 4.5
Hz, 24H), 3.96 (s br, 24H), 3.80 (s br, 24H), 3.62 (s br, 24H), 3.53 (m,
84H), 3.19 (d, J = 9.6 Hz, 18H), 1.98 (m, 48H), 1.15 (m, 120H), 0.69 (m,
1
28.04, 128.01, 125.7, 124.93, 124.86, 124.7, 124.6, 121.83, 121.77,
20.5, 120.3, 120.1, 117.2, 116.9, 116.1, 115.6, 114.8, 111.9, 111.8,
05.0, 95.0, 84.07, 84.01, 66.1, 65.9, 55.3, 49.8, 49.5, 45.9, 45.7, 40.3,
5.9, 23.1, 13.9, 12.43, 12.40. HRMS (ESI) calcd for C70
H
69
N O S
2 4 2
+
-1
[
(M+H) ] m/z 1065.46933, found 1065.4691. ATR-FTIR (cm ): 3363,
2
1
926, 2189, 2037, 1685, 1596, 1507, 1446, 1397, 1349, 1256, 1221,
182, 1158, 1136, 1074, 1020, 946, 885, 806, 751.
120H). 13C NMR (CDCl
145.72, 144.23, 141.71, 140.65, 135.75, 132.68, 130.52, 129.56, 128.95,
3
): 160.31, 156.04, 151.50, 151.11, 147.34,
1
1
9
3
28.23, 127.95, 127.19, 125.59, 125.50, 124.72, 124.58, 122.29, 121.73,
21.37, 120.41, 120.33, 120.02, 117.08, 116.95, 115.03, 114.75, 111.84,
4.92, 83.94, 65.75, 65.56, 55.17, 49.58, 45.76, 45.63, 40.18, 34.21,
3.07, 31.75, 30.31, 25.90, 23.05, 21.18, 13.85, 12.34, 12.31. 31P
4
-[2-[2-(2-Methoxyethoxy)ethoxy]ethoxy]phenol (12). In a solution of
triethylene glycol monomethyl ether (6.00 g, 36.58 mmol) in CH
mL) at 0°C, was added Et N (5.83 mL). The mixture was cooled below
°C. Stirring vigorously and maintaining the reaction mixture at that
temperature, a solution of p-toluenesulfonyl chloride (3.25 mL; 42.07
mmol) in 3mL of CH Cl was added dropwise. Stirring was continued
overnight at room temperature. A white precipitate was filtered and the
filtrate was washed with NaHCO (2 × 10mL). The aqueous layer was
extracted with CH Cl2. Organic layers were combined, dried over Na SO
then evaporated under reduce pressure to yield 8.46 g (95%) of yellow oil.
2 2
Cl (10
3
3
(CDCl ) 64.41, 63.30, 8.66.
5
2
2
Dendrimer 2-G
of 12 (12 mg, 46.50 mol) in distilled THF (2mL), was added Cs
mg, 89.28 mol) and stirred overnight at room temperature. 1-G
mg, 1.86 mol) in 1.5 mL of distilled THF was then added to the mixture
2
. The reaction was shielded from the light. To a solution
CO (29
(30.1
2
3
3
2
2
2
4
31
and stirred 5 days at room temperature. (monitored by P NMR). The
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201805617
FULL PAPER
solution was filtered, and dendrimer was precipitated in pentane (200
without serum and phenol red. For confocal imaging, a Zeiss LSM 780
microscope equipped with DIC plan-apochromat 63x/1.4 NA oil
immersion objective was used. Nuclear staining was excited at 405 nm
mL). The resulting powder was washed with Et
2
O to removed excess of
12 then solubilized in THF and centrifuged to remove cesium traces.
Dendrimer was again precipitated in pentane, filtered under argon, and
2
and 2-G at 548 nm. On the other hand, cells were incubated 3 h with
dried under reduce pressure to yield 30 mg (75%) of orange powder.
dendrimer 2-G
2
(50 µg/ml). Thirty minutes before the end of incubation,
1
-1
M.p: 178 °C. H NMR (CDCl
3
) 7.68-7.28 (m, 168H), 7.13-6.52 (m, 317H),
cells were loaded with 1 mg.mL CellMask for plasma membrane stains.
For multi-photon imaging, a Zeiss LSM 780 microscope with tuneable
Chameleon Ultra II laser (Coherent, mode- locked Ti: sapphire laser,
4
(
.16-3.16 (m, 558H), 1.97 (m, 48H), 1.11 (m, 120H), 0.66 (m, 120H). 31P
CDCl ) 64.59, 8.61.
3
680-1080 nm) and equipped with DIC plan-apochromat 63x/1.4 NA oil
2
immersion objective was used. 2-G and plasma membrane stains were
excited at 750 nm.
Photophysical studies
UV-Vis absorption and emission spectroscopy. All photophysical
measurements have been performed with freshly-prepared solutions in
air-equilibrated THF at room temperature (298 K). UV/Vis absorption
spectra were recorded on a Jasco V-570 spectrophotometer. Steady-
state and time-resolved fluorescence measurements were performed on
dilute solutions (ca. 10-6 M chromophore concentration, optical density <
Two-photon excited photodynamic therapy. For experiments of 2PE-
PDT, MCF-7 breast cancer cells were seeded in a 384 multi-well plate
2
with 0.17 mm glass bottom and incubated with or without 2-G (50 g.
-
1
mL ) for 20 hours. After incubation, cells were washed, maintained in
fresh culture medium and then submitted or not to laser irradiation. Two-
photon irradiation was carried out on MRI platform. PDT was then
0.1) contained in standard 1 cm quartz cuvettes using an Edinburgh
performed on
a multi-photon confocal microscope Zeiss LSM 780
Instruments (FLS920) spectrometer in photon-counting mode. Emission
spectra were obtained, for each compound, under excitation at the
wavelength of the absorption maximum. Fluorescence quantum yields
were measured according to literature procedures using quinine bisulfate
equipped with tuneable Chameleon Ultra II laser (Coherent, 680-1080
nm) generating 140 fs wide pulses at 80 MHz rate. The laser beam was
focused by a microscope objective lens (10x, NA 0.4). The wells were
as a standard (quantum yield  = 0.546).[38] The lifetime
irradiated at 760 nm by 3 scans of 1.57 sec each at an average power of
in 0.5 M H SO
2 4
2
80 mW. The surface of the scanned areas was 1.5 x 1.5 mm (mean
values were obtained from the reconvolution fit analysis (Edinburgh F900
analysis software) of decay profiles obtained using the FLS920
instrument under excitation with a nitrogen-filled nanosecond flash-lamp.
The quality of the fits was evidenced by the reduced ² value (² <1.1).
-
2
energy of 16.6 J cm ). Two days after irradiation, the percentage of living
cells was determined by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl) -2-(4sulfophenyl)-2H-tetrazolium) enzymatic
assay. Briefly, cells were incubated in the presence of 0.5 mg. mL MTS
for 4 h to determine mitochondrial enzyme activity. Absorbance was read
at 492 nm with a spectrophotometer Thermoscientific Multiskan FC.
-
1

Measurements of singlet oxygen quantum yield ( ). Measurements
were performed on a Fluorolog-3 (Horiba Jobin Yvon), using a 450 W
Xenon lamp. The emission at 1272 nm was detected using a liquid
nitrogen-cooled Ge-detector model (EO-817L, North Coast Scientific Co).
Daylight study. For experiments of daylight toxicity, MCF-7 cancer cells
were seeded in a 96 multi-well plate and incubated with or without 2-G
2
Singlet oxygen quantum yields 

 were determined in dichloromethane
-
1
(
20 g. mL ) for 5 hours. 2-G
2
was previously solubilized in THF at 5
solutions, using tetraphenylporphyrin (TPP) in dichloromethane as
[TPP] = 0.60) and were estimated from 1O
mg/mL and sonicated. After incubation, cells were washed, maintained in
fresh culture medium and then submitted or not to daylight for 4 hours.
The percentage of living cells was determined by MTT (3-(4,5-
dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide) enzymatic assay.
reference solution (

2
luminescence at 1272 nm.
Two-photon absorption. Two-photon absorption cross sections (
obtained from the two-photon excited fluorescence (2PEF) cross sections
2
) were
-
1
Briefly, cells were incubated in the presence of 0.5 mg. mL MTT for 4
hours to determine mitochondrial enzyme activity. Then medium was
removed and purple precipitate was dissolved in 150 l ethanol/DMSO
(
2
) and the fluorescence emission quantum yield (). 2PEF cross
-
4
sections in toluene (10 M chromophore concentration) were determined
using a Ti-sapphire laser delivering 150 fs excitation pulses, according to
the experimental protocol established by Xu and Webb.[39] This
experimental protocol allows avoiding contributions from excited-state
absorption that are known to result in largely overestimated 2PA cross-
sections. Fluorescein in 0.01 M NaOH, whose 2PEF cross-sections are
well-known,[39] served as the reference, taking into account the
necessary corrections for the refractive index of the solvents.[40] The
quadratic dependence of the fluorescence intensity on the excitation
intensity was verified for each data point, indicating that the
measurements were carried out in intensity regimes in which saturation
or photodegradation do not occur. More details about the experimental
setup have been previously published.[31]
(
1:1) solution and stirred 30 min. Then absorbance was read at 540 nm.
Acknowledgements
We acknowledge financial support from CNRS and Région
Bretagne for a fellowship to AS (ARED, Biphobio grant). We
wish to thank Vincent Hugues for his contribution to two-photon
excited fluorescence experiments. We thank MRI (Montpellier
RIO Imaging platform) for confocal facilities.
Keywords: dendrimers • photophysics • fluorescence • imaging
Cellular imaging. One- and two-photon imaging have been performed
on Montpellier RIO Imaging (MRI) platform. For cell culture, MCF-7
human breast cancer cells (ATCC) were routinely cultured in Dulbecco's
Modified Eagle Medium F-12 Nutrient Mixture (Ham) supplemented with
glutamax, 10% foetal bovine serum, and 1/100 antibiotics (penicillin/
streptomycin). Cells were allowed to grow in humidified atmosphere at
•
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This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201805617
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Entry for the Table of Contents
Layout 2:
FULL PAPER
A. Sourdon, M. Gary-Bobo, M.
Maynadier, M. Garcia, J.-P. Majoral, A.-
M. Caminade, O. Mongin,* M.
Blanchard-Desce*
Page No. – Page No.
Dendrimeric nanoparticles for two-
photon photodynamic therapy and
imaging: synthesis, photophysical
properties, innocuousness in daylight
and cytotoxicity under two-photon
irradiation in the NIR
New dendrimers combining large two-photon brightness and photosensitizing
properties are efficient cell death inducers under two-photon irradiation in the NIR,
while being non toxic in the dark and under daylight exposition.
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