Fluorescent periodic mesoporous organosilica nanoparticles dual-functionalized: Via click chemistry for two-photon photodynamic therapy in cells

Article abstract of DOI:10.1039/c6tb00638h

The synthesis of ethenylene-based periodic mesoporous organosilica nanoparticles for two-photon imaging and photodynamic therapy of breast cancer cells is described. A dedicated two-photon absorbing fluorophore possessing four triethoxysilyl groups and ha

Full text of DOI:10.1039/c6tb00638h

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Picard, D. Aggad, M. Klausen, C. Mauriello-Jimenez, M. Maynadier, O. Mongin, G. Clermont, E. Genin, X.
Cattoën, M. Wong Chi Man, L. Raehm, M. Garcia, M. Gary-Bobo, M. Blanchard-Desce and J. Durand, J.
Mater. Chem. B, 2016, DOI: 10.1039/C6TB00638H.
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Fluorescent Periodic Mesoporous Organosilica Nanoparticles
Dual-Functionalized via Click Chemistry for Two-Photon
Photodynamic Therapy in Cells
Jonas G. Croissant,a Sébastien Picard,b Dina Aggad,e Maxime Klausen,b Chiara Mauriello
Jimenez,a Marie Maynadier,f Olivier Mongin,c Guillaume Clermont,b Emilie Genin,b Xavier
Cattoën,d Michel Wong Chi Man,a Laurence Raehm,a Marcel Garcia,e Magali GaryꢀBobo, *e
Mireille BlanchardꢀDesce,b*JeanꢀOlivier Durand.a*
a) Institut Charles Gerhardt Montpellier, UMRꢀ5253 CNRSꢀUM2ꢀENSCMꢀUM1, case courrier 1701 Place
Eugène Bataillon, Fꢀ34095, Montpellier Cedex 05, France. *durand@univꢀmontp2.fr
b) Univ. Bordeaux, Institut des Sciences Moléculaires, UMR CNRS 5255, 351 Cours de la Libération, Fꢀ33405.
Talence Cedex, France. mireille.blanchardꢀdesce@uꢀbordeaux.fr
c) Institut Des Sciences Chimiques de Rennes, CNRS UMR 6226 Université Rennes 1 Campus Beaulieu,
Rennes Cedex, France.
d) Institut NEEL, CNRS, and Université Grenoble Alpes, Fꢀ38042 Grenoble, France.
e) Institut des Biomolécules Max Mousseron UMR 5247 CNRS; UM 1; UM 2 ꢀ Faculté de Pharmacie,
Université Montpellier, 15, Avenue Charles Flahault, 34093 Montpellier Cedex 05, France. magali.garyꢀ
bobo@inserm.fr
f) NanoMedSyn, Faculté de Pharmacie, 15, Avenue Charles Flahault, 34093 Montpellier Cedex 05, France.
J.G.C. and S.P. contributed equally to this work.1
Introduction
Periodic mesoporous organosilica (PMO) nanoparticles (NPs) obtained from the sole
condensation of bridged organo(alkoxysilanes) represent an emerging area of research in
porous hybrid nanomaterials.1ꢀ11 The uniqueness of PMO materials arises from the
maximization of the organic content constituting the pores (“100%”, i.e. absence of silica
source)5 which no longer act as a doping agent but rather as the main constituent of the
nanomaterial.1 Thus, the choice of the organic moiety(ies) allows one to tune the
13
hydrophilicity/hydrophobicity and interactions that govern the surface chemistry,12,
mechanical and optical properties,13 as well as the stability of the material’s framework upon
hydrolysis14 and thermal decomposition.15 As a result, PMO NPs very recently inspired
several pioneering works in drug2, 3, 6, 7, 16, gene,17 and protein deliveries,18 twoꢀphoton
17, 19
photodynamic therapy,3 high intensity focused ultrasound imaging and therapy,11,
antibiotics adsorption,20 heterogeneous catalysis,21ꢀ25 carbon dioxide capture,26 and
1 Authors J.C. and S.P. contributed equally to the work.
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superacidic antimicrobial nanomaterials.27 Significant scientific reports in the area of
nanomedicine describes the advantages of PMO NPs when compared to mesoporous silica
NPs such as low hemolytic behavior,14 increased biocompatibility (depending on the organic
group), higher loading capacities,2 tunable physicoꢀchemistry of the pores, and designed
biodegradability.2, 28
The soꢀcalled “click chemistry” popularized by Barry Sharpless29 describes versatile
reactions that are high yielding with a very wide substrate scope. Besides, such reactions are
to be simple to conduct in easily removable or benign solvents, and stereospecific. One of the
most used click coupling is the copperꢀcatalyzed azideꢀalkyne cylcoaddition (CuAAC), which
was widely applied on mesoporous silica NPs.30 Jian Liu and coworkers have reported click
coupling on bulk ethaneꢀbased PMO material in order to introduce chemical functionalities to
increase the material adsorption properties.20 Only one study has been reported on nanoscaled
PMO involving click coupling by J. Deka et al. for heterogeneous catalysis via clicked
MacMillan catalysts.22
Scheme 1. Representation of the design of the EN2ꢀClick NPs for FRETꢀmediated TPEꢀPDT.
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Mesoporous organosilica and PMO nanomaterials have been engineered for twoꢀ
photonꢀtriggered diagnostic and therapeutic applications.3, 31ꢀ41 Twoꢀphoton excitation (TPE)
in the biological spectral window provides several advantages for biorelated applications such
as improved penetration depth in tissues in relation with reduced scattering as well the
possibility to selectively address confined volumes.38, 42 As such TPE is of interest for cancer
therapy as it offers the possibility for photoactivation of therapeutic effects only at the tumor
site in particular if the tumor site can be easily imaged via fluorescence,.43, 44 Various twoꢀ
photon absorbing systems have been designed in view of TPE drug delivery38, 39, 45, 46 and
photodynamic therapy34, 36, 43 (PDT) including organicallyꢀdoped mesoporous silica NPs. We
recently reported TPEꢀexcited drug delivery and PDT via PMO NPs,3 which appeared more
attractive than mesoporous silica nanoframeworks for two reasons: (1) the drug loading
capacities were severalꢀfold higher due to the hydrophobic pore walls of organically bridged
silsesquioxane structure of PMO NPs,2, 32 (2) silsesquioxane bridges also promote strong
adsorption of drugs without passive leakage, which allowed their transportation without the
need of the pore capping agents or nanomachines.1
Herein we report a novel alkoxysilylated twoꢀphoton fluorophore (N2F) and its coꢀ
condensation with bis(triethoxysilyl)ethane and 3ꢀazidopropyltriethoxysilane for the
preparation of clickable twoꢀphotonꢀsensitive ethyleneꢀbridged PMO (EN2C) NPs. The EN2C
NPs were then used as a multifunctional platform for anticancer drug release upon acidic
trigger. Moreover, the nanomaterial was clicked with a dual role fluorescent photosensitizer
(8ꢀBrꢀquinoline derivative) that is able to generate both fluorescence and singlet oxygen upon
irradiation (either under oneꢀphoton excitation in the near UV or TPEꢀNIR)47 in view of
combining direct TPEꢀPDT and Förster resonance energy transfer (FRET) from the N2F
fluorophores to the clicked photosensitizer upon TPE (see Scheme 1). Furthermore, we
present for the first time PMO NPs for fluorescent imaging and spatially selective TPEꢀPDT
on cancer cells in the NIR region by combining direct and FRETꢀmediated excitation of a
nonꢀporphyrinoid surface photosensitizer thanks to the large twoꢀphoton absorption response
of the combination of TPA fluorophore and sensitizer.
Results and discussion
The synthesis of the tetrapropargylated twoꢀphoton fluorophore N2F precursor is
depicted in Scheme 2. First, the preparation of the divinyl fluorene intermediate
3
was
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achieved in very good overall yield following a twoꢀstep sequence starting from the diiodo
fluorene 1.48 Alkylation with 1ꢀbromononane in the presence of potassium hydroxide in
DMSO provided the compound
afford the derivative by means of a double CorriuꢀKumada coupling. Double Mizorokiꢀ
Heck coupling of the divinyl fluorene with the bromo derivative , prepared by bromination
of the commercially available ꢀphenyldiethanolamine , led to the pushꢀpush quadrupole
2 which was then reacted with vinylmagnesium bromide to
3
3
5
N
4
6.
Finally, the clickable twoꢀphoton fluorophore N2F precursor was obtained by the
propargylation of the four terminal hydroxyl groups in the presence of propargyl bromide
with NaH and a catalytic amount of KI in DMF. The synthesis of the Click precursor was
achieved from the 8ꢀBrꢀquinoline derivative
7 (recently reported as a new sensitizer
combining twoꢀphoton induced fluorescence and singlet oxygen generation)47 by
propargylation of the hydroxyl moiety with moderate yield.
Scheme 2 Reagents and conditions: i) KOH, KI, 1ꢀbromononane, DMSO, rt, 79%; ii) Vinyl
magnesium bromide, PdCl2(dppf), THF, 4h, 94%; iii) NBS, CH2Cl2, rt, 89%; iv) P(oꢀTolyl)3,
Pd(OAc)2, DMF, Et3N, 90 °C v) NaH, propargyl bromide, KI, DMF, rt, 80%; vi) NaH, propargyl
bromide, KI, THF, rt, 31%.
The two chromophores show intense absorption in the near UVꢀvisible region in the case of
N2F precursor and in the near UV region for the bromoquinoline click precursor (see Table 1
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and Fig. S1). Both compounds emit fluorescence in the visible region (blue emission for N2F
and green emission for the bromoquinoline chromophore). Chromophore N2F shows a much
larger fluorescence quantum yield than the bromoquinoline, in relation with high singlet
oxygen generation ability of the bromoquinoline derivative. Furthermore N2F shows large
twoꢀphoton absorption (2PA) crossꢀsection in the NIR region with a peak 2PA crossꢀsection
(
σ2max) over 1000 GM while compound
7 shows about 7ꢀfold smaller 2PA crossꢀsections.
Hence N2F is a good twoꢀphoton fluorophore while the bromoꢀquinoline chromophore is a
good sensitizer generating singlet oxygen under either oneꢀphoton excitation (in the near UV)
or twoꢀphoton excitation (in the NIR region). These two chromophoric modules thus show
complementary optical properties for the design of functionalized PMO nanoparticles that can
both be imaged by twoꢀphoton fluorescence imaging and deliver singlet oxygen under twoꢀ
photon excitation. Furthermore, energy transfer from N2F to the bromoquinoline may occur
when the two chromophoric subunits are in close proximity (typically at distance of less than
10 nm) as the emission spectrum of N2F and the absorption spectrum of the quinoline show a
small overlap (see Fig. S1)
Table 1. Photophysical and 2PA characteristics of N2F precursor and click precursor models in
ethanol.
max
max
max
max c
λ abs
εmax
λem
λ2PA
(nm)
σ2
a
b
Compound
ΦF
Φꢀ
(nm) (Mꢀ1 cmꢀ1) (nm)
(GM)
1100
154
N2F precursor model
392
373
83000
34000
468
540
0.61
ꢀ
740
770
0.03 0.62
7
a Fluorescence quantum yield determined relative to quinine in H2SO4 0.5 M (ΦF=0.546).
b Singlet oxygen determined relative to tetraphenylporphyrin in toluene (Φꢀ[TPP]=0.68 in toluene).
c In toluene.
The N2F precursor was then clicked with (3ꢀazidopropyl)triethoxysilane via CuAAC
to obtain the N2F reactant (ESI). Finally, the solꢀgel elaboration of the EN2C nanomaterial
was performed via the coꢀcondensation of the alkoxysilylated N2F with
bis(triethoxysilyl)ethylene and azidopropyltriethoxysilane in an aqueous mixture of the
cetyltrimethylammonium bromide (CTAB) surfactant with sodium hydroxide catalyst at 80
°C (ESI).
The size and morphology of the surfactantꢀfree EN2C NPs were examined via
transmission and scanning electron microscopies (TEM, SEM). The material was found to be
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of spherical shape with an average diameter of 80 nm (see Fig. 1AꢀB). Besides, the uniformity
in shape and the relatively narrow size distribution of the NPs were confirmed by the SEM
micrograph (Fig. 1C), the latter being also supported by dynamic light scattering analysis
(Fig. 1D).
The texture and structure of the nanomaterial were then investigated. Nitrogen
sorption measurement and the BrunauerꢀEmmettꢀTeller (BET) theory revealed a surface area
of 661 m².gꢀ1 with pores of 3.6 nm in diameter (Figs. 1E and S2A). The isotherm can be
described as the typical type IV curve of mesoporous compounds, with wormlike network of
mesopores as displayed in high magnification TEM micrograph (see Fig. 1B). The porous
mesostructure was confirmed by the peak at 2 degrees in the small angle Xꢀray diffraction
pattern, which corresponds to a Bragg dꢀspacing of 44 Å (Fig. S2B). The relatively low
intensity of the XRD peak correlates the lower ordering of the material, which may result
from the multiple species used in the coꢀcondensation process.
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Figure 1. TEM and SEM images (AꢀB and C respectively), DLS size distribution (D), and N2ꢀ
adsorptionꢀdesorption isotherm of EN2C NPs (E). UVꢀvisible and FTIR spectra of EN2C and EN2ꢀ
Click NPs (F and G respectively).
The composition and chemical functionalities were then analyzed by various
techniques. The successful incorporation of the N2F fluorophores in the EN2C NPs was first
demonstrated by the absorption band at λmax of 400 nm in the UVꢀvisible spectrum (see Figs.
1F and S3), and various carbon environments in the solid state 13C nuclear magnetic
resonance (NMR) crossꢀpolarization magic angle spinning (CPMAS) spectrum (see Fig.
S4A). The incorporated content of N2F was calculated to be of 1.12 ꢁmol per gram of EN2C
NPs via the BeerꢀLambert law. The ethenyleneꢀbridged organosilica framework was then
examined via the 13C CPMAS NMR spectrum, which exhibited the presence of the ethenylene
fragment (Fig. S4A, see δ = 145 ppm and the E PMO NPs control therein). Moreover, Fourier
transform infrared (FTIR) spectroscopy displayed the νSiꢀC and νC=C ethenylene vibration
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modes at 1193 and 1636 cmꢀ1 respectively (Fig. 1G). The azidopropyl groups were clearly
observed by the carbon signals of the propyl in the 13C spectrum at 9, 23, and 58 ppm (see
Fig. S4A),22 and the νN=N stretching of the azide functions at 2110 cmꢀ1 in the FTIR spectrum
(Fig. 1G). Note that the 29Si CPMAS spectrum validated the formation of the siloxane
network with the major proportion of the T2 and T3 signals (Fig. S4), which is also supported
by the shift of the νSiꢀO stretching modes towards 1150 cmꢀ1, typical of highly condensed
frameworks. Besides, most SiꢀC linkages are preserved during the solꢀgel process as revealed
by the scarce Q3 and Q4 environments around ꢀ100 ppm in the silicon spectrum of EN2C. The
high organic content of the EN2C NPs obtained from several organoalkoxysilanes was
validated by the energy dispersive spectroscopy analysis (EDS, Fig. S5) leading to 44 weight
percent of carbon (Table S1).
The mesoporosity of the EN2C nanoplatform was then exploited for two kinds of
applications. First, PMO pores were loaded with the doxorubicin (DOX) anticancer drug for
cargo release application. The NPs were stirred with the DOX in an acidic (pH 5.5) aqueous
media for two days, the solution was then neutralized and the material was centrifuged and
washed several times (ESI). We previously reported the importance of loading ethenyleneꢀ
bridged PMO NPs in acidic media in order to increase the diffusion of DOX cargos
throughout the porous cavities.2 Finally, DOXꢀloaded NPs were placed in a plastic cuvette in
water and the pH of the solution was modified to mimic the increased acidity arising from the
lysosomal compartment (from pH 7.4 to pH 5.5). The maximum absorption of the DOX
molecule (495 nm) was plotted as a function of time to obtain the release profile (Fig. 2). On
the one hand, the absence of premature release at neutral pH suggests the robustness of this
nanovehicle for biological transportation. The instantaneous and sustained release of cargos
upon acidic stimulus (with up to 14.2 wt% of DOX) validated the potential of EN2C NPs as
efficient drug delivery nanovehicle. PMO NPs are positively charged at pH 5. Electrostatic
repulsions are responsible for the release of positivelyꢀcharged DOX from PMO NPs.49, 50
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Figure 2. Release capability of DOXꢀloaded EN2C NPs via acidic stimulus. The last point
corresponds to the maximum release (14.2 wt% of DOX).
Secondly, the clickable EN2C PMO material was functionalized via click chemistry
with a photosensitizer affording EN2ꢀClick NPs (see Scheme 1 and Fig. S1A). The interest of
this coupling step are several: (i) circumvent the drawbacks of the chemical and thermal
stabilities of the photosensitizer during the solꢀgel process,51 (ii) photosensitizers combining
large singlet oxygen generation ability with high twoꢀphoton σ2 in the NIR are scarce and
often quite large (such as extended porphyrins),44 therefore combining a bright fluorophore
showing high σ2 with an efficient photosensitizer, acting as complementary FRET donorꢀ
acceptor pair, would allow FRETꢀmediated TPEꢀPDT, (iii) click chemistry enables a
quantitative covalent grafting of photosensitizers on the surface of the pores, favoring spatial
proximity required to ensure efficient FRET as well as easy reaction of dissolved oxygen and
subsequent generation of toxic singlet oxygen to the cells after twoꢀphoton excitation (either
direct or FRET mediated). The successful CuAAC coupling in EN2C NPs induced the
modification of the UVꢀvisible spectrum, a decrease of the absorption band of the N2F at 400
nm along with the appearance of another band at around 301 nm in EN2ꢀClick NPs (see Figs.
1F and S3). Besides, the complete disappearance of the azide vibration mode in the FTIR
spectrum unambiguously demonstrates the quantitative coupling of the photosensitizers (see
Fig. 1G).
The emission properties of EN2C and EN2ꢀClick NPs were then studied in ethanol
(see Table 2 and Figure 3). The shape of the emission band of EN2C NPs is similar to that of
their corresponding N2F precursor, except for a bathochromic shift of about 30 nm observed
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in the NPs (in relation with environment effects). A decrease of the fluorescence quantum
yield is also observed when going from the isolated chromophore (0.61, see Table 1) to the
NPs (0.15, see Table 2). This phenomenon could be related to quenching effects in relation
with an increase of the polarity of the chromophore environment (as suggested by the redꢀ
shifted emission) and/or with its high concentration within the NPs.
In contrast, the emission spectrum of EN2ꢀClick NPs is very similar to that of the
photosensitizer
of EN2C (0.15), but higher than that of
at 435 nm, wavelength at which does not absorb any more and N2F precursor strongly
absorbs, the emission spectrum of EN2ꢀClick is not modified and is still almost the same than
that of , in agreement with FRET from N2F chromophore to within EN2ꢀClick NPs. In
addition, under twoꢀphoton excitation, the twoꢀphoton emission spectrum is also
superimposable with the oneꢀphoton emission spectra of both EN2ꢀClick and , whatever the
7
they incorporate. Their fluorescence quantum yield (0.06) is lower than that
7
(0.03). It should also be noticed that, upon excitation
7
7
7
7
excitation wavelength between 700 and 900 nm (see Fig. S6). These observations indicate that
an efficient resonant energy transfer is operative from the quadripolar fluorophores towards
the photosensitizers.
The 2PA properties of EN2C and EN2ꢀClick NPs were then examined by investigating
their twoꢀphoton excited fluorescence (2PEF) in the NIR range (Table 2 and Figs. S6ꢀ7).
EN2C NPs exhibit large 2PA crossꢀsections (360 GM per N2F chromophore), even if a
marked decrease is observed in comparison with the isolated N2F chromophore in solution
(1100 GM, see Table 1), presumably in relation with the high chromophore loading of the
NPs and resulting deleterious interactions between neighboring chromophores. Grafting of
photosensitizing chromophores via click chemistry on EN2C leads to EN2ꢀClick NPs
exhibiting much higher 2PA crossꢀsections (2300 GM per N2F chromophore). The number of
clicked quinoline chromophores per N2F chromophore is not accurately known, but we can
estimate that the photosensitizer / N2F ratio is comprised between 15 and 30, which would
lead to a 2PA crossꢀsection of the clicked photosensitizers ranging between 60 and 130 GM,
which is consistent with the value obtained for the isolated chromophore
7 in solution (154
GM). Hence, it appears clearly that EN2ꢀClick NPs are much stronger twoꢀphoton absorbing
NPs than their EN2C NPs precursors. Both types of chromophores are responsible for the
twoꢀphoton absorption response of the EN2ꢀClick NPs: the clicked quinoline photosensitizers
contribute to about 80% of the global absorption while N2F chromophores contribute to about
20%. Finally, it should also be noticed that, whereas EN2ꢀClick exhibits a 2.5 lower
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fluorescence quantum yield than that of EN2C, its twoꢀphoton brightness (σ2.ΦF) is more than
2.5 times larger than that of EN2C, thanks to the additive contribution of the numerous
clicked photosensitizer moieties.
Table 2. Photophysical and 2PA properties of EN2C and EN2ꢀClick nanoparticles in ethanol.
max
max
max
max b
λ abs
λem
λ2PA
(nm)
σ2
a
NPs
ΦF
(nm)
420
406
(nm)
499
540
(GM)
EN2C
0.15
0.06
730
740
360
EN2ꢀClick
2300
a Fluorescence quantum yield determined relative to quinine in H2SO4 0.5 M (ΦF=0.546).
b 2PA crossꢀsections per quadrupolar fluorophore.
Figure 3. Emission spectra of EN2C and EN2ꢀclick (solid lines) NPs excited at 435 nm and of their
molecular precursors (dashed lines) in ethanol.
The photosensitizerꢀclicked twoꢀphotonꢀsensitive nanoplatforms were thus applied for
twoꢀphoton theranostic applications. The biocompatibility of the NPs was first evaluated on
the MCFꢀ7 breast cancer cell line. The NPs before and after click couplings were incubated in
cells at concentrations from ten to a hundred micrograms per milliliter, which revealed them
to be biocompatible (see Figs. S8 and S9). The endocytosis of the NPs was then assessed after
20 h of incubation at 40 µg.mLꢀ1 via a twoꢀphoton Carl Zeiss confocal microscope at low
power (3%, input power 3W, excitation at 760 nm). Bright fluorescent dots are observed with
EN2C and EN2ꢀClick NPs, which were found to colocalize with the cell interior, thus proving
nanoparticles uptake (Fig. 4). As indicated in Figure S10, the NPs accumulate in the
lysosomes after endocytosis.
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Figure 4. In-vitro twoꢀphoton fluorescence imaging of EN2C (A) and EN2ꢀClick NPs (B) incubated
20 h at 40 µg.mLꢀ1. Scale bar of 10 µm. Cell membranes stained by Cell Mask®.
The nanomaterials were eventually tested for in-vitro TPEꢀPDT. The confocal laser
was settled at maximum laser power and three scans of 1.57 seconds in four different areas
without overlaps between irradiated areas were performed.38 The cell survival was assessed
48 h later by MTT assay. In a control experiment such parameters of irradiation were found to
be harmless for the cells (Fig. 5). Besides, EN2C NPs did not induce signification cytotoxicity
before or after laser irradiation, which confirmed that the N2F moiety acts as a fluorophore
rather than a photosensitizer. Conversely, EN2ꢀClick NPs produced 40% of cell killing via
TPEꢀPDT. Hence, these results strongly suggest that N2F fragments transferred their TPEꢀ
absorbed energy by FRET mechanism, to excite the clicked photosensitizers for the
production of singlet oxygen. Moreover, concomitant direct twoꢀphoton excitation of the
clicked photosensitizers significantly contributes to cell death.
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Laser OFF
103
Laser ON
109
120
100
80
60
40
20
0
95
100
90
*
62
Control
Figure 5. In-vitro TPEꢀPDT experiment via EN2C and EN2ꢀClick NPs incubated 20 h at 40 µg.mLꢀ1.
Cell survival measurements by MTS assay 48 h after irradiation. Data are mean values ± standard
deviation from three independent experiments. * Statistically significant versus nonꢀirradiated NPs (p
< 0.05 from Student’s t test).
Conclusion
In conclusion, we described highly twoꢀphotonꢀsensitive tripleꢀfunctionalized PMO
NPs combining potent fluorophores and photosensitizers for promising for theranostics
applications on cancer cells. A novel fluorophore was first prepared and then incorporated
into ethyleneꢀazidopropylꢀbridged PMO NPs via solꢀgel synthesis. The PMO platform could
be used for loading and pHꢀtriggered release of drugs. Clickable functions into PMO NPs
were further used to covalently postꢀgraft photosensitizers within the NPs for PDT. Thanks to
combined directꢀ and indirect FRETꢀmediated TPEꢀPDT and TPEꢀfluorescence, these PMO
NPs were shown to enable simultaneous imaging and therapy of breast cancer cells under NIR
irradiation. These results demonstrate the versatile and useful chemical functionalities which
could be incorporated into PMO NPs for multiple applications in nanomedicine and twoꢀ
photon theranostics for localized treatments.
ACKNOWLEDGMENTS
We acknowledge financial support from Agence Nationale pour la Recherche (Grant
ANRꢀ10ꢀNANOꢀ022ꢀ01 and ANRꢀ12ꢀNANOꢀ0017). M.B.ꢀD. is grateful to the Conseil
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Régional d’Aquitaine for financial support (Chair of Excellence to M.B.ꢀD) and to the
University of Bordeaux (fellowship to MK).
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TOC Graphic
Abstract : The synthesis of ethenylene-based periodic mesoporous organosilica
nanoparticles for two-photon imaging and photodynamic therapy of breast
cancer cells is described. A dedicated two-photon absorbing fluorophore
possessing four triethoxysilyl groups and having large two-photon absorption in
the Near IR region, and azidopropyltriethoxysilane were incorporated in the
structure. The mesoporous nanoparticles of 100 nm diameter were further
functionalized by means of click chemistry with a propargylated fluorescent
bromo-quinoline photosensitizer able to generate singlet oxygen. The
photophysical properties and two-photon absorption properties of the
nanoparticles were investigated evidencing complementary contribution of the
two dyes. Both dyes contribute to the two-photon absorption response of the
mesoposous nanoparticles while efficient FRET from the two-photon
fluorophore to the quinoline sensitizer is observed. The dual-functionalized
nanoparticles were incubated with MCF-7 breast cancer cells. Two-photon
confocal imaging demonstrated the endocytosis of the nanoparticles within
cancer cells. Moreover, brief two-photon irradiation (3 scans of 1.57s ) at 760
nm at high laser power (3W) was shown to induce 40% of cancer cells death
demonstrating the potential of the dual-functionalized mesoporous organosilica
nanoparticles for two-photon photodynamic therapy.