Two-Photon Imaging, Therapy and Drug Delivery. and Core ... · ┴ Institut Charles Gerhardt...

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Article

Mixed Periodic Mesoporous Organosilica Nanoparticlesand Core-Shell Systems, Application to in Vitro

Two-Photon Imaging, Therapy and Drug Delivery.Jonas Croissant, Damien Salles, Marie MAYNADIER, Olivier Mongin, Vincent

Hugues, Mireille Blanchard-Desce, Xavier Cattoën, Michel Wong Chi Man, AudreyGallud, Marcel Garcia, Magali Gary-Bobo, Laurence Raehm, and Jean-Olivier Durand

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Mixed Periodic Mesoporous Organosilica Nanoparticles and

Core-Shell Systems, Application to in Vitro Two-Photon Imaging,

Therapy and Drug Delivery.

Jonas Croissant,┴ Damien Salles, ┴ Marie Maynadier, ┬ Olivier Mongin, ╥ Vincent Hugues,§

Mireille Blanchard-Desce*,§ Xavier Cattoën,#┴ Michel Wong Chi Man, ┴ Audrey Gallud, ┬ Marcel Garcia, ┬ Magali Gary-Bobo*, ┬ Laurence Raehm, ┴ and Jean-Olivier Durand*.┴ ┴ Institut Charles Gerhardt Montpellier, UMR-5253 CNRS-UM2-ENSCM-UM1cc 1701, Place Eugène Bataillon F-34095 Montpellier cedex 05. (France) ╥ Institut Des Sciences Chimiques de Rennes, CNRS UMR 6226 Université Rennes 1 Campus Beaulieu F-35042 Rennes Cedex, (France) § Univ. Bordeaux, Institut des Sciences Moléculaires, UMR CNRS 5255, 351 Cours de la Libération, F-33405 Talence Cedex, (France) # Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble, France and CNRS, Inst NEEL, F-38042 Grenoble, France. ┬ Institut des Biomolécules Max Mousseron UMR 5247 CNRS; UM 1; UM 2 - Faculté de Pharmacie, 15 Avenue Charles Flahault, 34093 Montpellier Cedex 05, France. Keywords: Nanoparticles, Periodic Mesoporous Organosilica, Gold, Core-Shell, Cancer Nanomedicine.

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1. INTRODUCTION

Periodic mesoporous organosilica (PMO) hybrid organic-inorganic materials have

recently reached the nanoscale and are starting to be applied in the fields of nanotechnology.1-

6 PMO matrices are powerful tools constructed via sol-gel processes involving solely bis and

multi-organoalkoxysilane, without silica precursor (e.g. tetraethoxysilane), in order to obtain

the highest impact of the organic fragments on the properties of the hybrid material. PMO

nanoparticles (NPs) have been recently described from simple, low-molecular-weight

organosilane precursors.7 Using cetyltrimethylammonium bromide (CTAB) as the surfactant,

the group of Kuroda2 reported 20 nm diameter ethylene-based wormlike structured

monodisperse PMO NPs and the group of Huo5 prepared highly ordered PMO NPs based on

methane, ethane, ethylene, and benzene organic moieties. Another strategy consists of using a

silica NP core as the template and to condense the bridged organosilane precursor at the

surface of the silica NP to afford hollow organosilica NPs after etching the silica core.8 This

strategy allowed designing hollow PMO NPs for biological applications in vitro and in vivo,

9,10 and yolk-shell structures for catalysis applications.11 The last strategy consists of

combining a silica core with perfluorocarbon (FC-4) and CTAB dual surfactant systems.

Using this method, Qiao and co-workers obtained yolk-shell silica core PMO shell NPs for

catalytic application through incorporation of gold, platinum, and palladium NPs via removal

of the hard silica template.1 Among the next challenges in this field, the preparation of

metallic core PMO shell systems, and the formation of PMO NPs incorporating large

functional organic fragments appear very promising, notably for theranostic applications. We

recently reported biodegradable ethylene-disulfide based PMO NPs for efficient pH-triggered

drug delivery in cancer cells,12 and Jianli Shi et al. reported glutathione-responsive hollow

PMO NPs for high intensity focused ultrasound,13 and hollow PMO NPs for pH-triggered

drug and gene deliveries.14 Herein, we report two-photon-sensitive multifunctional PMO and

gold core PMO shell NPs for anti-cancer applications in vitro: two-photon fluorescence

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imaging, two-photon PDT, and synergistic drug delivery (see scheme 1). Such properties were

attained in the material via co-condensation of bis(triethoxysilyl)ethylene, or

bis(triethoxysilyl)benzene with high ratios and a previously reported two-photon

photosensitizer (2PS),15 leading to the so-called Mixed PMO (MPMO) NPs. Furthermore, the

gold core PMO/MPMO shell nanocarriers were fabricated in a remarkably efficient one-pot

process.

Scheme 1. TPE-nanotherapy application of PMO nanomaterials, such as Au@MPMO and

drug-loaded Au@MPMO NPs endocytosed in cancer cells (a and b respectively), TPE-PDT

(c), autonomous drug delivery (d), and TPE-PDT combined with synergistic drug delivery (e).

2. EXPERIMENTAL SECION

2.1 Materials. Tetraethoxysilane, cetyltrimethylammonium bromide (CTAB), sodium

hydroxide, dimethylsulfoxide, camptothecin, doxorubicin hydrochloride, and ammonium

nitrate were purchased from Sigma-Aldrich. Absolute ethanol was purchased from Fisher

Chemicals. Hydrochloric acid was purchased from Anal. R. Norma Pure. 1,2-

bis(triethoxysilyl)ethylene and 1,2-bis(triethoxysilyl)benzene were purchased from ABCR.

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2.2 Methods. Absorption spectra were recorded on a Hewlett-Packard 8453

spectrophotometer and fluorescence data were collected on a Perkin-Elmer LS55 fluorimeter.

Dynamic light scattering analysis were performed using a Cordouan Technologies DL 135

Particle size analyzer instrument. 29Si and 13C CPMAS solid state NMR sequences were

recorded with a VARIAN VNMRS300, using Q8MH8 and adamantane references

respectively. TEM images were recorded with a JEOL instrument. SEM images were

recorded with a FEI instrument. Energy dispersive spectroscopy was performed via an FEI

scanning electron microscope.

2.3 Syntheses of MPMO NPs

E2 MPMO NPs. A mixture of CTAB (250 mg, 6.86*10-1 mmol), distilled water (120 mL),

and sodium hydroxide (875 µL, 2 M) was stirred at 80°C for 50 minutes at 700 rpm in a 250

mL three necks round bottom flask. Then, 1,4-bis(triethoxysilyl)ethylene (1 mL, 2.63 mmol)

was added along with the 2PS (178 mg, 1.29*10-1 mmol, in 900 µL of anhydrous ethanol),

and the condensation process was conducted for 2 h. Afterwards, the solution was cooled to

room temperature while stirring; fractions were gathered in propylene tubes and the NPs were

collected by centrifugation during 15 minutes at 21 krpm. The sample was then extracted

twice with an alcoholic solution of ammonium nitrate (6 g.L-1), and washed three times with

ethanol, water, and ethanol. Each extraction involved a sonication step of 30 minutes at 50°C;

the collection was carried out in the same manner. The as-prepared material was dried for few

hours under vacuum.

B2 MPMO NPs. A mixture of CTAB (250 mg, 6.86*10-1 mmol), distilled water (120 mL),

and NaOH (875 µL, 2 M) was stirred at 80°C for 50 minutes at 700 rpm in a 250 mL three

necks round bottom flask. Then, 1,4-bis(triethoxysilyl)benzene (1 mL, 2.52 mmol) was added

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along with the 2PS (178 mg, 1.29*10-1 mmol, in 900 µL of anhydrous ethanol), and the

condensation process was conducted for 2 h. Afterwards, the solution was cooled to room

temperature while stirring; fractions were gathered in propylene tubes and the NPs were

collected by centrifugation during 15 minutes at 21 krpm. Extraction and the following steps

were identical as those described for E2 MPMO NPs.

AE PMO NPs. A mixture of water (25 mL), ethanol (10 mL), and CTAB (160 mg, 4.40*10-1

mmol) in a 50 mL three neck round bottom flask was stirred at 70°C. Then an aqueous

solution of potassium tetrachloroaurate (13 mg, 3.45*10-2 mmol in 1 mL) was injected, and

NaOH (100 µL, 2 M) was injected to induce the instantaneous nucleation of the nanoparticles.

After 30 seconds, hydrochloric acid (18 µL, 2 M) was added to the solution to produce 8 nm

monodisperse gold nanoparticles at lower pH, suitable for a controlled sol-gel process. The

nanoparticles growth was conducted for 12 minutes under a 600 rpm stirring, and the

temperature was set at 80°C. Afterwards, 1,2-bis(triethoxysilyl)ethene (100 µL, 2.62*10-1

mmol) was added dropwise to grow the porous PMO shell on the gold nanocrystals. The

condensation process was conducted for 1 h 30 minutes. Afterwards, the solution was cooled

to room temperature while stirring; fractions were gathered in propylene tubes and the NPs

were collected by centrifugation for 15 minutes at 21 krpm. Extraction and the following steps

were identical as those described for E2 MPMO NPs.

AB PMO NPs. A mixture of water (25 mL), ethanol (10 mL), and CTAB (160 mg, 4.40*10-1

mmol) in a 50 mL three neck round bottom flask was stirred at 70°C. Then an aqueous

solution of potassium tetrachloroaurate (13 mg, 3.45*10-2 mmol in 1 mL) was injected, and

NaOH (100 µL, 2 M) was injected to induce the instantaneous nucleation of the nanoparticles.

After 30 seconds, hydrochloric acid (18 µL, 2 M) was added to the solution to produce 8 nm

monodisperse gold nanoparticles at lower pH, suitable for a controlled sol-gel process. The

nanoparticles growth was conducted for 12 minutes under a 600 rpm stirring, and the

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temperature was set at 80°C. Afterwards, 1,4-bis(triethoxysilyl)benzene (100 µL, 2.52*10-1

mmol) was added dropwise, followed by sodium hydroxide (50 µL, 2 M) to grow the porous

PMO shell on the gold nanocrystals. The condensation process was conducted for 2 h.

Afterwards, the solution was cooled at room temperature while stirring; fractions were

gathered in propylene tubes and collected by centrifugation for 15 minutes at 21 krpm.

Extraction and the following steps were identical as those described for E2 MPMO NPs.

AE2 MPMO NPs. A mixture of water (25 mL), ethanol (10 mL), and CTAB (160 mg,

4.40*10-1 mmol) in a 50 mL three neck round bottom flask was stirred at 70°C. Then an

aqueous solution of potassium tetrachloroaurate (13 mg, 3.45*10-2 mmol in 1 mL) was

injected, and NaOH (100 µL, 2 M) was injected to induce the instantaneous nucleation of the

nanoparticles. After 30 seconds, hydrochloric acid (18 µL, 2 M) was added to the solution to

produce 8 nm monodisperse gold nanoparticles at lower pH, suitable for a controlled sol-gel

process. The nanoparticles growth was conducted for 12 minutes under a 600 rpm stirring,

and the temperature was set at 80°C. Afterwards, 1,4-bis(triethoxysilyl)benzene (100 µL,

2.62*10-1 mmol) and the 2PS (89 mg, 6.45*10-2 mmol, in 900 µL of anhydrous ethanol) were

added dropwise, followed by sodium hydroxide (50 µL, 2 M) to grow the porous PMO shell

on the gold nanocrystals. The condensation process was conducted for 2 h. Afterwards, the

solution was cooled to room temperature while stirring; fractions were gathered in propylene

tubes and collected by centrifugation during 15 minutes at 21 krpm. Extraction and the

following steps were identical as those described for E2 MPMO NPs.

AB2 MPMO NPs. A mixture of water (25 mL), ethanol (10 mL), and CTAB (160 mg,

4.40*10-1 mmol) in a 50 mL three neck round bottom flask was stirred at 70°C. Then an

aqueous solution of potassium tetrachloroaurate (13 mg, 3.45*10-2 mmol in 1 mL) was

injected, and NaOH (100 µL, 2 M) was injected to induce the instantaneous nucleation of the

nanoparticles. After 30 seconds, hydrochloric acid (18 µL, 2 M) was added to the solution to

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produce 8 nm monodisperse gold nanoparticles at lower pH, suitable for a controlled sol-gel

process. The nanoparticles growth was conducted for 12 minutes under a 600 rpm stirring,

and the temperature was set at 80°C. Afterwards, 1,4-bis(triethoxysilyl)benzene (100 µL,

2.52*10-1 mmol) and the 2PS (89 mg, 6.45*10-2 mmol, in 900 µL of anhydrous ethanol) were

added dropwise, followed by sodium hydroxide (50 µL, 2 M) to grow the porous PMO shell

on the gold nanocrystals. The condensation process was conducted for 2 h. Afterwards, the

solution was cooled to room temperature while stirring; fractions were gathered in propylene

tubes and collected by centrifugation during 15 minutes at 21 krpm. Extraction and the

following steps were identical as those described for E2 MPMO NPs.

2.4 Loading of MPMO NPs with anti-cancer drugs.

CAMPTOTHECIN LOADING IN PMO NPs. A mixture of dimethylsulfoxide (DMSO, 1

mL), camptothecin (3 mg), and PMO NPs (25 mg) was prepared in a 5 mL round bottom flask

and stirred at 40°C for two days. The preparation was centrifuged at 10000 rpm in 40 mL

propylene tubes, and the supernatant was removed. Then, the nanomaterial was washed once

with DMSO (without sonication), and twice with deionized water, and dried under vacuum.

DOXORUBICIN LOADING IN PMO NPs. A mixture of water (250 µL), doxorubicin (1

mg), and PMO NPs (10 mg) was prepared in an eppendorf tube, sonicated for 30 minutes, and

stirred overnight at room temperature. The preparation was centrifuged at 10000 rpm in 40

mL propylene tubes, and the supernatant was removed. The nanomaterial was washed twice

with water (10 mL), and dried under vacuum. The loading capacities were deduced by the

titration of doxorubicin in the supernatant fractions.

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3. RESULTS AND DISCUSSION

3.1 Syntheses of MPMO NPs

First, PMO nanomaterials were designed according to three strategies shown in

scheme 2. The 1,2-bis(triethoxysilyl)ethylene (E) or 1,4-bis(triethoxysilyl)benzene (B)

precursors were mixed with the 2PS to lead to E2 or B2 MPMO NPs respectively (Scheme

2a). This synthetic process involved a mixture of water/ethanol containing CTAB, and sodium

hydroxide as the catalyst (see ESI). The presence of EtOH allowed to solubilize the precursors

and to control the sol-gel condensation in order to obtain the NPs with desired size and

morphology. Core-shell Au@PMO NPs AE and AB were prepared in only 2 h at 80°C in one

pot. Au NPs were first generated in situ,16 and then bis(triethoxysilyl)ethylene (E) or

bis(triethoxysilyl)benzene (B), were condensed respectively (Scheme 2b). Again the presence

of EtOH was necessary to solubilize the precursors and to control the reaction. The same

procedure was used with 2PS mixed with bis(triethoxysilyl)ethylene (E) or

bis(triethoxysilyl)benzene (B) to synthesize core shell gold core@ethylene-2PS-based PMO

shell (AE2) or gold core@benzene-2PS-based PMO shell (AB2) respectively, which feature

gold cores and a mixed PMO shells (Scheme 2c). Besides, our syntheses of PMO NPs were

performed in only 2 h, which is remarkably fast compared to the few reported methodologies

(2 to 6 h of reaction, plus an additional 24 h of aging).5,17

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Scheme 2. Design of E2 or B2 Mixed PMO NPs, composed of the 2PS and either

bis(triethoxysilyl)ethylene (E), or bis(triethoxysilyl)benzene (B), respectively (a). One-pot

synthesis of AE or AB gold core PMO shell NPs, respectively composed of either the E or the

B moiety (b). One-pot synthesis of AE2 or AB2 gold core Mixed PMO shell NPs, composed

of either 2PS and E (AE2), or 2PS and B (AB2 NPs) (c).

3.2 Characterisations of MPMO NPs

The structures of MPMO NPs were readily visible with transmission electron

microscopy (TEM, Fig. 1). E2 and B2 MPMO NPs were respectively of 200 and 100 nm in

size, and monodisperse (Fig. 1a-b). Au@PMO NPs and Au@MPMO NPs were typically of a

hundred nanometers in diameter, and composed of a couple of 15 nm gold nanospheres in

their center (Fig. 1c-f). The mesoporous organization of the nanomaterial frameworks was

further confirmed by high magnification TEM micrographs (Fig. S1a-f). Scanning electron

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microscopy (SEM) images as well as dynamic light scattering (DLS) analyses displayed the

monodispersity of the aforementioned PMO NPs (Fig. S1g-l, and m-r respectively).

Figure 1. Characterization of the structure of the PMO NPs. a-f, Schematic representations

and TEM images of E2, B2, AE, AB, AE2, and AB2 NPs (a-f respectively).

Besides, the accessibility of the mesoporous structure of the MPMO NPs was

demonstrated by N2-adsorption and desorption technique, with high BET surface areas

ranging from 900 to 1100 m².g-1 (Fig. 2a-f). The BJH pore size distribution was generally

centered at 2.5 nm, which is also confirmed with the low angle peak in the X-ray

diffractograms (Fig. 2g-l). Note that E2 was structured in a 2D-hexagonal arrangement, as

revealed by the presence of harmonics from 4 to 5 degrees (2θ, CuKα) in the X-ray

diffractograms, and by the observation of parallel channels in the TEM image (Fig. S1a).

Interestingly, AE and AE2 materials synthesized from the same precursor (E) and surfactant,

but in the presence of gold cores exhibited a radial porosity, whereas B2, AB and AB2

possessed a worm-like porous framework (see Fig. S1b-f). It was observed from the panel of

characterizations than the ethylene moiety leads to better structured materials than the

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benzene ones. This is in agreement with the TEM images as well as the XRD measurements

which display significantly more distinct peaks for materials obtained with E (Fig. 2g,i,k) than

for those with B (Fig. 2h,j,l).

The chemical composition of the MPMO NPs was then characterized by multiple techniques.

On one hand, the solid state nuclear magnetic resonance (NMR) 29Si cross-polarization magic

angle spinning (CPMAS) spectra of the PMO NPs evidenced the formation of the siloxane

framework with the major proportion of the T2 and T3 signals (Fig. S2), while the ethylene (-

145 ppm), benzene (-134 ppm) and 2PS moieties were identified via NMR 13C CPMAS

spectra (see Fig. S3). UV-visible spectra also showed the 2PS absorption band (λmax≈ 390 nm)

of MPMO NPs (Fig. 2m,n,q,r), as well as the benzene groups (λmax≈ 265 nm) of B2, AB, and

AB2 NPs (Fig. 2n,p,r). On the other hand, the presence of gold NPs in AE, AB, AE2, and

AB2 PMO and MPMO NPs was clearly identified both by UV-visible absorption spectra (see

Fig. 2m-r) and by the wide angle XRD patterns (see Fig. S4), specifically through the peak at

38° corresponding to the 111 crystallographic planes, which is absent in E2 and B2 NPs (Fig.

S4a,b). Besides, high resolution TEM images of the gold cores inside AE2 and AB2

confirmed the crystalline structure of such nanoparticles with the typical 0.22 nm interatomic

distance in the gold NPs (Fig. S5). Inductively coupled plasma and energy dispersive

spectrometry analyses determined 0.8 to 4.6 wt% of gold in the NPs (Table S1). The 2PS

content of the NPs was determined by elemental analyses of nitrogen (14 atoms per 2PS

moiety) and found to be of 24 and 27 weight percent (wt%) respectively for E2 and B2 NPs,

as well as 42 and 46 wt% for AE2 and AB2 NPs respectively (see Table S3). Note that, the

overall preparation yields of the surfactant-free PMO and MPMO NPs were generally ranging

from 40 to 60% (see Table S2), though lower in AE2 (30-40%) and higher in B2 (80-95%).

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Figure 2. N2-adsorption-desorption isotherms (a-f), diffraction patterns at low angles (g-l), and UV-

visible spectra (m-r) of E2, B2, AE, AB, AE2, and AB2 NPs respectively.

The optical and photophysical properties of the MPMO NPs were then investigated. A

non-alkoxysilylated two-photon reference (2PS Ref) molecule6 was used (see structures and

absorption spectra of the 2PS and 2PS ref in Fig. S6a). The E2 and B2 MPMO NPs had

higher quantum yields than Au@MPMO NPs (Table S4), and higher bathochromic shifts of

the 2PS bands were observed with core-shell systems, reflecting higher aggregation states18

of the 2PS during the core-shell syntheses. The two-photon absorption cross-sections of the

PMO nanomaterials were measured and an important decrease of the σ2 compared to 2PS ref

was observed for MPMO and Au@MPMO nanosystems from 100 GM to less than 10 GM

per fluorophore (Fig. S6). Nevertheless, MPMO and Au@MPMO NPs were exploited for

TPE imaging and therapy of MCF-7 breast cancer cells.

3.3 Application of MPMO NPs in two-photon cancer imaging and therapy

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The two-photon-sensitive PMO nanomaterials library was then tested for two-photon

nanomedical applications. An in-vitro study was conducted on the MCF-7 breast cancer cell

line, and the laser excitation was performed with a Carl Zeiss two-photon confocal

microscope.19,20 First, the cellular uptake was assessed via two-photon fluorescence imaging

to track the nanocarriers. The cell walls were stained with CellMaskTM 15 minutes before the

imaging experiment. As displayed in Figure 3a-f, the two-photon confocal microscopy

confirmed the successful cellular-uptake, through efficient fluorescent properties of all PMO

NPs composed of the 2PS (Fig. 3a,b,e,f). Besides, the internalization of AE and AB NPs

possessing only the gold core was also detected, showing that these NPs were efficient for

TPE (Fig. 3c,d), which is probably due to the aggregation of some gold nanospheres in the

core thus leading to a plasmon resonance in the NIR and a behaviour typical of gold

nanorods.21

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Figure 3. Two-photon confocal microscopy images (λex = 760 nm) of incubated E2, B2, AE, AB,

AE2, and AB2 NPs (a-f respectively). Scale bars 10 µm. Demonstration of the synergistic effect of

two-photon photodynamic therapy (PDT) and drug delivery, either with the camptothecin (CPT) or the

doxorubicin (DOX) drugs, via drug-free and drug-loaded E2 and B2 NPs (g). Demonstration of the

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synergistic effect of two-photon photodynamic therapy (PDT) and drug delivery, via DOX-free

Au@PMO a,d Au@MPMO NPs, and DOX-loaded Au@MPMO NPs (h).

Drug-free PMO NPs were then studied for TPE-PDT under the same conditions. No

cancer cell killing was observed with E2 and only 15% of cancer cell death was observed with

B2 (Fig. 3g). However, when gold NPs were encapsulated in the MPMO matrices, AE2 and

AB2 showed 40% and 27% cancer cell killing respectively after irradiation (Fig. 3h). It is

noteworthy that AE and AB NPs showed no cancer cell death under the same conditions, thus

two-photon PDT attributable to the gold cores are not sufficient to kill cancer cells.

Furthermore two-photon photothermal effects have been shown to be negligible with gold

nanorods under two-photon excitation.22 Although we did not notice an enhancement of σ2 of

2PS in the presence of gold, a synergy between gold nanocrystals and 2PS moieties was

clearly operative, and produced an important enhancement of cancer cell death.

We then examined E2 and B2 MPMO NPs as vectors for camptothecin (CPT) and

doxorubicin (DOX) autonomous drug deliveries. The hydrophobic CPT was loaded in the

pores in DMSO, remained in them aqueous medium after loading, and was released in DMSO

(1.1-1.3 wt%, see Fig. S7). DOX release experiments were first carried out at pH 7 in water,

with doxorubicin-loaded MPMO and Au@MPMO NPs (Fig. S8). No release of DOX was

observed in these conditions showing that important interactions between the drug and the

organosilica matrix occurred.12 The release was triggered at pH 5.5 (lysosomal pH), and all

nanocarriers had high loading capacities of DOX, typically of 10 wt% (Table S5). With non-

irradiated MCF-7 cancer cells, incubated MPMO NPs showed efficient cancer cell killing

with CPT and DOX, reaching up to 45% with DOX-loaded MPMO NPs (Fig. 3g, see white

bars).

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Finally, the unprecedented combined action of drug delivery and TPE-PDT on cancer

cells was studied for MPMO NPs loaded with DOX. Interestingly, an important synergy of

these therapeutic features was noticed for MPMO, as more cell death was observed for all the

irradiated cells than without irradiation, with up to 58 and 76% cancer cell killing with

E2+DOX and B2+DOX respectively. Concerning core-shell MPMO NPs composed of

inserted 2PS moieties inside the framework, the efficiency of the materials was further

increased up to 76% cancer cell death with both AE2 and AB2. Hence, the most promising

synergistic treatment is obtained with the AE2 nanocarrier since it provided both an efficient

two-photon spatiotemporally-controlled cell killing (40% without drug), while the TPE-

enhanced DOX delivery further decreased the cell survival.

4. CONCLUSION

In summary, a library of PMO NPs was described for the first time for two-photon-

triggered nanomedicine. MPMO and Au@MPMO NPs were designed in efficient one-pot

processes of 2 to 3 h. The PMO nanomaterials showed remarkably high specific surface areas,

which made them suitable for drug transportation. All the NPs were endocytosed by MCF-7

cancer cells, as shown by TPE fluorescence imaging at low laser power. Based on these

results, an unprecedented dual therapeutic approach was designed using drug-loaded MPMO

and Au@MPMO NPs which led to synergistic cancer cell killing by TPE-PDT combined with

drug delivery. Autonomous DOX delivery was tailored without irradiation, and 76% of

synergistic cell death was obtained via TPE-PDT and enhanced-DOX delivery. Therefore the

library of NPs herein described are highly promising for theranostic applications with an

excellent spatiotemporal accuracy.23

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ASSOCIATED CONTENT

Supporting Information

Characterizations of the prepared nanoPMOs, high magnification TEM, SEM, DLS, solid-

state NMR, X-rays, elemental analyses, photophysical properties, drug release, two-photon

experimental settings. “This material is available free of charge via the Internet at

http://pubs.acs.org.”

AUTHOR INFORMATION

Corresponding authors

*Fax: +33-467-143-852, E-mails: [email protected], [email protected], [email protected].

ACKNOWLEDGEMENTS

We thank ANR P2N Mechanano for funding. Technological support from the Rio Imaging Platform is gratefully acknowledged. MBD gratefully acknowledges Conseil Regional d’Aquitaine (CRA) for financial support. VH received a fellowship from CRA. D. Cot is acknowledged for the SEM imaging, and X. Dumail for his help in the synthesis of 2PS. REFERENCES

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