Screening of Photosensitizers by ... - ACS Publications

Sep 2, 2016 - ... the formation dynamics of 1O2. 1O2-based chemiluminescence (CL) is a suitable method to directly monitor the generated amount of 1O2...
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Screening of Photosensitizers by Chemiluminescence Monitoring of Formation Dynamics of Singlet Oxygen during Photodynamic Therapy Fengjuan Zou, Wenjuan Zhou, Weijiang Guan, Chao Lu, and Ben Zhong Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02611 • Publication Date (Web): 02 Sep 2016 Downloaded from http://pubs.acs.org on September 3, 2016

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Screening of Photosensitizers by Chemiluminescence Monitoring of Formation Dynamics of Singlet Oxygen during Photodynamic Therapy Fengjuan Zou,† Wenjuan Zhou,† Weijiang Guan, † Chao Lu,*, † and Ben Zhong Tang‡



State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical

Technology, Beijing 100029, China ‡

Department of Chemistry, The Hong Kong University of Science & Technology, Clear Water

Bay, Kowloon, Hong Kong Fax/Tel.: +86 10 64411957. E−mail: [email protected]

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ABSTRACT: Finding an efficient photosensitizer is crucial in ensuring therapeutic effect of photodynamic treatment. Currently, screening of photosensitizers during photodynamic therapy is achieved by evaluating the total yield of singlet oxygen (1O2), rather than monitoring the formation dynamics of 1O2. 1O2-based chemiluminescence (CL) is a suitable method

to

directly

monitor

the

generated

amount

of

1

O2 .

Herein,

the

tetraphenylethene-sodium dodecyl sulfonate surfactant with aggregation-induced emission characteristics can remarkably amplify the intrinsic CL emission from 1O2 by integrating its micellar microenvironment with CL energy acceptor effect in a cage-like structure. We present a new luminescence platform for the rapid screening of photosensitizers by monitoring the formation dynamics of 1O2 during photodynamic therapy. This study will not only be critical in optimizing the irradiation time during photodynamic therapy, but also open a new door to the discovery of efficient photosensitizers.

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INTRODUCTION Photodynamic therapy (PDT) is a minimally invasive approach for theatment of various cancers and other diseases.1 In a typical PDT process, a photosensitizer upon photoirradiation in the aerobic condition can transfer energy to 3O2, which generates 1O2 to destroy tumors or diseased cells by chemical oxidation.2-5 Considering the fact that 1O2 is the ultimate cytotoxic agent required for effective PDT, we thought of its necessity to employ a probe for monitoring the formation dynamics of 1O2 in order to screen efficient photosensitizers. So far, a number of strategies have been attempted to monitor the production of 1O2.6-12 Much investigation has been carried out on fluorescence probes for 1O2 by taking advantage of the specific binding between them, but this lacks enough information involving in the formation dynamics of 1

O2.13-16 Therefore, development of innovative approaches for rapid evaluation of

photosensitizers by virtue of the real-time monitoring of 1O2 is one of the biggest challenges. Real-time monitoring of 1O2 with its intrinsic chemiluminescence (CL) is a specific and noninvasive method, providing the most direct information on the formation dynamics of 1O2. Unfortunately, this method suffers from typically weak luminescence signals with a cumbersome liquid N2-cooled InGaAs detector.17-20 Nowadays, several strategies have been implemented to amplify the weak intrinsic CL from 1O2. In general, cypridina luciferin analogs (CLA) and its derivatives are employed to improve the detection sensitivity for 1O2 by a redox reaction between CLA and 1O2.21 However, such an amplified CL-based detection of 1O2 is limited to the cross-interference from other reactive oxygen species. On the other hand, micellar microenvironment is often employed as a unique reaction medium to partially amplify the CL emission of reactive oxygen species.22-25 In addition, fluorescent molecules are used as CL energy acceptors of reactive oxygen species (ROS).26-29 However, the concentration of fluorescent dyes in solution is limited to a low level for preventing the notorious aggregation-caused quenching (ACQ) at high concentrations.30 Such a limitation of 3

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improvement of CL resonance energy transfer (CRET) efficiency between 1O2 donors and fluorescent molecules should be overcome by means of the aggregation-induced emission (AIE)-active fluorophores, which are generally non-fluorescent in solution but induced emission highly in the aggregated state or solid state.31-32 In this contribution, it was found that a tetraphenylethene-sodium dodecyl sulfonate anionic surfactant (TPE-SDS)33-34 with AIE characteristics could form a cage-like structure during the self-assembly process, improving the CRET efficiency between 1O2 donor and TPE-SDS acceptor by shortening the distance between 1O2 and TPE luminophor. Therefore, TPE-SDS can act as a micellar microenvironment and a CL energy acceptor to amplify the CL emission of 1O2 (Scheme 1). To the best of our knowledge, this is the first report that enables the real-time observation of the formation dynamics of 1O2 during PDT. Finally, the fabricated luminescence platform has been used for screening three photosensitizers (Rose Bengal, Rhodamine 101 and Riboflavin) by monitoring the formation dynamics of 1O2 using a light-emitting diode light source.

Scheme 1. Schematic illustration of the CRET-based probe for the real-time monitoring of the formation dynamics of 1O2 during PDT.

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EXPERIMENTAL SECTION Chemicals and Materials. All chemicals used in experiment were analytical grade and used without further purification. Sodium dodecyl sulfate (SDS) was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). 2,2,6,6-Tetramethylpiperidine (TEMP) and 9,10-anthracenediyl-bis(methylene)

dimalonic

acid

(ABDA)

were

purchased

from

Sigma-Aldrich Chemical Co. (St. Louis, MO, U.S.A.). Fluorescein sodium was purchased from Mackin Biochemical Co. Ltd. NaIO4, NaClO, FeSO4, ascorbic acid (AA), KO2, and ethanol were purchased from Beijing Chemical Reagent Company. Thiourea and sodium azide (NaN3) were purchased from Tianjin Fuchen Chemical Reagents Factory (Tianjin, China). Nitro blue tetrazolium chloride (NBT) was purchased from Nacalai Tesque Inc. (Tokyo Japan). 9,10-Diphenylanthracene (DPA) was obtained from Aldrich (Steinheim, Germany). Rose Bengal and Rhodamine 101 were purchased from Aladdin. Riboflavin was obtained Beijing HWRK Chem Co., LTD. (Beijing, China). TPE-SDS was synthesized by the procedures according to our previous work.33 Working solution of 0.2 M aqueous NaIO4 solution was prepared by dissolving solid NaIO4 in deionized water. 0.1 M aqueous H2O2 solution was prepared freshly by diluting 30% (v/v) H2O2 (Beijing Chemical Reagent Company, China) with deionized water. ClO− was derived from diluting NaClO solution in deionized water and the final concentration was determined by the UV-vis absorbance at 292 nm (ε = 350 M−1cm−1).35 Hydroxyl radical (•OH) was prepared from on-line Fenton reaction (Fe2+/H2O2=10) and its final concentration was determined by H2O2.35 The on-line reaction of H2O2-HCl and NaNO2 solutions was used to prepare peroxynitrite (ONOO−, 100 µM), and the final concentration was determined by the UV-vis absorbance at 302 nm (ε = 1670 M−1cm−1).35 Superoxide anion (O2•−) was prepared by dissolving KO2 in the anhydrous dimethyl sulfoxide solution and the final concentration was determined by the UV-vis absorbance at 550 nm (ε = 21.6 mM−1cm−1).36 1O2 was provided by the reaction between H2O2 5

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and NaIO4 and the final concentration was determined by H2O2.8 Deionized water (18.2 MU cm, Milli Q, Millipore, Barnstead, CA) was used for the preparation of all solutions throughout the experiments. The chemical structures of three photosensitizers (Rose Bengal, Rhodamine 101, Riboflavin), fluorescein sodium, SDS, and ABDA were shown in Figure S1. Apparatus. A biophysics chemiluminescence (BPCL) luminescence analyzer (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China) was used for the CL detection. The fluorescence spectra were carried out using a Hitachi F−7000 fluorescence spectrophotometer (Tokyo, Japan) at a slit of 5.0 nm with a scanning rate of 1500 nm/min. The CL spectra were obtained using an Edinburgh FLS 980 fluorescence spectrophotometer at a step of 2 nm and dwell of 0.100 s with the excitation lamp off. ESR measurements were performed on a JES-FA200 spectrometer (JEOL, Tokyo, Japan). The UV−visible spectra were acquired on a Shimadzu UV-2401 PC spectrophotometer (Tokyo, Japan). Mass spectrometry (MS) was carried out with a Quattro microtriple quadrupole mass spectrometer (Waters, USA). CL Measurements. CL signals in the presence of the different kinds of materials including fluorescein sodium, SDS, TPE-SDS, TPE, the mixture of fluorescein sodium and SDS, the mixture of TPE and SDS were measured. A volume of 100 µL material solution was mixed with 50 µL of NaIO4 solution (0.2 M) in a CL quartz vial adjacent to the photomultiplier tube (PMT). Then 50 µL of 0.1 M H2O2 solution was injected to the mixture, and the CL signal was detected by the PMT (−1000 V) and exported to the computer for data acquisition. The profile of CL intensity was analyzed by BPCL software combined with Windows 7 at an interval of 0.1 s. Computational Details. A combination of the hybrid B3LYP and the 6-31G(d) basis set was used to fully optimize the HOMO and LUMO energies of the TPE-SDS. The spectral simulation of excited-state characteristics was carried out by time-dependent density functional theory (TD-DFT) using optimized ground state geometries. According to 6

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calculations that TD-DFT in combination with the B3LYP hybrid functional method and the 6-31G(d) basis set, the error of Chemical

Trapping

of

△EST 1

values for TPE-SDS is less than 0.1 eV.10,37

O2

and

Mass

Spectrometry/Ultraviolet

spectrum

Measurement. DPA (60 mM), NaIO4 (0.2 M), and H2O2 (0.1 M) aqueous solution was mixed at room temperature and incubated in the dark for 1 h in a biphasic system composed of chloroform and H2O (1:3, v/v). For comparison, 60 mM DPA was added to the IO4−−H2O2 system in the presence of TPE-SDS (60 µM). The mass spectra of DPA and its endoperoxide (DPAO2) were recorded in the positive mode.38,39 H2O2 (0.1 M) was added to the mixture of and NaIO4 (0.2 M) and TPE-SDS (60 µM) solution in the presence of ABDA (20 µM). Then the absorbance of ABDA at 378 nm was recorded.7 For further comparison, we performed a controlled experiment by recording the absorbance of ABDA at 378 nm without adding NaIO4 and TPE-SDS. Monitoring the Formation Dynamics of 1O2 during PDT. Rose Bengal, Rhodamine 101 and Riboflavin were used as photosensitizers, a 532 nm light-emitting diode (LED) was used as the light source for 1O2 generation test for Rose Bengal and Rhodamine 101, while a 440 nm LED was used for Riboflavin samples.40-42 The irradiance of LEDs are 80 mW/cm2. The work voltage of CL detection was −950 V and the interval of the BPCL analyzer was set at 0.1 s per spectrum. Firstly, 1.25 mL of photosensitizer (50 µM ) was added into 1.25 mL of TPE-SDS aqueous solution (500 µM) in the quartz vial. After continuous O2 gas bubbling for several minutes until the amount of O2 reaching saturation, the light was turned on and the CL signals were measured in real time on a BPCL luminescence analyzer.

RESULTS AND DISCUSSION Remarkable CL Enhancement of TPE-SDS towards 1O2. The molecule structure of TPE-SDS was shown in Figure S2A. On the basis of time-dependent density functional theory, 7

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the energy gap (△EST value between S1 and T1 levels) of TPE-SDS was calculated to be 0.42 eV (Figure S2B). This small energy gap could overcome competitive non-radiative decay pathways, resulting in high photoluminescence efficiency of TPE-SDS.43 With these superior luminescence properties, TPE-SDS can be used for an ideal CL energy acceptor. The IO4−−H2O2 system was used to generate 1O2 in a static-injection CL setup (Figure S3).44 Note that the diagram of energy flow with 1O2 and 3O2 was displayed in Figure S421,45. As shown in Figure 1A, the TPE-SDS micelle solution could induce a significant increase in the CL intensity of IO4−−H2O2 system. In contrast, no remarkable CL enhancement was observed in the presence of tetraphenylethene (TPE, AIE fluorophore), SDS micelles and the mixture solution of TPE and SDS. On the other hand, it was observed that traditional fluorescence dyes (e.g. fluorescein sodium) as CRET acceptor could exhibit an obvious CL enhancement.46-48 Note that the enhancement effect was just slightly improved by the micellar microenvironment when fluorescein sodium was mixed with SDS. In addition, there was no change in fluorescence properties of fluorescein sodium (Figure S5), indicating little penetration of fluorescein sodium into the hydrophobic core of micelles. Therefore, the long distance between 1O2 donor and fluorescence dye acceptor resulted in the low CRET efficiency.49-53 The special structure of TPE luminophor in TPE-SDS makes it easy to form a cage-like structure when the concentrations of TPE-SDS are higher than its critical micelle concentration during the self-assembly process. Such a cage-like structure could facilitate the stabilization of 1O2.54 Generally, water penetration does not extend significantly beyond the first 2−4 methylene groups adjacent to the surfactant headgroup.55 More importantly, the mean travel distance of 1O2 in water (~ 200 nm) is much longer than the typical size of the TPE-SDS micelles (~10 nm).56 Therefore, 1O2 is easy to diffuse freely through the charged interfacial region to penetrate into the interior of the TPE-SDS micelles within its lifetime (4.4 8

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µs).57,58 It was concluded that the high CRET efficiency between 1O2 donor and TPE-SDS acceptor was achieved by shortening the distance between 1O2 and TPE luminophor (Figure 1B). On the other hand, in the IO4−−H2O2 system, the TPE-SDS micelles showed bright cyan fluorescence (λem = 498 nm) with a maximum excitation wavelength at 366 nm (Figure 1C). It was noted that 1O2 emitted light at about 362 nm in the SDS micelle solution (Figure 1D). The spectrum overlaps between the emission of 1O2 donor and the excitation of the TPE-SDS acceptor indicated the easy occurrence of CRET.59 The results showed that a new strong emission peak of the IO4−−H2O2 system appeared at about 510 nm in the presence of TPE-SDS micelles, corresponding to the emission of TPE luminophor (Figure 1C and 1D).

Figure 1. (A) CL intensity of the IO4−−H2O2 system in the presence of (a) H2O, (b) TPE, (c) SDS, (d) the mixture of TPE and SDS, (e) fluorescein sodium, (f) the mixture of fluorescein sodium and SDS, and (g) TPE-SDS micelles. (B) The location of 1O2 in the TPE-SDS micelles. (C) Excitation and emission spectra of TPE-SDS micelles. (D) CL spectra of the IO4−−H2O2 system in the presence of TPE-SDS and SDS. The concentrations of SDS, TPE, fluorescein sodium, TPE-SDS and 1O2 were 0.01 M, 60 µM, 60 µM, 500 µM, and 0.1 M, respectively. CL spectra were obtained using fluorescence spectrophotometer without an excitation light.

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Reactive Intermediates of the IO4−−H2O2 CL System in the Presence of TPE-SDS. To confirm the exact emitting species of the IO4−−H2O2 system in the presence of TPE-SDS, the effects of trapping agents for different ROS on the CL intensity were investigated. As shown in Figure S6, the CL intensity was totally quenched by the addition of 5.0 mM NaN3 (scavenger of 1O2), meaning that the strong CL was initiated from 1O2.60 5.0 mM thiourea (•OH scavenger) and 1.0 mM NBT (O2•− scavenger) could induce an obvious decrease in the CL intensity, which was due to the fact that a small number of •OH and O2•− generated in the IO4−−H2O2 system contributed to 1O2 generation.51 In addition, 1O2 could rapidly react with 9,10- diphenylanthracene (DPA) (kr = 1.3 × 10−6 M-1 s-1) to form a stable DPA endoperoxide (DPAO2).61 Therefore, the generated 1O2 in the IO4−−H2O2 system was further confirmed using the DPA chemical probe. The mass spectrum of DPA recorded in the positive mode exhibited a major [M]+ ion at m/z 330, corresponding to the positively charged molecular ion. In comparison, an intense [M+H]+ ion at m/z 363 (DPAO2) was observed in the IO4−−H2O2 system (Figure 2A and S7), meaning the formation of 1O2. Finally, electron spin resonance (ESR) spectra were also used as the most direct evidence to identify the generated 1O2 in the IO4−−H2O2 system. 2,2,6,6-Tetramethylpiperidine (TEMP) was employed to capture 1O2 to form 2,2,6,6-tetramethyl-4-piperidine-N-oxide (TEMPO) adduct, a stable nitroxide radical.[3] As shown in Figure 2B, the ESR signals showed 1:1:1 triplet signal (g = 2.003), which corresponded to those of TEMPO nitroxide radical. The ESR signal intensity of TEMPO was increased in the presence of TPE-SDS, indicating the high yield of 1O2 in the CL system. In addition,

a

high

1

O2

level

was

detected

in

the

CL

system

using

9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA) as a 1O2 indicator (Figure 2C).10 These results demonstrated that TPE-SDS micelles could stabilize and promote production of 1

O2 in the IO4−−H2O2 system.

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Figure 2. (A) Mass spectra of DPA (60 mM) and DPAO2. All solutions were incubated in the dark at room temperature for 1 h under stirring, in a biphasic system composed of chloroform and H2O (1:3, v/v). (B) ESR signals of TEMP−1O2 adduct generated in the IO4−−H2O2 system in the absence or presence of TPE-SDS. Microwave frequency: 9.055 GHz, power: 4.00 mW, modulation amplitude: 2.003 G, modulation frequency: 100 kHz. (C) Chemical trapping measurements of 1O2: UV absorption spectra of ADBA, ADBA in the IO4−−H2O2 system, and ABDA in the mixture of TPE-SDS and IO4−−H2O2.

Specific CL Response of TPE-SDS towards 1O2. Owing to the inherent energy transfer nature of 1O2, the TPE-SDS showed an excellent selectivity towards 1O2 through CRET. As depicted in Figure 3A, an ultrastrong CL emission of the IO4−−H2O2 system was induced in the presence of TPE-SDS. However, no obvious CL emission could be triggered by other ROS, including H2O2, •OH, O2•−, ClO−, and ONOO−. On the other hand, the fabricated CL platform exhibited good analytical performances for the detection of 1O2 with a wide linear range (1.0−100 µM), good reproducibility and stability (Figure S8).

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Screening of Photosensitizer by CL Monitoring of Formation Dynamics of 1O2 during PDT. Currently, almost all of the 1O2 indicators suffered depletion over time as a result of irreversible binding between them.6-12 Therefore, these methods could only provide the accumulation curve of 1O2 yield. Fortunately, the proposed CL probe for 1O2 could overcome this limitation. Furthermore, the present method exhibited very short response time, which facilitated the real-time monitoring of formation dynamics of 1O2 during PDT process. In order to simulate PDT in vitro, an LED light source was used to activate the photosensitizer with a selected wavelength, which matches the absorption spectrum of photosensitizer.41,62 The formation dynamics of 1O2 during PDT process was detected with a monochromatic filter (490 nm) between the CL quartz cell and the photomultiplier tube (Figure 3B). In this assay, the TPE-SDS solution was firstly mixed with a photosensitizer (e.g., Rose Bengal), followed by the real-time CL signals collection under the corresponding LED irradiation. The 1O2-induced CL signals were verified by adding NaN3 into the mixture of Rose Bengal and TPE-SDS during PDT. As displayed in Figure 3C, the CL intensity at 490 nm was kept at a continuous low-level after adding NaN3 under continuous irradiation for even 20 min. Furthermore, no CL signals were collected under a saturated N2 environment, while the remarkable CL signals were observed under O2 environment (saturated O2 and air). These results demonstrated that the CL emission of the proposed probe was originated from 1

O2 in the photosensitizing process. Note that no CL signals appeared without photosensitizers

(Figure S9). O2 was bubbled into a mixture of TPE-SDS and Rose Bengal after the continuous 20 min irradiation. It was found that the similar kinetics curve could be obtained (Figure S10), demonstrating the reusability of the probe in probing 1O2. Therefore, the proposed probe can be used for the real-time monitoring of the formation dynamics of 1O2 during PDT. The screening experiments were further carried out using three photosensitizers. The photosensitizers and LED irradiation had no obvious effect on the fluorescence properties of TPE-SDS (Figure S11−S13), displaying little bleaching of TPE-SDS during the 12

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photosensitizing process. Interestingly, the formation dynamics of 1O2 during PDT process was different for the tested three photosensitizers. The kinetics curves of three photosensitizers (Rose Bengal, Rhodamine 101 and Riboflavin) at different concentrations were investigated (Figure S14−S16). The CL signal in all of them rapidly reached a peak in the first three minutes, corresponding to a high yield of 1O2. More interestingly, it was noted that the produced 1O2 was extremely low in Rhodamine 101 and Riboflavin after about 10 min. However, for Rose Bengal, abundant 1O2 was continuously produced even under constantly irradiation for 20 min (Figure 3D). These results indicated that a suitable irradiation time during PDT should be considered for different photosensitizers, avoiding the damage of the normal tissues as a result of the long-time irradiation.

Figure 3. (A) CL intensity of the TPE-SDS in the presence of different ROS. The concentration of ROS was 100 µM. (B) CL configuration system for screening various photosensitizers using CRET-based TPE-SDS probe. (C) Kinetics curves for the formation dynamics of 1O2 in Rose Bengal under different oxygen conditions and in the presence of NaN3 (5.0 mM) in O2 atmosphere. (D) kinetics curves for the formation dynamics of 1O2 in different photosensitizers (each 50 µM). All experiments were performed at room temperature. The concentration of the TPE-SDS was 500 µM.

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CONCLUSIONS In summary, we have developed a luminescent screening method for photosensitizers by monitoring the formation dynamics of 1O2 during PDT. This approach presented herein makes a great breakthrough in successfully monitoring the real-time concentration of 1O2 generated during PDT, different from conventional systems that monitor the total yield of 1O2. We believe that the investigation of dynamic changes of 1O2 during PDT will help us to deeply understand the behaviors of photosensitizers, thereby providing valuable guidance for optimization of the irradiation time during PDT. We are currently attempting to refine our approach to include quantitative analyses of the formation dynamics of 1O2.

ASSOCIATED CONTENT Supporting Information Chemical structure of (A) Rose Bengal (B) Rhodamine 101 (C) Riboflavin (D) fluorescein sodium (E) ADBA, and (F) SDS; Chemical structure and HOMO–LUMO distributions of TPE-SDS; Schematic diagram of a static injection setup; Fluorescence spectra of fluorescein sodium before and after the addition of SDS; Diagram of energy flow with 1O2 and 3O2; The location of fluorescein sodium and 1O2 in SDS micellar microenvironment; Effects of ROS scavengers on the CL intensity of IO4––H2O2 system in the presence of TPE-SDS; Mass spectra of DPA and DPA in the IO4––H2O2 system; CL intensity of IO4––H2O2 system in presence of TPE-SDS by different concentrations of H2O2; Calibration curve for 1O2; CL signal of TPE-SDS under saturated O2; Reversibility of TPE-SDS for the real-time monitoring of 1O2 in Rose Bengal; Fluorescence spectra of TPE-SDS being mixed with Riboflavin before and after irradiation for 20 min; Fluorescence spectra of TPE-SDS being mixed with Rhodamine 101 before and after irradiation for 20 min; Fluorescence spectra of TPE-SDS being mixed with Rose Bengal before and after irradiation for 20 min; Kinetics curves for the 14

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formation dynamics of 1O2 in Rose Bengal with different concentrations; Kinetics curves for the formation dynamics of 1O2 in Rhodamine 101 with different concentrations; Kinetics curves for the formation dynamics of 1O2 in Riboflavin with different concentrations. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Fax/Tel.: +86 10 64411957. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Basic Research Program of China (973 Program, 2014CB932103), the National Natural Science Foundation of China (21575010 and 21375006), Innovation and Promotion Project of Beijing University of Chemical Technology, and the Innovation and Technology Commission (ITC-CNERC14S01).

REFERENCES (1) Celli, J. P.; Spring, B. Q.; Rizvi,I.; Evans, C. L.; Samkoe, K. S.; Verma, S.; Pogue, B. W.; Hasan, T. Chem. Rev. 2010, 110, 2795−2838. (2) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380−387. (3) Kovalev, D.; Fujii, M. Adv. Mater. 2005, 17, 2531−2544. (4) Ge, J. C.; Lan, M. H.; Zhou, B. J.; Liu, W. M.; Guo, L.; Wang, H. Q.; Jia, Y.; Niu, G. L.; Huang, X.; Zhou, H. Y.; Meng, X. M.; Wang, P. F.; Lee, C.-S.; Zhang, W. J.; Han, X. D. Nat. Commun. 2014, 5: 4596. 15

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