Near-Infrared AIE Dots for Image-Guided Photodynamic

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Far red/near infrared AIE dots for imageguided photodynamic cancer cell ablation Guangxue Feng, Wenbo Wu, Shidang Xu, and Bin Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06136 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on July 29, 2016

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ACS Applied Materials & Interfaces

Far red/near infrared AIE dots for image-guided photodynamic cancer cell ablation Guangxue Feng,†§ Wenbo Wu,†‡§ Shidang Xu† and Bin Liu†∥* †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering Drive 4, 117585, Singapore ‡

Department of Materials Science and Engineering, National University of Singapore, 7

Engineering Drive 1, 117574, Singapore

∥

Institute of Materials Research and Engineering (IMRE), 2 Fusionopolis Way, Innovis, 136834,

Singapore KEYWORDS: Photodynamic therapy, bioimaging, nanoparticles, aggregation-induced emission, theranostics.

ABSTRACT

We report a facile encapsulation approach to realize bright far red/near infrared (FR/NIR) fluorescence and efficient singlet oxygen (1O2) production of organic fluorogens with aggregation-induced emission (AIEgen) and intramolecular charge transfer (ICT) characteristics for image-guided photodynamic cancer cell ablation. The synthesized AIEgen BTPEAQ possesses donor-acceptor-donor structure, which shows bright fluorescence in solid state. Due to the strong ICT effect, BTPEAQ exhibits poor emission with almost no 1O2 generation in aqueous

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solution. Encapsulation of BTPEAQ by DSPE-PEG block co-polymer yields polymer-shelled dots which show enhanced brightness with a fluorescence quantum yield of 3.9%, and a 1O2 quantum yield of 38%. While upon encapsulation by silica, the formed SiO2-shelled dots show much improved fluorescence quantum yield of 12.1% but with no obvious 1O2 generation. This study clearly demonstrates the importance of encapsulation approach for organic fluorophores, which not only affects the brightness but also the 1O2 production. After conjugating the polymershelled AIE dots with cRGD peptide, the obtained BTPEAQ-cRGD dots show excellent photoablation towards MDA-MB-231 cells with integrin overexpression while keeping control cells intact.

INTRODUCTION Fluorescence imaging has become an indispensable platform for biological researchers, which provides versatile direct visualization of biological species and biological processes.1-2 As a major class of fluorescent materials, small organic fluorophores have been widely developed and applied in biological imaging, especially for dyes with far red or near infrared (FR/NIR) emission, which benefit from high penetration depth and low biological autofluorescence interference.3-4 Among different strategies to red-shift the absorption and emission of small organic dyes, the introduction of donor-acceptor (D-A) structure has been one of the most successful approaches.5 However, due to the strong intramolecular charge transfer (ICT) characteristics associated with the D-A system, these fluorophores usually exhibit strong fluorescence in non-polar solvents, but show largely quenched or even annihilated fluorescence in aqueous media.6-7 Moreover, encapsulation of these small organic dyes in non-polar interior of nano-capsules or nano-carriers does not work well, due to the aggregation-caused quenching


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(ACQ) effects.8 These limitations have largely affected the bioimaging applications of small organic dyes especially for those with FR/NIR emission. Moreover, this ICT process can not only quench the fluorescence, but also lead to reduced singlet oxygen (1O2) species generation for most organic photosensitizers (PSs),9 largely affecting their efficacy in photodynamic cancer cell ablation. In 2001, Tang et al. developed a new class of organic fluorogens which is exactly opposite to conventional organic dyes suffering from ACQ effects.10-11 These novel fluorogens showed very weak emission when molecularly dispersed in good solvent but can be induced to emit strong fluorescence upon aggregation in poor solvents or solid state, which are well-known as fluorogens with aggregation-induced emission (AIEgens).12-13 AIEgens usually possess nonplanar propeller-like structures, where the restriction of their intramolecular motions such as rotation or vibration will block non-radiative pathways, leading to largely intensified fluorescence.14 The development of AIEgens has opened new opportunities for biological sensing and imaging applications.15-17 Very recently, new AIEgens are reported to show strong photosensitizing ability under light irradiation.18-21 Different from conventional PSs which show largely decreased 1O2 generation upon aggregate formation, these AIE PSs still exhibit strong 1

O2 generation in aggregated state.22-25 Moreover, along with the development, many AIEgens

with red and FR/NIR emission have been developed for in vivo applications, which are mainly synthesized through the incorporation of D-A systems.26-27 As a consequence, the competition between the AIE and ICT effects determines the final brightness and 1O2 generation of AIEgens in aqueous media. For most of them, the negligible fluorescence and poor 1O2 production in aqueous solution largely compromise the final imaging and photodynamic therapy performance.


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In this contribution, we demonstrate a facile approach to bring back the bright fluorescence and high 1O2 generation in aqueous media for fluorogens with strong ICT characteristics, making them suitable for image-guided photodynamic cancer cell ablation. Taking BTPEAQ as an example, with two tetraphenylethene (TPE) peripheries as the donor and 9,10-anthraquinone (AQ) segment as the centered acceptor, it shows negligible emission when molecularly dispersed in THF but emits strong fluorescence in solid state. In addition, BTPEAQ showed largely decreased fluorescence along with the increase of solvent polarity, where negligible fluorescence was observed in aqueous solution even when they formed nanoaggregates. We used matrix encapsulation approach to fabricate two different AIE dots, polymer-shelled dots using DSPEPEG as the matrix, and SiO2-shelled dots with solid silica as the shell, respectively. As compared to pure BTPEAQ nanoaggregates in water, polymer-shelled and SiO2-shelled dots showed much improved brightness with fluorescence quantum yields of 3.9% and 12.1%, respectively. Moreover, along with its enhanced brightness, polymer-shelled dots also exhibited enhanced 1O2 generation ability with 1O2 quantum yield of 38%, while pure BTPEAQ aggregates and SiO2shelled dots showed weak 1O2 production. The polymer-shelled dots were further decorated with tumor targeting peptide, cRGD, for image-guided photodynamic ablation of integrin overexpressed cancer cells. RESULTS AND DISCUSSION


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Figure 1. A) Chemical structure of BTPEAQ, B) photographs of BTPEAQ in THF solution (left), or solid state (right) under UV (365 nm) lamp illustration. C) Absorption and D) PL spectra of BTPEAQ in solvents with different polarities. The synthetic route to BTPEAQ (Figure 1A) is shown in Scheme S1 in supporting information (SI), and its structure is confirmed by NMR (Figures S1 and S2, SI) and high resolution mass spectroscopy (Figure S3). BTPEAQ possesses two tetraphenylethene (TPE) peripheries as the electron donor, and 9,10-anthraquinone (AQ) center as the electron acceptor. When molecularly dispersed in solution, such as THF, it showed negligible fluorescence, while at solid state, it emitted bright orange emission under UV lamp (Figure 1B), indicating that the synthesized BPTEAQ is AIE-active. Possessing donor-acceptor-donor (D-A-D) structures, BTPEAQ exhibited strong ICT characteristics, which was revealed by measuring its absorption and photoluminescence (PL) spectra in solvents with different polarities. As shown in Figure 1C,


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changing the dielectric constant (ε) of solvent does not lead to much change of the absorption spectrum of BTPEAQ, where the absorption maximum is located at 335 nm. However, BTPEAQ exhibited remarkable bright fluorescence in solvents with low ε, such as Hexane (ε = 2.0) or Toluene (ε = 2.4), but negligible emission in tetrahydrofuran (THF) (ε = 7.5) or dichloromethane (DCM) (ε = 9.1). Moreover, when dispersed in water (ε = 80.4), because of ICT characteristics, BTPEAQ should show very weak emission, but as it is AIE-active, weak but detectable fluorescence can still be observed in aqueous solution (Figure 1D). We further confirmed its AIE property by dissolving BTPEAQ in water/THF mixture with different water fractions (Figure S4). Along with the increased water fraction, BTPEAQ showed intensified fluorescence with intensity enhancement of over 40-fold. The results clearly indicate that the AIE and ICT effects are competing with each other, while ICT dominates the fluorescence of BTPEAQ in water, leading to weak fluorescence.

Scheme 1. Schematic illustration of polymer and SiO2 AIE dots.


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To realize the bright fluorescence of BTPEAQ in aqueous solution, we designed nanoencapsulation approach to fabricate BTPEAQ based AIE dots. Encapsulation of BTPEAQ in constrained space not only restricts its molecular motion to realize AIE effects, but also provides non-polar microenvironment to reduce the ICT caused fluorescence quenching. Two different encapsulation matrices (polymer and silica) are used to fabricate BTPEAQ based AIE dots (Scheme 1). Firstly, polymer-shelled dots were fabricated via a nano-precipitation method.28 Block copolymer, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-Mal) was chosen as the encapsulation matrix due to its excellent encapsulation performance and biocompatibility.29 Upon transferring BTPEAQ and DSPE-PEGMal from THF to aqueous phase, the hydrophobic parts including BTPEAQ and DSPE segment will tangle with each other to form the core, while PEG chain will extend outside to form the protective shells. BTPEAQ SiO2-shelled dots were grown using triblock polymer, poly(ethylene oxide)-b-poly(propylene oxide)-b-poly-(ethyleneoxide) (F127), as the template via one-pot reaction according to our previous report.30-31 F127/BTPEAQ THF mixture was dried under nitrogen flow, which was further dissolved in hydrochloride solution under sonication. The micelles formed simultaneously under sonication, where the poly(propylene oxide) (PPO) segments and BTPEAQ form the core, while hydrophilic poly(ethylene oxide) (PEO) forms the shell, allowing for silica shell growth through tetraethyl orthosilicate (TEOS) hydrolysis. Silica shell growth was terminated by dimethyldimethoxysilane (DMDMS), and the SiO2-shelled dots were obtained through dialysis against water to remove excess reagents and then by passing through a 0.2 µm syringe filter.


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Figure 2. A) Size distribution and B) TEM image of polymer-shelled dots, C) size distribution and D) TEM image of SiO2-shelled dots. E) Absorption and PL spectra of BTPEAQ AIE dots. F) Fluorescence decays at 650 nm of BTPEAQ based AIE polymer-shelled (black) and SiO2shelled (red) dots in aqueous solutions. Instrument response (IRF) (blue) was also indicated. The sizes of BTPEAQ loaded SiO2-shelled and polymer-shelled dots were accessed by dynamic light scattering (DLS) (Figures 2A and C), which revealed an average hydrodynamic diameter of ~7.5 nm for SiO2-shelled dots and ~28.2 nm for polymer-shelled dots, respectively. High resolution transmission electron microscopy (HR-TEM) images show the similar sizes, where both are black spherical dots (Figures 2B and D). Figure 2E shows their absorption and emission spectra in aqueous suspension. Both BTPEAQ dots exhibited the same absorption spectrum, where the peak is located at 335 nm with a shoulder around 430 nm, while the emission maximum locates at 650 nm. SiO2-shelled dots show 3-fold higher fluorescence intensity than polymer-shelled dots, both are much higher than pure BTPEAQ aggregates in


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aqueous solution. The fluorescence quantum yields (η) are measured to be 12.1% for SiO2shelled dots, 3.9% for polymer-shelled dots, and 0.3% for BTPEAQ aggregates, respectively, using 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran in methanol (QY = 43%) as the reference. The enhanced brightness of both AIE dots as compared to pure BTPEAQ aggregates should be attributed to the protection by the encapsulation matrices, which not only confines the free motion of BTPEAQ molecules, but more importantly suppresses their direct contact with water to reduce the ICT caused quenching effects. The fluorescence difference between polymer-shelled and SiO2-shelled dots indicates that SiO2 as the shell provides a more compact environment and improved isolation of BTPEAQ from water molecular. We further investigated the fluorescence lifetimes (τ) to understand the higher fluorescence quantum yield of SiO2-shelled dots than polymer-shelled dots. As reported, τ and η are related to the radiative decay rate (kr) and nonradiative decay rate (knr) through the following equations: η = kr/(kr+knr) and τ = 1/(kr+knr).32 kr is an intrinsic property of a fluorophore, which in general is kept constant. Therefore, τ and η are changing in the same direction, and mainly affected by the nonradiative pathway. Figure 2F shows the fluorescence decay curves for both AIE dots, where SiO2-shelled dots exhibit elongated fluorescence lifetimes of 1.25 ns as compared to polymershelled dots (0.84 ns) (Table S1, SI). The lifetime difference clearly indicates the suppression of the nonradiative decay pathways of BTPEAQ aggregates inside SiO2-shelled dots, which should be attributed to the reduced interaction with water and closer packing of BTPEAQ within the solid silica shell than that in the polymer shell. It is reported that lowering the energy gap (∆Est) between the lowest singlet excited state (S1) and the lowest triplet excited state (T1) promotes the efficient intersystem crossing (ISC) process, which helps increase the 1O2 generation ability.21 The time-dependent density functional theory


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(TD-DFT) calculation of BTPEAQ was performed by using a suite of Gaussian 03 program to study its ∆Est. As shown in Figure S5, the lowest unoccupied molecular orbital (LUMO) of BTPEAQ is dominated by the orbitals from the centre AQ part, while the electron clouds of the highest occupied molecular orbital (HOMO) are mainly located on TPE peripheries. The difference in the electron cloud distribution shows intrinsic ICT properties, which is consistent with the fluorescence titration results. In addition, the energy gap between LUMO and HOMO is around 0.13 ev, such a small ∆Est should efficient promote the ISC process, benefiting its 1O2 generation. 1.0

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Figure 3. Absorption spectra of pure BTPEAQ nanoaggregates (A), SiO2-shelled dots (B), and polymer-shelled dots (C) in the presence of ABDA under light irradiation. D) The degradation of


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ABDA by BTPEAQ along with increased irradiation time. A0 is the absorbance of ABDA before light irradiation, A is the absorbance of ABDA after light irradiation for designated time. The white light power is 100 mWcm-2 provided by LB-150 Cold Light Illumination. To access 1O2 quantum yield, 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA) that quickly decomposes with decreased absorbance upon interaction with 1O2 is used as the indicator. It was found that the pure BTPEAQ aggregates in aqueous solution showed negligible 1

O2 generation, as the ABDA indicator showed only slight decrease in absorbance (Figure 3A).

The diminished fluorescence and negligible 1O2 generation of BTPEAQ aggregates in aqueous solution should be contributed to the strong ICT characteristics of BTPEAQ. Upon encapsulation by different matrices, the situation is quite different. Negligible 1O2 production is found for SiO2-shelled dots (Figure 3B), while the polymer-shelled BTPEAQ dots showed dramatically enhanced 1O2 generation with rapid degradation of ABDA under white light irradiation (Figure 4C). Using commercially available photosensitizer Rose Bengal (RB) as reference, the 1O2 quantum yields for BTPEAQ aggregates, SiO2-shelled and polymer-shelled dots were calculated to be 2%, 4%, and 38%, respectively. To eliminate the possibility of trapping 1O2 by SiO2-shelled dots, we co-encapsulated ABDA and BTPEAQ in the same SiO2shelled dots. The light irradiation does not cause much decomposition of the co-encapsulated ABDA (Figure S6) and the ABDA decomposition rate is similar to Si-shelled dots, indicating Si shell is not able to trap 1O2 and SiO2-shelled dots have very poor 1O2 generation ability. As the fluorescence and 1O2 generation are competing processes, with longer fluorescence lifetime, SiO2-shelled dots showed bright fluorescence at the expense of the triplet state and 1O2 generation. As ICT, fluorescence and 1O2 are competing processes, the protection of BTPEAQ by polymer or SiO2 shells will reduced the ICT effects, thus the fluorescence and 1O2 generation


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of polymer- and SiO2-shelled dots are enhanced as compared to pure BTPEAQ aggregates. Moreover, due to more compact packing, SiO2-shelled dots showed longer fluorescence time, which give much brighter fluorescence but weaker 1O2 generation as compared to polymershelled dots. This clearly demonstrates the ability to modulate the optical properties and 1O2 generation of PSs by different encapsulation matrices. Moreover, RB showed quickly decreased absorbance under light irradiation (Figure S7), while the absorbance of BTPEAQ remains very stable, indicating the high photostability of BTPEAQ. The relatively high brightness and efficient 1O2 generation make the polymer-shelled dots a promising candidate for image-guided photodynamic therapy, while the largely enhanced brightness and minimized photo-toxicity suits SiO2–shelled dots for non-invasive bioimaging. After demonstrating the ability to control the brightness and 1O2 generation ability of BTPEAQ with different matrices, we selected polymer-shelled dots for image-guided photodynamic cancer cell ablation as a demonstration for their biological applications. Cyclic arginine−glycine−aspartic acid tripeptide (cRGD) that can specifically target αβ integrin that overexpressed on many cancer cell surface was conjugated to polymer-shelled dots to yield BTPEAQ-cRGD dots.23 BTPEAQ-cRGD dots showed almost the same fluorescence and 1O2 quantum yields as compared to polymer-shelled dots, indicating that the surface functionalization does not affect the optical properties of encapsulated BTPEAQ. MDA-MB-231 breast cancer cells with integrin overexpression were selected as the target, while NIH-3T3 fibroblast normal cells and HeLa cancer cells were chosen as negative controls. These cells were treated with BTPEAQ-cRGD dots for 2 h, and the fluorescence images were accessed by confocal laser scanning microscopy (CLSM). The confocal images show that bright red fluorescence originated from BTPEAQ-cRGD dots was clearly observed in the cytoplasm (nucleus stained with Hoechst


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33342 with blue emission) in MDA-MB-231 cells (Figure 4). While very weak red fluorescence can be found inside both NIH-3T3 and HeLa cells due to their low or lack expression of integrin. Collectively, the imaging results clearly demonstrate the high selectivity of BTPEAQ-cRGD dots for targeting MDA-MB-231 cancer cells.

Figure 4. Confocal images of MDA-MB-231 cells, NIH-3T3 and HeLa cells after incubation with BTPEAQ-cRGD dots for 2 h. The nuclei were stained with Hoechst 33342 (5 µg/mL, 10 min) to provide blue emission (B, E, H). All the images share the same scale bar of 30 µm. The killing effects of BTPEAQ-cRGD dots towards MDA-MB-231 breast cancer cells and NIH-3T3 normal fibroblast cells through photodynamic treatment were further evaluated by standard MTT assays. The BTPEAQ-cRGD dots showed very low dark toxicity towards both cell lines even at high concentration of 200 mg/L based on BTPEAQ mass concentration, where the viabilities of both cell are still over 90% (Figure 5A). However, large difference in cell


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viability was observed when PDT treatment was applied. After incubation of MDA-MB-231 cells with BTPEAQ-cRGD dots for 2 h followed by light irradiation (100 mWcm-2) for 10 min, the cell viability dramatically decreased along with increased incubation concentration (Figure 5B), where over 80% of cells are damaged at BTPEAQ concentration of 100 mg/mL while almost all cells are dead at BTPEAQ concentration of 200 mg/mL. On the other hand, the same treatment causes minimal effects to NIH-3T3 cells whose cell viability remains over 90% for all the experiments (Figure 5B).

Figure 5. Cell viabilities of BTPEAQ-cRGD dots treated MDA-MB-231 cells and NIH-3T3 cells with (A) or without (B) white light irradiation (100 mWcm-2, 10 min) provided by LB-150 Cold Light Illumination. (C) Live/dead staining of BTPEAQ-cRGD dots treated MDA-MB-231 cells with varied light irradiation times. The live cells were stained by fluorescein diacetate (green, 50 µg/mL for 10 min), while dead cells were stained by PI (red, 100 µg/mL for 10 min).


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We also performed the live/dead staining with fluorescein diacetate (green for live cells) and propidium iodide (red for dead cells), for direct visualization the PDT effects with different light irradiation time (Figure 5C). Without light treatment, obvious and bright green fluorescence with negligible red emission was observed from all MDA-MB-231 cells, indicating the excellent biocompatibility of the BTPEAQ-cRGD dots in dark. In contrast, along with the increase of light irradiation time, the population of red fluorescent cells increased at the expense of green emissive cells, indicating that the MDA-MB-231 cells can be effectively killed by BTPEAQcRGD dots upon light irradiation. These results further verify the excellent PDT therapeutic effect of BTPEAQ-cRGD under light irradiation in in vitro experiments. CONCLUSION In conclusion, we demonstrated an encapsulation approach to fabricate AIE dots to realize bright FR/NIR emission and efficient 1O2 generation of AIEgens with strong ICT characteristics and demonstrated its application in image-guided photodynamic cancer cell ablation. The new AIEgen BTPEAQ shows poor emission with almost no 1O2 production in aggregated state in aqueous solution due to the strong ICT characteristics attributed by its D-A-D structure. Encapsulated by DSPE-PEG-Mal block co-polymer, the resultant polymer-shelled AIE dots showed enhanced brightness with fluorescence quantum yield of 3.9%, and 1O2 quantum yield of 38%. While upon encapsulating BTPEAQ by silica shell, the formed SiO2-shelled dots showed much improved fluorescence quantum yield of 12.1% with negligible 1O2 generation. Moreover, the RGD functionalized polymer-shelled AIE dots showed excellent photo-ablation towards MDA-MB-231 cells, but not to NIH-3T3 cells. This study clearly demonstrates an efficient approach to enhance the brightness and control the 1O2 production of organic fluorophores and PSs, particularly those with D-A structures and strong ICT characteristics.


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EXPERIMENTAL SECTION Materials. DSPE-PEG-Mal was purchased from Laysan Bio Inc (AL, USA). Cyclic arginine−glycine−aspartic acid tripeptide (cRGD) was provided by GL Biochem Ltd (Shanghai, China). Compound S1 was prepared in our previous work, while its synthetic route was shown in Scheme S1. Compound S2 and all the other reagents were purchased from Sigma-Aldrich. Characterization. 1H spectra were measured on a Bruker AV 300 or 400 spectrometer in CD2Cl2 using tetramethylsilane (TMS; δ = 0) as internal reference. The UV-vis and PL spectra were measured on Shimadzu UV-1700and Perkin Elmer LS-55 spectrometer, respectively. Laser light scattering with Zetasizer Nano S (Malvern Instruments Ltd, Worcestershire, UK) was used to determine the hydrodynamic diameters of AIE dots. High-resolution transmission electron microscope (HR-TEM, JEM-2010F, JEOL, Japan) was used to study AIE dot morphologies. FluoTime 200 TCSPC fluorescence platform from Picoquant GmbH (Berlin, Germany) was used to study the fluorescence lifetime following the same procedures in our previous report.31 Synthesis of BTPEAQ. A mixture of compound S1 (96.0 mg, 0.22 mmol), S2 (36.6 mg, 0.10 mmol), potassium carbonate (303.6 mg, 2.2 mmol), 9 mL THF, 3 mL water and Pd(PPh3)4 (3 mol%) was carefully degassed and charged with nitrogen. Then the reaction mixture was stirred at 60 °C for 24 hours. After cooling to ambient temperature, the reaction was stopped by the addition of water, extracted with dichloromethane and washed with brine. The organic layer was dried over anhydrous magnesium sulfate and purified by column chromatography using nhexane/dichloromethane (v/v = 1/2) as eluent to afford BTPEAQ as red solid (81.1 mg, yield = 82.0 %). 1H NMR (400 MHz, CDCl3, 298 K),  (TMS, ppm): 3.75 (s, 6H, -OCH2-), 3.76 (s, 6H, -OCH2-), 6.63-6.69 (m, 6H, ArH), 6.93-7.19 (m, 24H, ArH), 7.51 (d, J = 8.4 Hz, 4H, ArH), 7.98 (m, 2H, ArH), 8.35 (d, J = 8.0 Hz, 2H, ArH), 8.51 (d, J = 1.6 Hz, 2H, ArH). 13C NMR (150


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MHz, CDCl3, 298 K),  (ppm): 182.90, 158.35, 158.21, 146.47, 145.28, 143.99, 141.05, 138.36, 136.13, 136.10, 134.00, 132.57, 132.14, 132.00, 131.81, 131.40, 127.96, 127.80, 126.51, 126.27, 125.16, 113.21, 113.03, 77.20, 76.98, 76.77, 55.07. HRMS (ESI), calcd for (C70H52N15O6): m/z [M+Na]+: 1011.3656; found: m/z 1011.3665. 1

O2 quantum yield measurement. The 1O2 quantum yield (η) of polymer-shelled and SiO2-

shelled BTPEAQ dots under light illumination was measured using ABDA as the indicator, and Rose Bengal (RB) as the standard reference. To conduct the experiment, ABDA is added into AIE dots or RB aqueous solution to make a working concentration of 10 µM. The mixture is exposed to white light (400-800nm, 100 mWcm-2) for designated time. The degradation of ABDA was access by the means of its absorbance changes at 378 nm. And the 1O2 quantum yield is calculated using the following equation:

1

O2 η of SiO2-shelled dots

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(1)

where KSi and KRB represent the decomposition rate constants of the photosensitizing process determined by the plot ln(Abs0/Abs) versus irradiation time, where Abs0 is the initial absorbance of ABDA, Abs is the ABDA absorbance at different irradiation time. ARB and ASi refer to the light absorbed by RB and SiO2-shelled dots, respectively, calculated by the integration of their absorption spectra from 400 to 800 nm, ηRB is the 1O2 η of RB, which is 75%. Preparation of Polymer-shelled dots. BTPEAQ (0.5 mg) and DSPE-PEG-Mal (1.0 mg) was dissolved in 1 mL THF to make a homogenous solution. The mixture was then added into 10 mL MilliQ water followed by 2 min ultrasound sonication with a microtip probe sonicator at 12 W output (XL2000, Misonix Incorporated, NY). The mixture was further stirred in dark in fume


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hood for THF evaporation at 600 rpm overnight, and concentrated by centrifugation with molecular cut-off of 30 kDa. To fabricate BTPEAQ-cRGD dots, cysteine modified cGRD peptide was treated with the obtained polymer-shelled dots at cRGD/DSPE-PEG-Mal molecular ratio of 1.5/1. After 4 h reaction, the unreacted cRGD is removed by dialysis against water with a membrane with molecular cut-off of 6-8 kDa for 3 days. Preparation of SiO2-shelled dots. BTPEAQ SiO2-shelled dots were synthesized according to previous reported experiments. Firstly, a 1.5 mL homogenous THF solution containing 100 mg of F127 and 0.5 mg of BTPEAQ was dried by gentle nitrogen flow to obtain a solid membrane at 20 mL glass vial bottom. 1.6 mL of 0.85 M HCl solution was then added into the solid residue, followed by 10 min sonication to obtain a homogeneous suspension of F127 micelles. Subsequently, 180 µL of TEOS was added into the suspension, and the mixture was further stirred at room temperature for 2 h. The silica shell growth was terminated by addition DMDMS (30 µL) to the mixture, which is kept stirring for another 24 h. The obtained SiO2-shelled dots were then dialysis against water using membrane with molecular cut-off of 12-14 kDa to remove HCl and unreacted reagents. The pure SiO2-shelled dots were obtained after purification through a 200 nm syringe filter. Confocal imaging. NIH-3T3 normal cells, HeLa and MDA-MB-231 breast cancer cells were cultured in 8-well chamber (LAB-TEK, Chambered Coverglass System) at 37 °C in humidified environment containing 5% CO2. After 80% confluence, the cell culture medium was replaced with BTPEAQ-cRGD dot suspended in culture medium at BTPEAQ concentration of 20 mg/L. After 2 h incubation, the cells were washed twice with 1XPBS and the nuclei were stained by Hoechst for 10 min to provide blue emission. After washing, the cells were immediately imaged by confocal laser scanning microscope (CLSM). The red fluorescence from BTPEAQ was


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acquired above 560 nm upon excitation at 488 nm. The blue fluorescence from Hoechst was obtained between 430 to 470 nm upon excitation at 405 nm. Cell ablation study. The viabilities of NIH-3T3 and MDA-MB-231 cells before and after photodynamic therapy were evaluated using methylthiazolyldiphenyltetrazolium bromide (MTT) assays. NIH-3T3 and MDA-MB-231 cells were seeded in 96-well plates (Costar, IL, USA) at an intensity of 4 × 104 cells/mL, respectively. After overnight culturing, the cells were treated with BTPEAQ-cRGD dots in DMEM suspension at various concentrations for 2 h. BTPEAQ-cRGD dot suspension was then replaced by fresh cell culture medium. The selected wells were exposed to light irradiation (100 mWcm-2, 10 min), and further cultured for 24 h. In the parallel experiment, both cell lines were treated with BTPEAQ-cRGD dots for 24 h in dark. The cells treated with light or under dark were then incubated with freshly prepared MTT solution (0.5 mg/mL, 100 µL/well) for 3 h. After removing MTT solution, 100 µL of filtered DMSO was added into each well to dissolve all the crystals formed. The cell viability was accessed by the means of MTT absorbance at 570 nm recorded using Microplate reader (Genios Tecan), where cells incubated with culture medium only was arbitrarily determined to have 100% cell viability. Live/dead cell staining. The MDA-MB-231 cells were seeded in 8-well chamber. After reaching 80% confluence, the cell culture medium was replaced by DMEM containing BTPEAQ-cRGD dots (20 mg/L based on BTPEAQ mass concentration). After 2 h incubation, the cells were washed and replaced with new DMEM. Selected cells were treated with light irradiation for designated time. After light treatment, the cells were then incubated with fluorescein diacetate (50 µg/mL) and PI (100 µg/mL) for 10 min each in sequence. After washing, the cells were imaged by CLSM. The green fluorescence from fluorescein diacetate is


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collected from 505 to 525 upon excitation at 488 nm, and the red fluorescence from PI is collected above 560 upon excitation at 543 nm.

ASSOCIATED CONTENT Supporting Information. The synthesis of the AIEgen in details, 1H and 13C NMR spectra, computational model result of BTPEAQ. Degradation of ABDA in ABDA/BTPEAQ coencapsulated SiO2-shelled dots, and degradation of ABDA by RB under light irradiation. The supporting information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions § These authors contributed equally to this work. ACKNOWLEDGMENT We thank the Singapore National Research Foundation (R-279-000-444-281, Institute of Materials Research and Engineering of Singapore (IMRE/14-8P1110) for financial support. REFERENCES (1). Weissleder, R.; Pittet, M. J., Imaging in the Era of Molecular Oncology. Nature 2008, 452, 580-589. (2)

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Fluorescence Imaging. Nat. Methods 2006, 3, 891-893.


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(20) Zhang, C.-J.; Hu, Q.; Feng, G.; Zhang, R.; Yuan, Y.; Lu, X.; Liu, B., Image-Guided Combination Chemotherapy and Photodynamic Therapy Using A Mitochondria-Targeted Molecular Probe with Aggregation-Induced Emission Characteristics. Chem. Sci. 2015, 6, 45804586. (21). Xu, S.; Yuan, Y.; Cai, X.; Zhang, C.-J.; Hu, F.; Liang, J.; Zhang, G.; Zhang, D.; Liu, B., Tuning the Singlet-Triplet Energy Gap: A Unique Approach to Efficient Photosensitizers with Aggregation-Induced Emission (AIE) Characteristics. Chem. Sci. 2015, 6, 5824-5830. (22) Feng, G.; Qin, W.; Hu, Q.; Tang, B. Z.; Liu, B., Cellular and Mitochondrial DualTargeted Organic Dots with Aggregation-Induced Emission Characteristics for Image-Guided Photodynamic Therapy. Adv. Healthcare Mater. 2015, 4, 2667-2676. (23) Yuan, Y.; Feng, G.; Qin, W.; Tang, B. Z.; Liu, B., Targeted and Image-Guided Photodynamic Cancer Therapy Based on Organic Nanoparticles with Aggregation-Induced Emission Characteristics. Chem. Commun. 2014, 50, 8757-8760. (24) Feng, G.; Yuan, Y.; Fang, H.; Zhang, R.; Xing, B.; Zhang, G.; Zhang, D.; Liu, B., A Light-Up Probe with Aggregation-Induced Emission Characteristics (AIE) for Selective Imaging, Naked-Eye Detection and Photodynamic Killing of Gram-Positive Bacteria. Chem. Commun. 2015, 51, 12490-12493. (25) Jin, G.; Feng, G.; Qin, W.; Tang, B. Z.; Liu, B.; Li, K., Multifunctional Organic Nanoparticles with Aggregation-Induced Emission (AIE) Characteristics for Targeted Photodynamic Therapy and RNA Interference Therapy. Chem. Commun. 2016, 52, 2752-2755.


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Near-Infrared AIE Dots for Image-Guided Photodynamic

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