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ATP-Activatable Photosensitizer Enables Dual Fluorescence Imaging and Targeted Photodynamic Therapy of Tumor Yizhong Shen, Qian Tian, Yidan Sun, Jing-Juan Xu, Deju Ye, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017

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Analytical Chemistry

ATP-Activatable Photosensitizer Enables Dual Fluorescence Imaging and Targeted Photodynamic Therapy of Tumor Yizhong Shen, Qian Tian, Yidan Sun, Jing-Juan Xu, Deju Ye,* and Hong-Yuan Chen* State Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China ABSTRACT: Targeted delivery of intracellular stimuli-activatable photosensitizers (PSs) into tumor cells to achieve selective imaging and on-demand photodynamic therapy (PDT) of tumors has provided a vital opportunity for precise cancer diagnosis and therapy. In this paper, we report a tumor targeting and adenosine triphosphate (ATP)-activatable nanophotosensitizer AptHyNP/BHQ2 by modifying hybrid micellar nanoparticles with both nucleolin-targeting aptamer AS1411 and quencher BHQ2labeled ATP-binding aptamer BHQ2-ATP-apt. We demonstrated that both the fluorescence emissions at 555 and 627 nm were quenched by BHQ2 in Apt-HyNP/BHQ2, resulting in low PDT capacity. After selective entry into tumor cells through nucleolinmediated endocytosis, the high concentration of intracellular ATP could bind to BHQ2-ATP-apt and trigger Apt-HyNP/BHQ2 dissociation, leading to turning “on” both fluorescence and PDT. The “off-on” fluorescence emissions at both 555 and 627 nm were successfully applied for dual color fluorescence imaging of endogenous ATP levels and real-time monitoring of intracellular activation of Apt-HyNP/BHQ2 in tumor cells. Moreover, imaging-guided precise PDT of tumors in living mice was also demonstrated, allowing for selective ablation of tumors without obvious side effects. This study highlights the potential of using a combination of tumor-targeting and ATP-binding aptamers to design ATP-activatable PSs for both fluorescence imaging and imaging-guided PDT of tumors in vivo.

mM) are distinctly different to that in extracellular environment (< 0.4 mM), and compared to normal cells, the ATP levels in many malignant tumor cells are also significantly elevated due to the increased glycolysis.27-29 This unique phenomena can provide an attractive opportunity to design ATPactivatable probes and ATP-responsive nanocarriers for sensitive imaging and selective treatment of tumors. For example, Noji et al. have taken the specific binding between ATP and the ε subunit of bacterial FoF1-ATP synthase to design several fluorescence resonance energy transfer (FRET)-based indicators for real-time measuring of ATP level in single HeLa cells.30 Zhu et al. have employed an aptamer-ATP binding mechanism to develop ATP-responsive mesoporous silica nanoparticles for targeted delivery and controlled release of anti-cancer drug doxorubicin (DOX), allowing for real-time monitoring of intracellular DOX release and selective cytotoxicity against HeLa cells.31 This strategy has been recently taken by Wang et al. to design activatable silver nanoclusters beacon for the detection of ATP in MCF-7 cells.32 Meanwhile, Ju et al. have utilized MoS2 nanoplates to design ATPactivatable PSs for the control of singlet oxygen (1O2) production, resulting in enhanced PDT of HeLa cells in vitro.33 Despite these progresses, targeted delivery of ATP-activatable PSs into tumor cells capable of specific turning “on” fluorescence and PDT with remarkable 1O2 generation is necessary for effective cancer theranostics in vivo, but remains unexplored. Herein, we reported the development of a new tumortargeting and ATP-activatable nanophotosensitizer Apt-

INTRODUCTION Stimuli-activatable photosensitizers (PSs) that show both enhanced fluorescence and photodynamic therapy (PDT) within tumor cells have offered a vital opportunity for real-time detection and specific therapy of tumors, which are highly demanded for precise cancer theranostics.1-5 Compared to traditional “always-on” PSs that are always fluorescent and phototoxic to nontarget tissues, activatable PSs can only show fluorescence and phototoxicity upon interaction to a specific stimuli in tumor cells.6-9 This can generally improve the sensitivity and specificity to distinguish tumor tissues from normal tissues.10-15 Moreover, the selective activation of PSs to control generation of reactive oxygen species (ROS) within tumor cells can be allowed to directly kill tumor cells with reduced side toxicity to normal tissues. Therefore, there are a myriad of activatable PSs that are response to different biological stimuli, including pH,11,16 biothiols,12,17-20 metal ions,10 and enzymes,2,3,21 have been actively reported, aiming to improve cancer treatment. An ideal activatable PS for tumor PDT generally possesses the characteristics, such as targeted delivery and accumulation in tumor cells, selective activation by intracellular biomolecules, weak fluorescence and low dark toxicity before activation, and strong fluorescence along with high phototoxicity after activation by the biomolecules. Adenosine 5'-triphosphate (ATP) is one of the most important biomolecules in living organisms, playing a crucial role in controlling various cellular functions and processes.22-26 It has been recognized that the intracellular ATP levels (1-10 1

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HyNP/BHQ2 by incorporating two different aptamers into a hybrid micellar nanoparticle, and demonstrate its high potential for fluorescence imaging of ATP levels and controllable PDT of tumors in vivo. The design of Apt-HyNP/BHQ2 was illustrated in Figure 1a, consisting of an amine-functionalized hybrid semiconductor QDs micellar nanoparticle NH2-HyNP, a nucleolin-targeting aptamer AS1411, a BHQ2-labeled ATPbinding aptamer BHQ2-ATP-apt, and a complementary oligonucleotide c-TA for ATP-apt. The NH2-HyNP was used because of its efficient fluorescence emission at both 555 nm and 627 nm, enabling high sensitivity for detection and imaging of ATP both in vitro and in vivo. More importantly, NH2-HyNP possessed a high 1O2 generation quantum yield (~0.91) under light irradiation,34 ensuring remarkable 1O2 production to induce effective PDT of cancer. Quencher BHQ2 was used because of its high absorption from 500 to 700 nm capable of efficiently quenching both the fluorescence emissions (555 and 627 nm, Figure S1) and 1O2 production of Apt-HyNP. AS1411 has been demonstrated as an efficient tumor-targeting aptamer capable of highly specific binding with nucleolins that are overexpressed on the surface of many tumor cells,35,36 allowing for selective delivery of Apt-HyNP/BHQ2 into tumor

cells. The application of BHQ2-ATP-apt as the ATPrecognitive aptamer was due to its high binding affinity with ATP,31,37 and the binding with ATP could trigger its dissociation from Apt-HyNP/BHQ2. We demonstrated that AptHyNP/BHQ2 was fluorescence and PDT “off” initially owing to the quenching effect of BHQ2. Upon entering into tumor tissues, the interaction between AS1411 and nucleolins could triggered efficient endocytosis and selective accumulation of Apt-HyNP/BHQ2 in the lysosomes of tumor cells (Figure 1b). The abundant intracellular ATP could specifically bind to the BHQ2-ATP-apt and thus trigger the release from AptHyNP/BHQ2, liberating Apt-HyNP with remarkable recovery of both fluorescence emissions (555 and 627 nm) and 1O2 production capacity. The activatable fluorescence emissions were successfully applied for dual color fluorescence imaging of ATP in tumor cells. Guided by the fluorescence imaging, irradiation of tumor cells to trigger remarkable 1O2 generation was conducted, resulting in rapid lysosome rupture and ultimate cell death both in vitro and in vivo. As a result, AptHyNP/BHQ2 could serve as a tumor-targeting and ATPactivatable PS with improved tumor selectivity for imagingguided precise PDT of tumors.

Figure 1. General design of tumor-targeting and ATP-activatable photosensitizer Apt-HyNP/BHQ2 for fluorescence imaging and PDT of tumors. (a) Illustration of the procedure for the synthesis of Apt-HyNP/BHQ2. (b) Schematic illustration of the mechanism of action of Apt-HyNP/BHQ2 for nucleolin-mediated endocytosis and intracellular ATP-activated fluorescence and PDT for tumor imaging and PDT.

HyNP/BHQ2 and 2.0 mM free AS1411 aptamer at 37.0 °C for 2 h. To investigate intracellular ATP-dependent activation of fluorescence, HeLa cells were incubated with 2.0 µM AptHyNP/BHQ2 and 100 mM IAA at 37.0 °C for 2 h. After incubation, the cells were washed with cold PBS (pH 7.4) three times, and the epifluorescence images were acquired under an IX73 optical microscope (Olympus, Japan). For colocalization assay, the cells were washed with cold PBS (pH 7.4) three times, and further incubated with 1.0 µM Lyso-Tracker@Red DND-99 at 37.0 °C for 20 min. The medium was replaced with fresh culture medium containing 2.0 µg/mL Hoechst

EXPERIMENTAL SECTION Synthesis. The preparation of Apt-HyNP/BHQ2 is described in the Supporting Information. Fluorescence Imaging of ATP in Cancer Cell Using AptHyNP/BHQ2. ~100K of HeLa cells and NIH 3T3 cells were seeded into 35.0 mm confocal dishes (Glass Bottom Dish) and incubated at 37.0 °C for 24 h. The medium was then replaced with fresh culture medium containing 2.0 µM AptHyNP/BHQ2. To investigate the nucleolin-dependent cell uptake, HeLa cells were co-incubated with 2.0 µM Apt2


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Analytical Chemistry 33342 and incubated at 37.0 °C for another 20 min. Then, the cells were washed with PBS for three times again, and the fluorescence images were acquired under an IX73 optical microscope (Olympus, Japan). The fluorescence of R6G in AptHyNP/BHQ2 was acquired with excitation at 530 to 550 nm, and emission from 570 to 600 nm. The fluorescence of TMPyP-Zn-QDs in Apt-HyNP/BHQ2 was acquired with excitation at 430 nm, and emission collected from 600 to 650 nm. Hoechst 33342 was excited at 340 to 390 nm, and emission was collected from 420 to 460 nm. Lyso-Tracker@Red DND99 was excited at 540 to 580 nm, and the emission was collected from 600 to 650 nm. Fluorescence Imaging of Intracellular 1O2 Levels. The intracellular 1O2 levels upon light irradiation were examined using a cell permeable 1O2 indicator DCFH-DA. HeLa cells in 35.0 mm Glass bottom dishes were incubated with 2.0 µM Apt-HyNP/BHQ2 under dark for 2 h. The medium was then removed, washed with fresh medium once, and 1.0 mL of fresh medium containing DCFH-DA (30.0 µM) was then added into the cells. After incubation at 37 °C for 20 min, the cells were irradiated with white light (LED lamp, 400 nm long pass filter, 20 mW/cm2) for 180 s. After irradiation, the cells were washed with 1×PBS (pH 7.4) three times, and the fluorescence images were acquired with FITC and R6G filters. Fluorescence Imaging of Tumor with Apt-HyNP/BHQ2 in Vivo. HeLa tumor-bearing female nude mice were i.v. injected with 200 µM Apt-HyNP/BHQ2 in 100 µL saline. The in vivo fluorescence imaging was then performed using an IVIS Lumina XR III in vivo Imaging System (excitation, 540 nm; emission, 620 nm long pass) at 0, 0.5, 1, 2, 4, 12, 24, 48, 96 and 120 h post injection. The tumor fluorescence intensities were quantified by region of interest (ROI) measurement using living image software (PerkinElmer, MA, USA). Tumor PDT with Apt-HyNP/BHQ2 in Vivo. PDT experiments in vivo were performed in HeLa tumor-bearing mice. After the tumor volume grew to ~120 mm3 in female nude mice, 12 mice were randomly divided into four groups shown as group 1 (saline only), group 2 (saline + light irradiation), group 3 (Apt-HyNP/BHQ2 only), and group 4 (AptHyNP/BHQ2 + light irradiation). For groups 1 and 2, 100.0 µL saline was directly injected into tumor in each mouse. For groups 3 and 4, 200.0 µM Apt-HyNP/BHQ2 in 100.0 µL saline was injected into tumor in each mouse. After 4 h, mice in groups 2 and 4 were irradiated with a xenon lamp (400 nm long pass filter, 120 mW/cm2) for two consecutive exposures of 15 min each, with a 20-min interval between the two irradiations. The tumor volume and body weight in each mouse were measured in every two days, and monitored up for 18 days. The relative tumor volumes were calculated for each mouse as V/V0 (V0 was the tumor volume when the treatment was initiated), and the relative body weight (m) was normalized to that of initial treatment. Statistical Analysis. Results were expressed as mean ± SD unless otherwise stated. Statistical comparisons between two groups were determined by Student’s t test. P < 0.05 was considered to be statistical significance. The statistical tests were run with Prism 6 (Prism GraphPad Software, Inc., San Diego).

Synthesis and Characterization of Apt-HyNP/BHQ2. We firstly synthesized the hybrid nanoparticle NH2-HyNP through encapsulating pre-assembled TMPyP-Zn-QD nanocomplexes and rhodamine 6G (R6G) with DSPE-PEG2000-OMe and DSPE-PEG2000-NH2. Dynamic light scattering (DLS) analysis showed that NH2-HyNP possessed monodisperse size, with an average hydrodynamic diameter of ~46.0 nm (Figure S2a). A mixture of aptamer AS1411 and oligonucleotide c-TA at a molar ratio of 1 : 9 were then covalently conjugated to the surface of NH2-HyNP to afford the aptamer-functionalized nanoparticle (Apt-HyNP). Apt-HyNP showed reduced zeta potential (-18.2 ± 1.4 mV) compared to that of NH2-HyNP (5.3 ± 1.1 mV) due to the presence of negative oligonucleotides (Figure S2b), which was also demonstrated by the agarose gel electrophoresis analysis (Figure S2c). Finally, BHQ2-ATP-apt was introduced to the surface of AptHyNP to prepare Apt-HyNP/BHQ2 through the specific complementary hybridization with c-TA. Figure 2a showed that Apt-HyNP/BHQ2 exhibited enhanced absorption ranging from 500 to 700 nm due to the incorporation of BHQ2 into Apt-HyNP. As expected, both the fluorescence emissions at 555 nm (R6G) and 627 nm (TMPyP-Zn-QDs) in Apt-HyNP/BHQ2 were dramatically quenched by BHQ2 (Figure 2b), confirming that BHQ2-ATPapt was successfully introduced into Apt-HyNP/BHQ2. The presence of BHQ2-ATP-apt on the surface of AptHyNP/BHQ2 led to a more netative zeta potential (-38.6 ± 1.1 mV) in relative to that of Apt-HyNP (-18.2 ± 1.4 mV) (Figure S2b). DLS analysis demonstrated that the average hydrodynamic size of Apt-HyNP/BHQ2 was around 58.0 nm in acqueous solution (Figure 2c), which was also revealed by transmission electron microscopic (TEM) analysis (Figure 2d). Notably, Apt-HyNP/BHQ2 was very stable under physiologically relevant conditons, with little change of hydrodynamic size after incubation in acqueous solution for over 5 days (Figure S3).

Figure 2. (a) Absorption spectra of Apt-HyNP (red) or AptHyNP/BHQ2 (black) in PBS buffer (pH 7.4). (b) Fluorescence spectra of 6.5 µM of Apt-HyNP (red) or Apt-HyNP/BHQ2 (black) in PBS buffer (λex=443 nm). (c) DLS of Apt-HyNP/BHQ2 in H2O. (d) TEM image of Apt-HyNP/BHQ2 stained with uranyl acetate.

RESULTS AND DISCUSSION 3



Figure 3. (a, b) Fluorescence spectra (a) and time-dependent fluorescence intensity at 627 nm (b) of 6.5 µM Apt-HyNP/BHQ2 in the absence or presence of 2.0 mM ATP in PBS buffer (pH 7.4). (c) Fluorescence spectra of 6.5 µM Apt-HyNP/BHQ2 following incubation with varying concentration of ATP (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, and 2.2 mM) in PBS buffer for 60 min. (d) The relationship corresponding to the fluorescence enhancement (F/F0) at 627 nm vs. the concentration of ATP ranging from 0 to 2.2 mM. Insert: the linear relationship between F/F0 at 627 nm and the concentrations of ATP. (e, f) Fluorescence spectra (e) and fluorescence enhancement (F/F0) at 627 nm(f) of 6.5 µM Apt-HyNP/BHQ2 following incubation with 2.0 mM ATP, TTP, CTP, GTP or UTP in PBS buffer for 60 min. F0 is the initial fluorescence intensity of Apt-HyNP/BHQ2 at 627 nm, and F is the fluorescence intensity of AptHyNP/BHQ2 at 627 nm following incubation with indicated analyses. All the fluorescence experiments were performed with the excitation at 443 nm.

ranging from 0–1.4 mM (Figure 3d and S5). The detection limit was found to be ~0.3 µM (signal-to-noise, S/N=3), which was comparable to other reported methods.38-41 The selectivity for ATP over other thymidine triphosphate analogues was also examined by comparing the fluorescence intensity (627 nm) of Apt-HyNP/BHQ2 (6.5 µM) following incubation with 2.0 mM ATP, TTP, CTP, GTP or UTP for 60 min. As shown in Figures 3e and 3f, only ATP could activate Apt-HyNP/BHQ2 and cause a significant ~24-fold fluorescence enhancement. Neither TTP, CTP, GTP nor UTP could interact with Apt-HyNP/BHQ2 to induce obvious fluorescence turn-on, indicating a high specificity of AptHyNP/BHQ2 for ATP detection. ATP-Triggered Activation of Apt-HyNP/BHQ2 to Enhance 1O2 Generation. The 1O2 generation capacity of Apt-HyNP/BHQ2 under irradiation in the absence or presence of ATP in PBS buffer was firstly monitored using 2,2,6,6tetramethylpiperidine (TEMP) as a 1O2 trapper. TEMP could be oxidized by 1O2 to form 2,2,6,6-tetramethylpiperidine-Noxyl (TEMPO), leading to significantly enhanced electron spin resonance (ESR) signals.42 As shown in Figure 4a, irradiation of Apt-HyNP/BHQ2 in the absence of ATP with white light (400 nm long-pass filter) at a power of 20 mW/cm2 for 3 min showed only slight increased ESR signals, indicating very little 1O2 production. However, irradiation of Apt-HyNP/BHQ2 in the presence of ATP under the same conditions induced very strong ESR signals indicative of remarkable 1O2 production. We also found that the ESR signals could be completely quenched when sodium azide (NaN3), an

Fluorescence Response of Apt-HyNP/BHQ2 towards ATP. We first investigated the response of Apt-HyNP/BHQ2 towards ATP in PBS buffer (pH 7.4). As shown in Figure 3a, Apt-HyNP/BHQ2 (6.5 µM) displayed weak fluorescence emissions at both 555 and 627 nm initially. After incubation with ATP (2.0 mM) for 60 min, strong fluorescence emissions at both 555 and 627 nm were observed, which were similar to that of Apt-HyNP (Figure S4). The fluorescence turn-on ratio of Apt-HyNP/BHQ2 induced by ATP at 555 and 627 nm was found to be ~17-fold and ~24-fold, respectively. Timedependent activation of Apt-HyNP/BHQ2 induced by ATP was then monitored in PBS buffer using a microplate reader, which showed that the fluorescence intensity at 627 nm gradually increased following incubation with ATP (2.0 mM) (Figure 3b). The fluorescence intensity of Apt-HyNP/BHQ2 reached a plateau after 60 min. In contrast, the incubation of Apt-HyNP/BHQ2 in the absence of ATP did not cause obvious fluorescence enhancement. These results indicated that AptHyNP/BHQ2 was stable in PBS buffer, while ATP could efficiently activate it, resulting in a fast turn-on fluorescence. We then evaluated the sensitivity of Apt-HyNP/BHQ2 (6.5 µM) to detect ATP by measuring its fluorescence spectra after incubation with different concentration of ATP (0–2.2 mM) in PBS buffer for 60 min. Figure 3c demonstrated that both the fluorescence emissions of Apt-HyNP/BHQ2 at 555 and 627 nm increased with the increasing concentration of ATP, indicating an ATP concentration-dependent activation of Apt-HyNP/BHQ2. The fluorescence intensity at either 555 or 627 nm was linearly proportional to ATP concentration 4


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Analytical Chemistry established quencher of 1O2, was added (Figure S6). We further verified the ATP-triggered activation of AptHyNP/BHQ2 to generate 1O2 using singlet oxygen sensor green (SOSG) as a fluorescence indicator. SOSG could be oxidized by 1O2 to form fluorescent SOSG endoperoxide (SOSG-EP, λem=525 nm), providing another indirect method to report 1O2 levels.43 As shown in Figure 4b, irradiation of Apt-HyNP/BHQ2 with ATP could trigger the SOSG oxidization, resulting in remarkably enhanced fluorescence at 525 nm in relative to that of Apt-HyNP/BHQ2 without ATP, which agreed well with that indicated by TEMP (Figure 4a). The fluorescence intensity of SOSG caused by AptHyNP/BHQ2 in the presence of ATP increased with irradiation time, indicating a light dose-dependent production of 1O2 (Figure 4c). It was notable that both the enhanced ESR signals and fluorescence intensity of SOSG at 525 nm were similar to that induced by the unquenched Apt-HyNP after irradiation (Figure 4a and b). These results suggested that ATP could efficiently activate Apt-HyNP/BHQ2, resulting in a remarkable recovery of 1O2 generation capacity. Moreover, it was also observed that the 1O2 generation capacity of Apt-HyNP/BHQ2 after activation by ATP at pH ranging from 5 to 7.4 was nearly the same (Figure 4d and S7).

investigated its potential for the detection of intracellular ATP in living HeLa cells. To optimize the incubation conditions, HeLa cells were incubated with varying concentration of AptHyNP/BHQ2 (0, 0.1, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 µM) for different times (0, 0.5, 1, 2, 4, 6 h), and the resulting intracellular fluorescence showed that Apt-HyNP/BHQ2 could light up HeLa cells in both concentration- and incubation time-dependent manners (Figure S8 & S9). The maximum intracellular fluorescence was observed after incubation with 2.0 µM of Apt-HyNP/BHQ2 for 2 h. Therefore, we selected the optimized incubation time of 2 h and concentration of 2.0 µM for the next studies.

Figure 5. Fluorescence images of HeLa cells incubated with 2.0 µM Apt-HyNP/BHQ2, 2.0 µM Apt-HyNP/BHQ2 + 2.0 mM AS1411 or 2.0 µM Apt-HyNP/BHQ2 + 100 mM IAA, and NIH 3T3 cells incubated with 2.0 µM Apt-HyNP/BHQ2 at 37 ºС for 2 h. Scale bar: 100 µm.

As shown in Figure 5, HeLa cells incubated with AptHyNP/BHQ2 (2.0 µM) at 37 ºC for 2 h exhibited strong intracellular fluorescence indicative of efficient cellular uptake and activation of Apt-HyNP/BHQ2. The intracellular fluorescence was significantly reduced in either HeLa cells pretreated with free AS1411 aptamer or in nucleolin-deficient NIH 3T3 cells.35,36 There was also much weaker fluorescence observed in HeLa cells after incubation with Apt-HyNP/BHQ2 (2.0 µM) at 4 ºC for 2 h (Figure S10). These results suggested that Apt-HyNP/BHQ2 could enter into HeLa cells via nucleolin-mediated endocytosis, resulting in bright intracellular fluorescence co-localized well with fluorescence from lysosomal tracker staining (Figure S11). To further demonstrate the selective activation of AptHyNP/BHQ2 by intracellular ATP, HeLa cells were pretreated with iodoacetic acid (IAA) that could inhibit glycolysis and thus reduce intracellular ATP concentrations,29,44 and then incubated with Apt-HyNP/BHQ2 (2.0 µM) for 2 h. We found that the intracellular fluorescence was significantly reduced by IAA, revealing that Apt-HyNP/BHQ2 was activated by intracellular ATP (Figure 5). Notably, we also found that both the fluorescence emissions of R6G and TMPyP-Zn-QDs in Apt-HyNP/BHQ2 were concomitantly activated by ATP, displaying an extensive overlap of green (R6G) and red (TMPyP-Zn-QDs) fluorescence distributed mainly in the lysosomes of HeLa cells (Figure S12). These results indicated that Apt-HyNP/BHQ2 was feasible for the dual color

Figure 4. (a) ESR spectra of TEMP (60 mM) with 6.5 µM of AptHyNP (red), Apt-HyNP/BHQ2 (black), or Apt-HyNP/BHQ2 together with 2.0 mM ATP (blue) in PBS buffer after irradiation with white light for 3 min. (b) The fluorescence intensity of SOSG (20.0 µM, λex/em=488/525 nm) in the presence of 6.5 µM of Apt-HyNP (red), Apt-HyNP/BHQ2 (black), or Apt-HyNP/BHQ2 together with 2.0 mM ATP (blue) in PBS buffer following irradiation with (Irradiated) or without (Non-irradiated) white light for 3 min. (c) Normalized fluorescence intensity and fluorescence images (insert) of 20.0 µM SOSG in the presence of 6.5 µM Apt-HyNP/BHQ2 + 2.0 mM ATP following continuous irradiation with white light for 0, 45, 90, 135 and 180 s. (d) Effect of pH on the fluorescence enhancement (∆ F= FLight - FDark at 525 nm) of 20.0 µM SOSG in the presence of 6.5 µM AptHyNP/BHQ2 + 2.0 mM ATP following irradiation white light for 3 min.

Fluorescence Imaging of ATP in Living Tumor Cells using Apt-HyNP/BHQ2. Having demonstrated the enhanced fluorescence of Apt-HyNP/BHQ2 in response to ATP, we then 5



fluorescence imaging of intracellular ATP in nucleolinpositive tumor cells. Activation of 1O2 Production to Induce Efficient PDT against HeLa Cells. The ability of ATP to activate AptHyNP/BHQ2 and control 1O2 generation in living HeLa cells was next investigated using 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) as an indicator. As shown in Figure 6, HeLa cells incubated with 2.0 µM Apt-HyNP/BHQ2 for 2 h but without irradiation with white light displayed the activated fluorescence of Apt-HyNP/BHQ2 only, indicating little 1O2 production. In contrast, irradiation of Apt-HyNP/BHQ2-load HeLa cells with white light for 3 min produced strong intracellular DCF fluorescence, which indicated that a large amount of 1O2 was generated. The intracellular DCF fluorescence could be abrogated by Vitamin C (VC), a known 1 O2 scavenger.15 Flow cytometric analysis of AptHyNP/BHQ2-treated HeLa cells showed a gradual increase in DCF fluorescence with prolonged irradiation time, indicating that the intracellular 1O2 levels were dependent on light dose. (Figure S13).

S1), but remarkable cell collapse and obvious blebs formation indicative of significant cell death were observed in AptHyNP/BHQ2-treated HeLa cells upon irradiation with white light for only 18 s (Movie S2). These results revealed that AptHyNP/BHQ2 hold a high ability to be activated by endogenous ATP and thus initiated efficient PDT against HeLa cells. Real-time Fluorescence Monitoring of ATP Levels in HeLa Tumors. The ability of Apt-HyNP/BHQ2 for real-time fluorescence imaging of ATP in HeLa tumor bearing mice was next examined. Apt-HyNP/BHQ2 (200 µM, 100 µL) was directly injected into HeLa tumors in living mice, and longitudinal whole-body fluorescence images were acquired at different time. As shown in Figure 7a & 7b, HeLa tumors injected with Apt-HyNP/BHQ2 were gradually lit up, and the fluorescence intensity reached the maximum at 4 h postinjection. The strong fluorescence in tumors could last for over 24 h, and most nanoparticles were then cleared out from tumors after 5 days. The strong fluorescence in HeLa tumors induced by Apt-HyNP/BHQ2 at 4 h was also confirmed by fluorescence imaging of HeLa tumor tissue slices ex vivo. We found that the tumor tissue slices resected from mice injected with Apt-HyNP/BHQ2 showed significantly enhanced intracellular fluorescence, indicating that Apt-HyNP/BHQ2 could efficiently enter into HeLa cells and activated by intracellular ATP after injection into HeLa tumors in living mice (Figure 7c). ATP-Activated PDT of HeLa Tumors in Living Mice. Guided by the fluorescence imaging results, we finally applied Apt-HyNP/BHQ2 to trigger in vivo PDT of HeLa tumors in living mice. HeLa tumors bearing mice received intratumoral injection of either saline or Apt-HyNP/BHQ2 (200 µM, 100 µL). After 4 h, half of the tumors were irradiated with a white light (400 nm long pass filter, 120 mW/cm2) for two consecutive exposures of 15 min each, and the other tumors left were not irradiated. The body weight and tumor size in each mouse were monitored in every two days, and continued for 18 days. As shown in Figures 8a and 8c, mice treated with either saline, saline with light irradiation, or Apt-HyNP/BHQ2 alone showed similar tumor growth rate, and the average tumor volume at day 18 was significantly ~18-fold greater than that at day 0. In sharp contrast, mice treated with AptHyNP/BHQ2 and light irradiation showed a remarkable reduction of tumor volume. The tumors were completely eliminated after 18 days, indicating that Apt-HyNP/BHQ2 was highly efficient to induce tumor PDT in living mice. Additionally, the relative body weight of mice was not obviously different among all the four groups (Figure 8b). These results suggested that Apt-HyNP/BHQ2 had low in vivo toxicity in dark, but exerted significant antitumor activity under light irradiation. To further verify the in vivo PDT efficiency of AptHyNP/BHQ2, hematoxylin-eosin (H&E) staining of tumor tissues resected at 24 h post irradiation was then conducted. Figure 8d showed that HeLa tumors treated with both AptHyNP/BHQ2 and light irradiation exhibited significant cell death in tumor tissues as compared to other three control groups. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining results also revealed a remarkable population of apoptotic cells in Apt-HyNPs/BHQ2 and light irradiation-treated tumor tissues, while there was

Figure 6. Fluorescence images of HeLa cells incubated with 2.0 µM Apt-HyNP/BHQ2 (pseudo red) and DCFH-DA (30.0 µM, pseudo green), following irradiation with white light (400 nm long pass filter, 20 mW/cm2) for 3 min, light with VC (2.5 mM) or without light. Scale bar: 100 µm. Red arrows showed the cell collapse after light irradiation.

With the enhanced 1O2 production induced by ATP and AptHyNP/BHQ2 in HeLa cells, we subsequently examined the PDT activity using MTT assay. The results showed that AptHyNP/BHQ2 exhibited negligible cytotoxicity in dark, with little cell death observed in HeLa cells following treatment with 2.0 µM of Apt-HyNP/BHQ2 for 24 h (Figure S14). In contrast, irradiation of Apt-HyNP/BHQ2-treated HeLa cells with white light (400 nm long pass filter, 20 mW/cm2) displayed concentrationand light dose-dependent cytotoxicity. When HeLa cells were treated with 2.0 µM of Apt-HyNP/BHQ2, nearly all cells were dead after irradiation for 180 s, which was also confirmed by Annexin V-/propidium iodide (PI) staining.45 More than 92% HeLa cells were apoptosis after treatment with Apt-HyNP/BHQ2 and light irradiation, while HeLa cells treated with either AptHyNP/BHQ2 or light irradiation only were little apoptosis/necrosis (Figure S15). The significant cell death was also examined by real-time monitoring of the morphology of Apt-HyNP/BHQ2-treated HeLa cells during light irradiation. It was found that continuous irradiation of blank HeLa cells for over 50 s could not induce cell morphology change (Movie 6


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Analytical Chemistry negligible cell apoptosis in tumor tissues following treatment with either saline, saline with irradiation, or Apt-HyNPs/BHQ2

without irradiation.

Figure 7. In vivo tumor imaging of HeLa tumor-bearing mice with Apt-HyNP/BHQ2. (a) Real-time in vivo fluorescence images of HeLa tumor-bearing mice after intratumoral injection of Apt-HyNP/BHQ2 for different times. Red arrows indicate the location of tumors in mice. (b) Quantitative analysis of the relative fluorescence intensity of tumor after injection of Apt-HyNP/BHQ2 for different times. Values are mean ± SD (n = 3). (c) Ex vivo fluorescence images of tumor tissue slices resected from HeLa tumor-bearing mice 4 h after injection with Apt-HyNP/BHQ2 and stained with DAPI. The blue fluorescence was acquired from 420 nm–460 nm upon excitation at DAPI channel, and the red fluorescence was acquired from 570 nm–600 nm upon excitation at R6G channel. Scale bar: 50 µm.

development of a tumor-targeting and ATP-activatable nanophotosensitizer Apt-HyNP/BHQ2, and demonstrated its utility for dual color fluorescence imaging and targeted PDT of tumors in vivo. We demonstrated that Apt-HyNP/BHQ2 could selectively enter into tumor cells via nucleolin-mediated endocytosis and specifically interact with endogenous ATP, resulting in efficient activation of both fluorescence and PDT efficacy. Apt-HyNP/BHQ2 was successfully applied for the detection of ATP levels both in living tumor cells and in vivo. Moreover, guided by the fluorescence imaging, selective and effective PDT of tumors was achieved, allowing for the complete removal of tumors under light irradiation. These results revealed that the design of tumor-targeting and ATPactivatable PSs hold great promise for fluorescence imaging and targeted PDT of tumors in terms of improved sensitivity and specificity. However, it should be mentioned that the procedure for the preparation of Apt-HyNP/BHQ2 was too tedious and difficult, and the cost of both aptamers were very high, which might present a substantial limitation for clinical applications. The design of more straightforward systems through using tumor-targeted small ligand (e.g., folic acid, RGD peptide) and/or other intracellular biomoleculeactivatable substrates may be desirable for precise theranostics of tumors in the future.

Figure 8. In vivo PDT of HeLa tumor with Apt-HyNP/BHQ2. (a, b) Changes in tumor volume (a) and body weight (b) in living mice following four indicated treatments. Values are mean ± SD (n=3, *** P < 0.001). (c) Photographs of HeLa tumor-bearing mice before (0 day) and 18 days after treatment with four indicated treatments. Red arrows indicate the location of tumors in mice. (d) H&E staining (left) and TUNEL staining (right) of HeLa tumor tissue slices resected from mice following four indicated treatments. Scale bars: 100 µm.

ASSOCIATED CONTENT Supporting Information Supplementary Figures, synthetic procedures and nanocharacterization. The Supporting Information is available free of charge on the ACS Publications website.

CONCLUSIONS

AUTHOR INFORMATION

In conclusion, we have employed two different aptamers to functionalize hybrid micellar nanoparticles for the

Corresponding Authors 7



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* Tel/Fax: +86-25-89681905; E-mail: [email protected]. * Tel/Fax: +86-25-89684862; E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thanked Prof. Yin Ding for her help with in vivo fluorescence acquirement. Financial supports from the National Natural Science Foundation of China (21775071, 21505070, 21327902 and 21632008), Natural Science Foundation of Jiangsu Province (BK20150567) and the Foundation Research Funds for the Central Universities (020514380096) were acknowledged.

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