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Enhanced Two-Photon Fluorescence Imaging and Therapy of Cancer Cells via Gold@Bridged Silsesquioxane Nanoparticles

Jonas Croissant , Marie Maynadier , Olivier Mongin , Vincent Hugues , Mireille Blanchard-Desce , * Arnaud Chaix , Xavier Cattoën , Michel Wong Chi Man , Audrey Gallud , Magali Gary-Bobo , * Marcel Garcia , Laurence Raehm , and Jean-Olivier Durand *

Two-photon excited photodynamic therapy (TPE-PDT)

offers promising perspective for cancer treatment [ 1 ] thanks

to the deep penetration of the two-photon light in the near-

infrared region and the unique spatial resolution it provides.

In particular, the use of two-photon excitable nanoparticles

(NPs) for cancer diagnosis and therapy would offer new

opportunities in nanomedicine. TPE-PDT combined with

NPs is indeed a new area of investigation which is still in

its infancy. [ 2,3 ] In most of the studies, either a porphyrin or

a chlorin derivative was indirectly activated by FRET from

an antenna designed for two-photon absorption. [ 4–11 ] Alter-

natively, a photosensitizer designed for its high two-photon

absorption cross-section can be incorporated within the

framework of silica-based nanoparticles. [ 12,13 ] However, this

approach is much less exploited due to the diffi culties in

Photodynamic Therapy

DOI: 10.1002/smll.201401759

J. Croissant, A. Chaix, Dr. X. Cattoën, Dr. M. Wong Chi Man, Dr. L. Raehm, Dr. J.-O. Durand Institut Charles Gerhardt Montpellier, UMR-5253 CNRS-UM2-ENSCM-UM1 cc 1701, Place Eugène Bataillon F-34095 , Montpellier cedex 05 , France E-mail: durand@um2.fr

Dr. O. Mongin Institut Des Sciences Chimiques de Rennes CNRS UMR 6226 Université Rennes 1 Campus Beaulieu F-35042 , Rennes Cedex , France

V. Hugues, Dr. M. Blanchard-Desce Univ. Bordeaux 1 Institut des Sciences Moléculaires UMR CNRS 5255, 351 Cours de la Libération F-33405 , Talence Cedex , France E-mail: m.blanchard-desce@u-bordeaux1.fr

Dr. M. Maynadier, A. Gallud, Dr. M. Gary-Bobo, Dr. M. Garcia Institut des Biomolécules Max Mousseron, UMR 5247 CNRS UM 1; UM 2 – Faculté de Pharmacie 15 Avenue Charles Flahault 34093 , Montpellier Cedex 05 , France E-mail: magali.gary-bobo@inserm.fr

designing and synthesizing the two-photon photosensitizers.

The low probability associated with TPE requires using

molecules with very high two-photon effi ciencies, and maxi-

mizing the density of the two-photon absorbing fragments in

the NPs. To this aim, we focused our effort on bridged silses-

quioxanes NPs (BSNPs) which allow the highest loading rate

of regularly and covalently linked organic functionalities [ 14 ]

and which are directly prepared by the sol-gel process from

a single molecular organosilane possessing two or more tri-

alkoxysilyl groups. To date, very few reports deal with the

preparation of BSNPs due to the diffi culties of precisely

controlling the growth of the NPs during the sol-gel hydrol-

ysis-condensation of the activated organosilanes. Shea and

co-workers’ pioneering work led to BSNPs with photore-

sponsive, [ 15,16 ] electrochromic [ 17 ] and electrochemical [ 18 ] prop-

erties and quite recently, Hammers and co-workers prepared

BSNPs from perylene diimide for photovoltaïc applications

using a modifi ed Stöber method. [ 19 ] To our knowledge, only a

single study has been reported concerning cancer therapy, [ 20 ]

with targeted BSNPs for platinum-based chemotherapy.

In the context of imaging, biodegradable BSNPs with high

Gd(III) payloads were recently designed for MRI of cancer

cells in vitro. [ 21 ] BSNPs thus constitute a promising platform

for cancer imaging and therapy.

In this work we therefore prepared novel BSNPs based

on the design of a two-photon photosensitizer possessing four

triethoxysilyl groups for TPE imaging and PDT of cancer

cells. Moreover, gold core BS shell NPs (Au@BSNPs) and

BSNPs decorated by gold nanospheres (BS@AuNPs) were

designed to increase the two-photon properties of BSNPs.

The precursor and the nanomaterials physico-chemical and

photophysical properties were fully characterized via ver-

satile techniques. Furthermore, TPE-PDT and fl uorescence

imaging on cancer cells revealed a remarkable exaltation of

the two-photon properties of the NPs, which correlated with

the photophysical studies.

The diaminodiphenylbutadiene core structure of the

two-photon photosensitizer (2PS) precursor depicted in

Scheme 1 a was known to selectively photo-activate electron

transfer in bio processes under TPE, [ 22–24 ] but this property

small 2014, DOI: 10.1002/smll.201401759

J. Croissant et al.

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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

has never been exploited for TPE-PDT. This 2PS molecule,

which exhibits four triethoxysilyl groups, was elaborated at

the gram scale in fi ve steps (see Figure S1), the last being the

[CuBr(PPh 3 ) 3 ]-catalyzed CuAAC click reaction with (3-azi-

dopropyl)triethoxysilane under mild conditions. [ 25 ] The 2PS

precursor was fully characterized via proton and carbon

nuclear magnetic resonance (NMR), Fourier-transform

infrared (FTIR), UV-visible, and mass spectroscopies (see SI,

and Figure S2). Besides, a non-alkoxysilylated reference of

the 2PS molecule (2PS Ref, see Figure 2 a and S3) was devel-

oped and fully characterized (Figure S4 and S5), in order to

study the photophysical properties of the 2PS fragment.

The BSNPs were prepared by a careful control of the

hydrolysis and polycondensation of the 2PS precursor in a

cetyltrimethylammonium bromide / sodium hydroxide / water

/ ethanol mixture at 60 °C for ten minutes. The presence of

EtOH in the procedure was critical to solubilize the 2PS and

to slow down the reaction kinetic in order to obtain and iso-

late the nanoparticles with the desired size and morphology.

The time and temperature of the reaction were also care-

fully optimized for this purpose. Notably, BSNPs could also

be obtained with a triethylamine catalyst in similar conditions

(ESI). Original BS@AuNPs were obtained by mixing gold

NPs [ 26 ] with these BSNPs, without the need of thiol linkers to

immobilize gold NPs on the surface of BSNPs. For the prep-

aration of Au@BSNPs, a one-pot procedure was developed.

First gold nanospheres were prepared in-situ [ 26 ] and stabilized

with a thin silica layer. The 2PS precursor was subsequently

added under the same conditions as for the BSNPs affording

the Au@BSNPs.

The BS, Au@BS and BS@Au nanostructures, compositions,

and properties were then characterized. Transmission elec-

tron microscopy (TEM) analysis on the three types of nano-

material revealed 150 nm nearly monodisperse spherical NPs

( Figure 1 a–c), as confi rmed by the scanning electron micros-

copy (SEM) images (Figure 1 d–e) and by dynamic light scat-

tering (DLS) size distributions (Figure S6). Au@BSNPs tended

to be slightly smaller and aggregated. The gold nanospheres

were readily visible on the surface of BS@AuNPs (Figure 1 b),

or within the BS nanoshells (Figure 1 c). Moreover, 29 Si and

13 C CP-MAS NMR spectra confi rmed both the formation of

the siloxane inorganic network with a high condensation of

the 2PS precursor and the preservation of the organic moiety,

as shown by the major proportion of T 3 silicon environ-

ments (Figure 1 g–h) and the carbon environments of the 2PS

(Figure S7). This is further supported by the FTIR spectra of

the BSNPs and Au@BSNPs nanomaterials with the red-shift of

the ν Si-O band from 1080 cm −1 in the 2PS precursor to 1137 cm −1

in the materials (Figure S8). The gold content of Au@BSNPs

and BS@AuNPs were assessed by induced-coupled plasma

and energy dispersive spectroscopy respectively and were

found to be of 24 and 25% in weight (Table S1).

small 2014, DOI: 10.1002/smll.201401759

Scheme 1. A novel two-photon sensitizer (2PS) (a) condensing via sol-gel chemistry in BSNPs, and in Au@BSNPs or BS@AuNPs with the combination of gold nanospheres (b). The presence of gold exalts the nanomaterials TPE-PDT and fl uorescence imaging (c).

Figure 1. Representation of BSNPs, BS@AuNPs, and Au@BSNPs along with their TEM (a–c) and SEM images (d–f). Solid state 29 Si CPMAS NMR spectra of BSNPs (g) and Au@BSNPs (h).

Enhanced Two-Photon Fluorescence Imaging and Therapy of Cancer Cells

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The optical and photophysical properties of the nanoma-

terials were then analyzed. To assess the optical properties

of the materials, the 2PS Ref model was utilized ( Figure 2 a).

The UV-visible absorption spectrum of BSNPs displayed

the 2PS absorption band, whereas the Au@BSNPs and BS@

AuNPs ones showed the gold NPs plasmon band as well

(Figure 2 b). The large plasmon bands are due to the gold NPs

core spatial proximity within the BS structure of Au@BSNPs,

or on the surface of BS@AuNPs. In the fl uorescence spectra

of the nanomaterials recorded in EtOH (Figure 2 c), an

important bathochromic shift was observed from the 2PS Ref

to the materials with a decrease of the fl uorescence quantum

yields ( Table 1 ). The red shifts are consistent with the aggre-

gation of the 2PS in the solid state with a reabsorption pro-

cess; therefore the fl uorescence quantum yields decreased

accordingly. [ 27 ] The two-photon absorption cross-sections

(σ 2 ) were determined per fl uorophore using the two-photon

excited fl uorescence (TPEF) method [ 12 ] and showed an

increase from 2PS Ref to the BSNPs, BS@AuNPs and Au@

BSNPs with maximum values of 100, 140, 210 and 260 GM

respectively at 700 nm (Figure 2 d). The enhancement of

the two-photon absorption cross-section

in the presence of gold NPs arises from

the plasmonic resonance upon TPE [ 28 ] of

the gold NPs with a spatial proximity. [ 29 ]

Comparing Au@BSNPs and BS@AuNPs,

the differences are small but the higher

enhancement of σ 2 in the former suggests

that the interactions of the photosensitizer

in the excited state with gold nanocrystals

in Au@BSNPs and BS@AuNPs are dif-

ferent and more effi cient with Au@BSNPs.

Two-photon imaging studies on living

MCF-7 breast cancer cells were then per-

formed with a Carl Zeiss confocal micro-

scope at low laser power (2.8% of the

input power in order not to damage living

cells) with BSNPs, Au@BSNPs, and BS@

AuNPs after 20 h of incubation. Indeed,

the internalization of the nanomaterials

was found to reach its maximum after

20 h of incubation (see Figure S9). Cell

membranes were stained with a specifi c

membrane marker (cell mask) in order to

enable the NPs tracking inside the cells

with two-photon fl uorescence. Figure 3

demonstrates the endocytosis of the NPs

small 2014, DOI: 10.1002/smll.201401759

Figure 2. The stable and soluble spectroscopic reference used to obtain the 2PS properties (a). Absorption and emission spectra of 2PS Ref (solution), BSNPs, Au@BSNPs, and BS@AuNPs in EtOH (b and c respectively). Two-photon cross-sections of 2PS Ref (solution in THF), BSNPs, Au@BSNPs, and BS@AuNPs in EtOH.

Table 1. Two-photon properties of the 2PS Ref and the nanomaterials.

NPs λ abs /λ em [nm]

Φ F a) σ 2 max b)

[GM]σ 2 max *Φ F

[GM]

2PS Ref 378/442 0.60 100 c) 60 c)

BSNPs 432/477 0.033 140 5

Au@BSNPs 425/478 0.12 260 30

BS@AuNPs 394/469 0.11 210 24

a) Quinine bisulfate standard at 0.5 M in H 2 SO 4 ; b) per chromophore; c) Analysis performed

in THF.

Figure 3. Two-photon imaging with confocal microscopy on living MCF-7 cells incubated for 20 h with 40 µg.mL −1 of BSNPs (a), Au@NPs (b), and BS@AuNPs (c).The cells were co-stained with a membrane marker (cell mask), and the membranes were visualized in green at 561 nm. NPs submitted to TPE emit fl uorescent dots stained in blue demonstrating the cellular uptake of the nanosystems, and the optical exaltation in the case of Au@BSNPs and BS@AuNPs. Scale bars of 10 µm.

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by cells. Comparing BSNPs, Au@BSNPs, and BS@AuNPs at

the same laser power, an enhancement of the TPE-excited

fl uorescence was observed with both gold-doped nanosys-

tems (Figure 3 b–c, see Figure S10). Indeed the quantum

yields and two-photon absorption cross-sections were higher

with the Au@BSNPs, and BS@AuNPs thus enhancing the

fl uorescence signal (see Table 1 ). Note that the lumines-

cence signal corresponds to the emission of the fl uorophore

(Figure S10), but a contribution of the gold NPs to the lumi-

nescence cannot be excluded.

Eventually, in vitro TPE therapy was performed on breast

cancer cells. MCF-7 cells were incubated for 20 h in the pres-

ence of BSNPs, Au@BSNPs or BS@AuNPs. No cytotoxicity

was observed without irradiation up to a concentration of

40 µg.mL −1 ( Figure 4 ) showing the biocompatibility of the pre-

pared nanomaterials. Additionally, we examined the infl uence

of the laser on epithelial normal cells (ARPE-19 cell line) which

confi rmed the harmlessness of the irradiation (see Figure S11).

On the contrary, when the MCF-7 cells were incubated with the

NPs and irradiated at 760 nm with 3 scans of 1.57 s at the max-

imum power of the laser (input power 3 W) and with a focused

laser beam, an important cancer cell death was observed at a

concentration of 40 µg.mL −1 where only 24%, 6% and 0% of

cancer cells survived respectively with BSNPs, Au@BSNPs,

and BS@AuNPs. The presence of gold again enhanced the effi -

ciency of the Au@BS and BS@AuNPs compared to BSNPs due

to a synergy between the 2PS and gold NPs. The cell survival

difference between Au@BSNPs and BS@AuNPs is not statisti-

cally signifi cant to assess the impact of the gold spatial position

in BSNPs on the PDT effect.

Note that 2PS Ref and the BSNPs were not able to pro-

duce singlet oxygen under irradiation (data not shown).

Therefore, we believe that a mechanism based on electron

transfer is involved in cancer cell killing with TPE-PDT. [ 30 ]

Photothermal effects have been shown to be negligible with

gold nanorods upon TPE-PDT, [ 2 ] but a contribution of pho-

tothermal therapy cannot be excluded with our systems.

In conclusion, a novel two-photon photosensitizer was

reported, and the subsequent sol-gel process with or without

gold NPs led to BSNPs and gold-doped BSNPs. The phys-

ico-chemical properties of the precursor and the resulting

nanomaterials were fully characterized via complemen-

tary techniques. The two-photon fl uorescence quantum

yields of the nanomaterials were measured and two-photon

absorption cross-sections were higher with all the synthe-

sized NPs than with the precursor. Furthermore, such NPs

were applied on cancer cells as powerful multifunctional

nanosystems combining both TPE-PDT and ultra-bright

fl uorescence imaging. The addition of gold nanospheres to

the BSNPs matrix induced a remarkable exaltation of the

in vitro fl uorescence correlating the photophysical studies.

Finally, a clear gold core mediated-enhancement of 20–25%

in TPE-PDT cell killing was obtained with gold-doped BS

nanosystems when compared to BSNPs. Consequently,

BSNPs and gold-doped BSNPs are very promising cancer

theranostics and work is in progress to further exploit these

nanomaterials.

Experimental Section

BSNPs (with NaOH Catalyst) : A mixture composed of water (25 mL), ethanol (10 mL), and cetyltrimethylammonium bromide (0.160 g, 4.40.10 −1 mmol) was stirred at 80 °C in a 50 mL round-bottom fl ask. Then a solution of the 2PS precursor in anhydrous ethanol (2PS: 44.4 mg, 2.7.10 −2 mmol; EtOH: 500 µL) was quickly injected. After 1 minute, sodium hydroxide (NaOH, 150 µL (2 M)) was added to trigger the sol-gel process. The reaction mixture was stirred for 2 minutes at 80 °C, and the solution was then neutral-ized by addition of hydrochloric acid (HCl (0.2 M), 1.38 mL). Then, the mixture was cooled down to 40 °C, and the nanomaterial was collected through centrifugation at 21000 rpm for 15 minutes. Each fraction was washed with ethanol (40 mL per fraction) and centrifuged for 10 minutes. This operation was repeated twice, and the compound was dried under vacuum for few hours.

Two-Photon Photodynamic Therapy : Human breast cancer cells MCF-7 (purchased from ATCC) were cultured in DMEM supple-mented with 10% fetal bovine serum and 50 µg.mL −1 gentamycin. All cells were allowed to grow in humidifi ed atmosphere at 37 °C under 5% CO 2 . For in vitro phototoxicity, MCF-7 cells were seeded into a 384 multiwell glass-bottomed plate (thickness 0.17 mm), with a black polystyrene frame, 2000 cells per well in 50 µL of cul-ture medium, and allowed to grow for 24 h. BSNPs were then dis-persed under ultrasounds in PBS at a concentration of 1 mg.mL −1 and cells were then incubated for 20 h with or without BSNPs at a fi nal concentration of 40 µg.mL −1 in DMEM. After incubation with BSNPs, cells were washed twice, maintained in fresh culture medium, and then submitted (or not) to laser irradiation; with the Carl Zeiss Microscope (laser power input 3W). Half of the well was irradiated at 760 nm by three scans of 1.57 s duration in 4 dif-ferent areas of the well. The laser beam was focused by a micro-scope objective lens (Carl Zeiss 10x/0.3 EC Plan-Neofl uar). The scan size does not allow irradiating more areas without overlap-ping. After 2 days, the MTS assay was performed (as previously described [ 12 ] and was corrected according to the following formula Abs control −2 × (Abs control - Abs BS ).

small 2014, DOI: 10.1002/smll.201401759

Figure 4. TPE-PDT on MCF-7 cancer cells. The cells were incubated 20 h with 40 µg.mL −1 of BSNPs, Au@BSNPs, and BS@AuNPs. * n = 3, *p < 0.05 for treatment with Au@BSNPs or BS@AuNPs and irradiation compared to treatment with BSNPs using Student's T test.

Enhanced Two-Photon Fluorescence Imaging and Therapy of Cancer Cells

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Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgment

ANR P2N Mechanano is gratefully acknowledged. We gratefully thank Montpellier RIO imaging platform.

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Received: June 17, 2014 Revised: July 28, 2014 Published online: