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Engineering of ATP-Powered Photosensitizer for Targeted Recycling Activatable Imaging of MicroRNA and Controllable Cascade Amplification Photodynamic Therapy Yizhong Shen, Tingting Wu, Qian Tian, Yu Mao, Junjie Hu, Xiliang Luo, Yingwang Ye, Hong-Yuan Chen, and Jing-Juan Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01692 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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Analytical Chemistry

Engineering of ATP-Powered Photosensitizer for Targeted Recycling Activatable Imaging of MicroRNA and Controllable Cascade Amplification Photodynamic Therapy Yizhong Shen,a Tingting Wu,a Qian Tian,b Yu Mao,a Junjie Hu,b Xiliang Luo,c Yingwang Ye,a * HongYuan Chen,b and Jing-Juan Xub * a

Engineering Research Center of Bio-Process, Ministry of Education, School of Food & Biological Engineering, Hefei University of Technology, Hefei, 230009, China b State Key Laboratory of Analytical Chemistry for Life Sciences and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 c Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China ABSTRACT: Owing to the low abundance of microRNAs (miRNAs) in living tumor cells, the development of intracellular cancerrelevant miRNAs stimuli-activatable photosensitizers (PSs) for accurate imaging and efficient photodynamic therapy (PDT) of tumors in vivo is extremely challenging. Herein, we engineered a tumor targeting and intracellular trace miRNA-activatable nanophotosensitizer Y-motif/FA@HyNPs based on endogenous ATP-powered strand-displacement cascade amplification strategy, which was prepared by assembly of quencher BHQ2-labeled Y-motif DNA structure (containing ATP-binding aptamer and target miRNA-binding complementary sequence) on the surface of folate (FA) and amine functionalized hybrid micellar nanoparticles. We showed that the fluorescence emissions both at 555 nm and 627 nm were effectively inhibited due to BHQ2 in Y-motif/FA@HyNPs, leading to negligible PDT efficacy. Once Y-motif/FA@HyNPs were selectively internalized into tumor cells via FA-receptormediated endocytosis, the intracellular trace target miRNA initiated the dissociation of the BHQ2-terminated sequences from Ymotif/FA@HyNPs by means of abundant endogenous ATP-powered strand-displacement reaction, causing remarkable fluorescence enhancement and cascade amplification PDT. The activated dual color fluorescence emissions at 555 nm and 627 nm were feasible to achieve real-time, high sensitive and specific imaging of trace target miRNA in living tumor cells. With the guidance of excellent imaging in living mice, Y-motif/FA@HyNPs exhibited the precise and efficient PDT of tumors as well as insignificant side effects in vivo. This work revealed the great potential of using an integration of receptor-mediated cell uptake and target-triggered recycling cascade amplification strategy to design early cancer-relevant stimuli-activatable PSs for both fluorescence imaging and PDT ablation of tumors in vivo, which could effectively facilitate the timeliness and precision of early cancer diagnosis and therapy.

treat cancer. Therefore, it is of great urgency to develop early cancer-relevant stimuli-responsive PSs for distinguishing cancerous and noncancerous tissues by monitoring the slight difference of these stimuli levels, achieving early precise localization-guided efficient PDT ablation of tumors. MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs that play significant roles in gene expression regulation.23-25 As previously reported, aberrant expression of miRNA is closely interrelated with multiple kinds of human cancers, and thus can be identified as valuable biomarkers in early cancer diagnostics and precise drug therapy.26, 27 Nevertheless, the extremely low abundance of miRNAs within the tumor cells, which in many cases is below the detection threshold that can effectively trigger the activation of sensitive imaging and selective treatment of tumors, seriously limits the development of miRNAs-activatable theranostic probes.28 Recently, the catalytic molecular imaging strategy on the

INTRODUCTION Stimuli-responsive phototherapy agents, with the functions of diagnosis and therapy integrated into a single matrix, provide promising avenues toward the goal of efficient and specialized anti-cancer treatment.1-3 Especially, the combination of tumor-targeting and stimuli-activatable properties in photosensitizers (PSs) is quite fascinating, as it exhibits enhanced imaging sensitivity and photodynamic therapy (PDT) efficacy of tumors through site-specific activation.4-6 Inspired by this point, various types of activatable PSs in response to different stimuli such as metal ions,7 nucleic acids,8 pH,9, 10 ATP,11, 12 biothiols,13-16 H2S,17, 18 and proteases,19-22 have been reported to maximize veracity of cancer theranostics, and meanwhile minimize potential off-target damage. However, these activatable PSs are obviously not sensitive enough for diagnosis and subsequent PDT of tumor at an early stage, which has been considered as the primetime to 1

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basis of strand-displacement reaction has been successfully utilized to increase signal-to-noise ratio for detecting intracellular target miRNAs, demonstrating great potential to design smart enough miRNA-activatable theranostic probes for precise and timely diagnosis of early cancer.29, 30 Inspired by these researches, some miRNAs-responsive drug delivery systems that take advantages of stranddisplacement amplification strategy have been exploited to realize sensitive imaging and imaging-guided chemotherapy or photothermal therapy of early tumors.3134 Despite these recent advancements, targeted delivery of cancer-related miRNA-activatable PSs into tumor cells enabling sufficiently sensitive cascade amplification imaging and PDT with prominent reactive oxygen species (ROS, i.e. singlet oxygen 1O2) production is still of significance for early cancer theranostics in vivo, but few relating researches have been published. Moreover, it has been proved that adenosine 5′-triphosphate (ATP) as an essential biogenic biomolecule possesses a high expression level (1-10 mM) in a living cell, exhibiting excellent potential to be endogenous powers and catalyst molecules.35, 36

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Through rational DNA design, carboxyl-terminated Ya single-strand DNA containing the complementary segment to Let-7a and ATP aptamer was covalently assembled to the surface of NH2/FA@HyNPs by amidation (Apt/FA@HyNPs), which afforded a convenient scaffold for specific duplex hybridization with complementary Yb and quencher BHQ2-terminated Yc single-strand DNA, obtaining stable Y-motif/FA@HyNPs. As expected, both the fluorescence emission and PDT activity of Ymotif/FA@HyNPs were effectively inhibited ascribed to fluorescence resonance energy transfer (FRET) from NH2/FA@HyNPs to BHQ2 (Figure S1). Notably, the spatial structure of ATP aptamer in Ya was closed when its terminals were stapled in rigid Y-motif DNA structure through complementary hybridization, resulting in forceless recognition of Y-motif/FA@HyNPs toward ATP.32 Once Y-motif/FA@HyNPs selectively accumulated into the lysosome of tumor cells through FA receptor-mediated efficient endocytosis, the endogenous Let-7a could selectively bind to the unpairing toehold region at Ya in Y-motif/FA@HyNPs, allowing for stranddisplacement reaction to liberate ATP aptamer from Ymotif DNA structure. Subsequently, the intracellular abundant ATP that served as the power molecule, could specifically trigger the conformation transformation of ATP aptamer to form stable hairpin structure on the surface of nanoparticle, releasing the initial Let-7a for next stranddisplacement reaction with recycling cascade amplification. As a result, BHQ2 was completely dissociated from nanoparticles, leading to remarkable activation of dual color fluorescence emissions (555 and 627 nm) and 1O2 generating ability. The activatable dual color fluorescence were successfully used for sensitive and specific imaging of trace Let-7a in tumor cells with targettriggered recycling cascade amplification. With the guidance of fluorescence imaging, the light irritation to tumor cells for inducing remarkable 1O2 production was also implemented, leading to rapid lysosome disruption and final cell death both in vitro and in vivo (Figure 1 & S2). To the best of our knowledge, this is the first development of tumor-targeting and cancer-relevant miRNA-activatable PS based on intracellular abundant ATP-powered recycling cascade strand-displacement strategy for sensitive enough imaging-guided efficient PDT of tumors, implying a great promise for early stage diagnosis and precise therapy of tumors.

Figure 1. Schematic illustration of the synthesis process of Ymotif/FA@HyNPs, and the mechanism of action of Ymotif/FA@HyNPs for folate receptor-mediated endocytosis and endogenous Let-7a miRNA-activated cascade amplification fluorescence and 1O2 generation for tumor imaging and PDT based on intracellular ATP-powered stranddisplacement reaction.

In this regard, we reported the demonstration of a cancerrelated Let-7a miRNA-activatable and intracellular abundant ATP-powered theranostic nanophotosensitizer Y-motif/FA@HyNPs, which was used for sensitive imaging of trace underexpressed Let-7a miRNAs and controllable PDT ablation of tumors in vivo depending on the strategy of target-triggered recycling cascade stranddisplacement amplification. Y-motif/FA@HyNPs was constructed by assembling rigid Y-motif DNA structure on the surface of a hybrid semiconductor QDs micellar nanoparticle NH2/FA@HyNPs that functionalized with folate (FA) and amine. Due to the excellent properties of high 1O2 quantum yield (~0.91) upon light irradiation, dual fluorescence emissions at 555 nm and 627 nm, as well as FA receptor-targeting group FA, NH2/FA@HyNPs was selected as the nanocarrier to ensure specific delivery of Ymotif/FA@HyNPs into tumor cells, sensitive fluorescence imaging for endogenous Let-7a, and remarkable 1O2 production to cause efficient PDT of tumor in vivo.37

EXPERIMENTAL SECTION Synthesis. The preparation of Y-motif/FA@HyNPs is described in the Supporting Information. Target-Triggered Recycling Cascade Amplification Imaging of Let-7a miRNA in Tumor Cells with Ymotif/FA@HyNPs. ~ 100 k MCF-7 cells were inoculated into five 35.0 mm confocal dishes, and then cultured at 37.0 °C in CO2 incubator for 24.0 h. After that, the culture mediums were removed, and all MCF-7 cells were washed with PBS buffer (pH = 7.4, 1X) for three times, which were then treated with the following four treatments: (1) incubation with 2.0 M Y-motif/FA@HyNPs for 2.0 h at 37.0 °C, (2) incubation with 2.0 M Y-motif/FA@HyNPs + 2.0 mM FA for 2.0 h at 37.0 °C, (3) incubation with 2.0 M Y-motif/FA@HyNPs + 100.0 mM IAA for 2.0 h at 37.0 °C, (4) transfection with 200.0 nM Let-7a inhibitor 2


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using lipofectamine3000 reagent at 37.0 °C for 6.0 h, and then incubation with 2.0 M Y-motif/FA@HyNPs for 2.0 h at 37.0 °C. After washing three times with PBS buffer (pH = 7.4, 1X), all cells were cultured with 1.0 mL fresh DMEM, which were followed to perform fluorescence imaging by an IX73 optical microscope. R6G in Ymotif/FA@HyNPs was excited at 530 to 550 nm, and emission from 570 to 600 nm. TMPyP-Zn-QDs in Ymotif/FA@HyNPs was excited at 400 to 440 nm, and emission from 600 to 650 nm. Specific Fluorescence Imaging of Let-7a in MCF-7 Tumor with Y-motif/FA@HyNPs In Vivo. When MCF-7 tumor volumes reached to approximately 120 mm3, 100.0 μL of 200.0 μM Y-motif/FA@HyNPs was intratumorally injected into five female BALB/c nude mice. The real-time fluorescence images of mice were captured at 0, 0.5, 1.0, 2.0, 3.0, 12.0, 18.0, 24.0, 48.0, 72.0, 96.0, and 108.0 h post injection using an IVIS Lumina XR III in vivo Imaging System (excitation, 540 nm; emission, 620 nm long pass). For ex vivo fluorescence imaging of Let-7a in MCF-7 tumor-tissue slices, the mice were euthanized 3.0 h after treatment with Y-motif/FA@HyNPs, and the MCF-7 tumors excised from the mice were acquired and cryosectioned onto slices at a 10 m thickness. Afterward, the slices were stained with DAPI for 20.0 min, and then washed with PBS buffer (pH = 7.4, 1X) for three times. Finally, the fluorescence imaging of stained tumor slices were conducted on an IX73 optical microscope. In Vivo PDT of Tumor with Y-motif/FA@HyNPs. For in vivo PDT treatments, twenty-five female BALB/c nude mice with approximately 120 mm3 MCF-7 tumor pieces were randomly divided into five groups with five mice in each group. Then the five group mice were subjected to the following treatments: group 1, PBS only; group 2, PBS + light irradiation; group 3, Y-motif/FA@HyNPs only; group 4, Ymotif/FA@HyNPs + light irradiation; and group 5, Ymotif/FA@HyNPs + Let-7a inhibitor + light irradiation. For group 1 and 2, 100.0 L PBS was intratumorally injected into each mouse. For group 3 and 4, 100.0 L of 200.0 M Ymotif/FA@HyNPs was intratumorally injected into each mouse. At 3.0 h post injection, irradiation with a xenon lamp (400 nm long pass filter) at a power of 120 mW/cm2 was performed on the tumors of mice groups 2 and 4 for three consecutive exposures of 10 min each, with a 20-min interval. For group 5, 100.0 L of 20.0 mM Let-7a inhibitor by means of lipofectamine3000 transfection reagent was first injected into tumor in each mouse. After injection of 12.0 h, 100.0 L of 200.0 M Y-motif/FA@HyNPs was also injected into tumor in each mouse. After injection of another 3.0 h, these mice in group 5 were irradiated with a xenon lamp (400 nm long pass filter) at a power of 120 mW/cm2 for three consecutive exposures of 10 min each, with a 20-min interval. The tumor volume (V) and the body weight (m) of the mice were monitored every other day for 14 days (day 0, 2, 4, 6, 8, 10, 12 and 14). The tumor volume (V) was calculated by the formula of V = (L  W2)/2, where L indicated the length of tumor, and W indicated the width of tumor.

assembled TMPyP-Zn-QD nanocomplexes, DSPE-PEG2000FA, DSPE-PEG2000-OMe, and DSPE-PEG2000-NH2. TEM image disclosed that the synthesized NH2/FA@HyNPs was well mono-dispersed (Figure S3), and possessed a hydrodynamic diameter of 42.5 ± 1.5 nm in aqueous solution according to DLS analysis (Figure 2a). By comparing the difference of NH2/FA@HyNPs with NH2@HyNPs in UV-vis absorption spectra, the enhanced absorbance at ~280 nm confirmed that FA has been successfully modified on the surface of nanoparticles, which was consistent with zeta potentials and agarose gel electrophoresis analysis (Figure S4). Subsequently, the carboxyl-terminated Ya DNA was covalently conjugated to the surface of NH2/FA@HyNPs via the amidation, followed by incubation with Yb and BHQ2terminated Yc to fabricate the Y-motif DNA structurefunctionalized Y-motif/FA@HyNPs theranostic nanoprobe by means of the specific complementary hybridization. Ymotif/FA@HyNPs displayed bigger hydrodynamic diameter of 67.7 ± 1.1 nm compared to that of NH2/FA@HyNPs (42.5 ± 1.5 nm) in aqueous solution because of the formation of Y-motif DNA structure on the surface of NH2/FA@HyNPs (Figure 2a), which was also validated by more negative zeta potential of 24.7 ± 0.5 mV in relative to that of NH2/FA@HyNPs (-2.0 ± 0.5 mV) (Figure 2b). The UV-vis absorption spectrum of Ymotif/FA@HyNPs presented enhanced absorption ranging from 500 to 700 nm ascribed to the BHQ2, further indicating that the Y-motif DNA structure was anchored on the surface of NH2/FA@HyNPs (Figure 2c). As a result, the fluorescence at both 555 nm (R6G) and 627 nm (pre-assembled TMPyP-ZnQDs) in Y-motif/FA@HyNPs were obviously quenched by BHQ2 (Figure 2d).

Figure 2. Characterization of the Y-motif/FA@HyNPs theranostic nanoprobe. (a) DLS, (b) zeta potentials, (c) UV-vis absorption and (d) fluorescence spectra of NH2/FA@HyNPs and Ymotif/FA@HyNPs.

To further verify the successful assembling of Y-motif DNA structure on NH2/FA@HyNPs, we performed the polyacrylamide hydrogel electrophoresis. The new and bright band observed in the mixture of free-labeled Ya', Yb, and Yc' lane was the powerful evidence to demonstrate the efficient formation of Y-motif DNA structure, which indirectly proved the successful construction of Ymotif/FA@HyNPs (Figure S5). Moreover, Ymotif/FA@HyNPs exhibited excellent stability under physiologically relevant conditions, with negligible change of hydrodynamic diameter or fluorescence change after incubation with different culture medium or nuclease (Figure S6 to S8). Notably, Y-motif/FA@HyNPs was also very stable under light irradiation, indicating the possibility

RESULTS AND DISCUSSION Preparation and Characterization of Ymotif/FA@HyNPs. We first adopted micelle encapsulation method to prepare hybrid semiconductor QDs micellar NH2/FA@HyNPs by mixing of rhodamine 6G (R6G), pre3



for implementation controllable on-demand PDT (Figure S9). Fluorescence Response of ATP-Powered Ymotif/FA@HyNPs toward Let-7a miRNA. To verify the capability of ATP-powered Y-motif/FA@HyNPs for detection of the Let-7a miRNA, we firstly studied the fluorescence response of Y-motif/FA@HyNPs to Let-7a in aqueous solution. Initially, Y-motif/FA@HyNPs emitted very weak fluorescence at both 555 nm and 627 nm, which could be attributed to the quenching effect of BHQ2. With the addition of 1.4 nM Let-7a at 37.0 °C for 15.0 min, both the fluorescence intensities at 555 nm and 627 nm of 2.0 M Y-motif/FA@HyNPs were gradually activated to ~2.1-fold and ~4.4-fold. On the contrast, 2.0 M Ymotif/FA@HyNPs incubation with 10.0 mM ATP exhibited a negligible fluorescence change. Once 2.0 M Y-motif/FA@HyNPs was incubated with the mixture of 1.4 nM Let-7a and 10.0 mM ATP, both the fluorescence intensities at 555 nm and 627 nm were rapidly amplified to ~6.7-fold and ~15.6-fold, revealing that the ATP-triggered aptamer conformation switching could efficiently promote the recycling use of the target Let-7a in the enzyme-free nucleic acid-activated cascade strand-displacement reactions (Figure S10 & 3a). Additionally, Ymotif/FA@HyNPs in the presence of ATP could response rapidly to the target Let-7a within 15.0 min and exhibited a well stability in aqueous solution from the kinetic studies (Figure S11 & 3b). Meanwhile, we found that kinetics of the fluorescence activation of Let-7a-triggered Ymotif/FA@HyNPs was dependent on the ATP concentration, and the maximum fluorescence activation efficiency was obtained at 10.0 mM ATP (Figure S12). Next, we explored the fluorescence responses of ATPpowered Y-motif/FA@HyNPs to varying concentrations of Let-7a in PBS buffer (pH = 7.4, 1X) at 37.0 °C for 15.0 min. The results showed that both the fluorescence intensities at 555 nm and 627 nm of 2.0 M Ymotif/FA@HyNPs in the presence of 10.0 mM ATP were increased with increasing concentrations of Let-7a from 0 to 1.6 nM (Figure 3c). When plotting the fluorescence intensities at 555 nm and 627 nm turn-on ratio (F/F0) of Ymotif/FA@HyNPs vs the concentration of Let-7a from 0 to 1.2 nM, the linear curve with a good correlation coefficient of R2 = 0.99 was obtained, and the detection limit was determined to be ~0.4 pM (signal-to-noise, S/N = 3), indicating that ATP-powered Y-motif/FA@HyNPs could achieve the recycling cascade amplification detection of Let-7a at a low level (Figure 3d & S13). The specificity of ATP-powered Y-motif/FA@HyNPs to Let-7a detection was evaluated by introducing other six kinds of miRNA sequences including Let-7b, Let-7c, Let7f, miRNA-21, miRNA-155, and miRNA-373. As shown in Figure S14 & 3e, only 1.4 nM Let-7a could efficiently activate the 2.0 M Y-motif/FA@HyNPs and initiate the 10.0 mM ATP-powered cascade amplification to cause a significant ~15.6-fold fluorescence enhancement at 627 nm. In contrast, any mismatch miRNA sequences caused similar fluorescence intensities to that of the blank at 627 nm, implying the high specificity of Y-motif/FA@HyNPs for Let-7a detecting. Furthermore, we also examined the effects of different thymidine triphosphate analogues such as CTP, TTP, UTP, and GTP on the powered ability to Let7a-triggered fluorescence cascade amplification of Y-

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motif/FA@HyNPs (Figure S15 & 3f). It was evident that only ATP could assist Y-motif/FA@HyNPs to achieve Let-7a miRNA-triggered recycling fluorescence cascade amplification, indicating that the response sensitivity of Ymotif/FA@HyNPs for Let-7a was specifically depended on ATP power. The negligible pH effect on ATP-powered Y-motif/FA@HyNPs for fluorescence response to Let-7a demonstrated that Y-motif/FA@HyNPs could be probably applied to detect Let-7a in acidic tumor microenvironment (Figure S16).

Figure 3. Fluorescence response of ATP-powered Ymotif/FA@HyNPs to Let-7a miRNA in aqueous solution. (a) Fluorescence enhancement ratio at 627 nm of 2.0 M Ymotif/FA@HyNPs before and after incubation with 10.0 mM ATP, 1.4 nM Let-7a, and 10.0 mM ATP + 1.4 nM Let-7a in PBS buffer (pH = 7.4, 1X) at 37.0 °C for 15.0 min. (b) Time-dependent fluorescence intensity at 627 nm of 2.0 M Y-motif/FA@HyNPs before and after incubation with 10.0 mM ATP, 1.4 nM Let-7a, and 10.0 mM ATP + 1.4 nM Let-7a in PBS buffer (pH = 7.4, 1X) at 37.0 °C for 15.0 min. (c) Fluorescence spectra of 10.0 mM ATPpowered 2.0 M Y-motif/FA@HyNPs respond to various concentrations of Let-7a ranging from 0 to 1.6 nM in PBS buffer (pH = 7.4, 1X) at 37.0 °C for 15.0 min. (d) Plot of the fluorescence enhancement ratio at 627 nm of 2.0 M Y-motif/FA@HyNPs with the assistance of 10.0 mM ATP vs the target Let-7a concentrations in PBS buffer (pH = 7.4, 1X) at 37.0 °C for 15.0 min. (e) Specificity of 10.0 mM ATP-powered 2.0 M Y-motif/FA@HyNPs toward Let-7a in PBS buffer (pH = 7.4, 1X) at 37.0 °C for 15.0 min. The concentrations of all miRNAs were 1.4 nM. (f) Fluorescence intensity at 627 nm of 2.0 M Y-motif/FA@HyNPs incubated with 1.4 nM Let-7a miRNA in the presence of 10.0 mM ATP and its analogues. F0 represents the initial fluorescence intensity at 627 nm of Y-motif/FA@HyNPs, and F represents the fluorescence intensity at 627 nm of Y-motif/FA@HyNPs following treated with indicated analyses. All fluorescence data were collected upon 443 nm excitation.

Evaluation the Capacity of Let-7a miRNA-Activatable Ymotif/FA@HyNPs to Induce 1O2 Cascade Amplification. The 1O2 production capacity of ATP-powered Ymotif/FA@HyNPs under irradiation in the absence and presence of Let-7a at an extremely low level was first evaluated by the fluorescence emission of singlet oxygen sensor green (SOSG), which could produce strong fluorescence at 525 nm after interaction with 1O2.38 As illustrated in Figure 4a & 4b, irradiation of NH2/FA@HyNPs upon a white light (LED, 400 nm longpass filter, 20 mW/cm2) for 3.0 min showed a strong fluorescence intensity at 525 nm (curve 1), whereas Ymotif/FA@HyNPs exhibited a weak fluorescence intensity at 525 nm (curve 2), implying that BHQ2-terminated Ymotif DNA structure could effectively inhibited the 1O2 generation capacity of NH2/FA@HyNPs. Compared with 4


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Analytical Chemistry light (400 nm long pass filter) at the power of 20.0 mW/cm2 for 3.0 min, respectively.

irradiation of alone Y-motif/FA@HyNPs, irradiation of Ymotif/FA@HyNPs in the presence of ATP caused an insignificant change in fluorescence of SOSG at 525 nm (curve 3), suggesting that ATP was incapable to active Ymotif/FA@HyNPs for 1O2 production in such circumstance as well. However, before (curve 4) and after (curve 5) addition of ATP, irradiation of Ymotif/FA@HyNPs in the presence of Let-7a could cause ~1.8-fold and ~4.5-fold fluorescence enhancement of SOSG at 525 nm with reference to irradiation of Ymotif/FA@HyNPs. By further introducing of Let-7a inhibitor, the florescence intensity of SOSG at 525 nm caused by irradiation of Y-motif/FA@HyNPs in the presence of ATP and Let-7a was dramatically inhibited (curve 6). These results indicated that Let-7a could specifically activate ATP-powered Y-motif/FA@HyNPs to achieve rapidly 1O2 cascade amplification, which were further validated by electron spin resonance (ESR) spectroscopy using 2,2,6,6-tetramethylpiperidine (TEMP) as an 1O trap.39 Initially, both irradiation of Y-motif/FA@HyNPs in 2 the absence and presence of ATP displayed very weak ESR signals. After addition of Let-7a, the ESR signals of Ymotif/FA@HyNPs upon irradiation was enhanced to ~2.1-fold, and the ESR signals of Y-motif/FA@HyNPs in the presence of ATP upon irradiation was enhanced to ~5.6-fold (Figure S17), which agreed well with that indicated by SOSG. In the presence of sodium azide (NaN3, 1O2 quencher),40 no ESR signals could be obtained from irradiation of Y-motif/FA@HyNPs mixed with Let-7a and ATP (Figure S18a). The ESR intensity ascribed to Y-motif/FA@HyNPs in the presence of Let-7a and ATP increased with irradiation time, suggesting a light dosedependent generation of 1O2 (Figure S18b & S18c). Notably, Ymotif/FA@HyNPs in the presence of Let-7a and ATP resulted in both fluorescence intensity of SOSG at 525 nm and ESR signals were closely to that induced by the uninhibited NH2/FA@HyNPs after irradiation. All of these results showed that Let-7a at an extremely low level could specifically and efficiently activate Y-motif/FA@HyNPs using ATP as a power to achieve 1O2 cascade amplification. In addition, we also observed that the effect of pH ranging from 5.0 to 7.4 on the 1O2 cascade amplification capacity of Let-7a-activatable Ymotif/FA@HyNPs in the presence of ATP was insignificant (Figure S19).

Specific Imaging of Y-motif/FA@HyNPs toward Endogenous Let-7a miRNA in Living Tumor Cells. Having verified that ATP-powered Y-motif/FA@HyNPs could realize the detection of Let-7a at an extremely low level in aqueous solution, we then explored the ability of Ymotif/FA@HyNPs to specifically image endogenous Let7a in living MCF-7 cells that expressed both folate receptor and Let-7a miRNA.41, 42 To obtain the optimal imaging conditions, MCF-7 cells were incubated with different concentrations of Y-motif/FA@HyNPs (0.0, 0.1, 0.5, 1.0, 2.0, and 4.0 M) for different times (0.0, 0.5, 1.0, 2.0, 4.0, and 6.0 h). The results showed that both the maximum fluorescence signals at R6G and TMPyP-Zn-QDs channels were observed in MCF-7 cells after incubation with 2.0 M Y-motif/FA@HyNPs for 2.0 h, implying the rapid uptake and efficient activation of Y-motif/FA@HyNPs by endogenous Let-7a (Figure S20 and S21). Co-localization assay displayed that the activation behavior of Ymotif/FA@HyNPs by endogenous Let-7a was carried out in the lysosome of living MCF-7 cells, owing to the nice fluorescence overlap of lysosomal tracker with both R6G and TMPyP-Zn-QDs (Figure S22).

Figure 5. Fluorescence images of MCF-7 cells treated with (from Left to Right) 2.0 M Y-motif/FA@HyNPs, 2.0 M Ymotif/FA@HyNPs + 2.0 mM FA, 2.0 M Ymotif/FA@HyNPs + 100.0 nM inhibitor, 2.0 M Ymotif/FA@HyNPs + 100.0 mM IAA, and MCF-10A cells treated with 2.0 M Y-motif/FA@HyNPs. Scale bar: 20 m.

To verify that the intracellular activatable fluorescence was originated from the folate receptor-mediated cellular uptake of Y-motif/FA@HyNPs, we performed a blocking study by FA. As demonstrated in Figure 5, MCF-7 cells incubated with 2.0 M Y-motif/FA@HyNPs and 2.0 mM FA exhibited negligible intracellular fluorescence. Meanwhile, the similar result was also observed in 2.0 M Y-motif/FA@HyNPs pretreated MCF-10A cells that negatively expressed the FA receptor.43 These results revealed that FA played a significant role in the targeted cellular uptake of Y-motif/FA@HyNPs. To confirm the specificity of Y-motif/FA@HyNPs for Let-7a imaging in living cells, MCF-7 cells collectively treated with Let-7a inhibitor and Y-motif/FA@HyNPs showed almost no fluorescence signal, indicating that Y-motif/FA@HyNPs could be specific activated by intracellular Let-7a. Subsequently, we studied the role of ATP to promote the fluorescence signal cascade amplification of endogenous

Figure 4. Evaluation the cascade amplification 1O2 of Let-7a miRNA-triggered Y-motif/FA@HyNPs in the presence of ATP in PBS buffer (pH = 7.4, 1X) at 37.0 °C. (a) Fluorescence spectra of 10.0 M SOSG incubation with the indicated treatments. (b) Comparison the fluorescence intensity of SOSG at 525 nm by different treatments of (a). The curves from 1 to 6 (irradiated group) and that from 7 to 12 (non-irradiated group) were indicated in turn the treatments of 2.0 M NH2/FA@HyNPs, 2.0 M Ymotif/FA@HyNPs, 2.0 M Y-motif/FA@HyNPs + 10.0 mM ATP, 2.0 M Y-motif/FA@HyNPs + 1.4 nM Let-7a, 2.0 M Ymotif/FA@HyNPs + 10.0 mM ATP + 1.4 nM Let-7a, and 2.0 M Y-motif/FA@HyNPs + 10.0 mM ATP + 1.4 nM Let-7a + 100 nM Let-7a inhibitor upon irradiation or non-irradiation with a LED 5



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in living MCF-7 cells upon light irradiation. The irradiation of MCF-7 cells collectively treated with 100 nM Let-7a inhibitor and 2.0 M Y-motif/FA@HyNPs showed that both DCF fluorescence and the dual fluorescence of R6G and TMPyP-Zn-QDs in Y-motif/FA@HyNPs were significantly inhibited, implying that intracellular 1O2 generation ascribed to Y-motif/FA@HyNPs was specifically activated by endogenous Let-7a. Interestingly, irradiation of MCF-7 cells that incubated with 2.0 M Ymotif/FA@HyNPs and 100.0 mM IAA displayed lower fluorescence intensities of DCF, R6G, and TMPyP-ZnQDs as compared with irradiation of Ymotif/FA@HyNPs-treated cells, indicating that intracellular ATP assisted endogenous Let-7a to activate the 1O2 cascade amplification of Y-motif/FA@HyNPs. Moreover, we also found that intracellular 1O2 level induced by Y-motif/FA@HyNPs was dependent on irradiation time by monitoring the DCF fluorescence on flow cytometer (Figure S23). Encouraged by the efficient 1O2 cascade amplification of Y-motif/FA@HyNPs in living MCF-7 cells upon irradiation, we subsequently investigated the cytotoxicity of Y-motif/FA@HyNPs to MCF-7 cells by MTT assays (Figure S24). The results showed that Ymotif/FA@HyNPs had almost no cytotoxicity in dark, while displayed high phototoxicity to MCF-7 cells upon irradiation (LED, 20.0 mW/cm2, 400 nm long pass filter). After pretreated with Let-7a inhibitor, little cytotoxicity of Y-motif/FA@HyNPs to MCF-7 cells upon irradiation was observed. Meanwhile, we also found that IAA reduced the cytotoxicity of Y-motif/FA@HyNPs to MCF-7 cells upon irradiation, resulting in that IC50 value of Ymotif/FA@HyNPs to MCF-7 cells was ~2.0-fold improved before and after treating with IAA (Figure S25). Similar results were also observed by Annexin V-/propidium iodide (PI) staining for necrotic/late apoptotic and early apoptotic cells, respectively.46 Over 92.0% MCF-7 cells treated with Y-motif/FA@HyNPs and irradiation were apoptosis, but negligible apoptosis for irradiation of MCF7 cells treated with Let-7a inhibitor and Ymotif/FA@HyNPs. Notably, the late cell apoptosis percentage was reduced from 48.7% to 14.9% by comparing the irradiation of Y-motif/FA@HyNPs treated MCF-7 cells in the absence and presence of IAA (Figure S26). All of these results revealed that PDT activity of Ymotif/FA@HyNPs with the assistance of endogenous ATP could be specifically activated by intracellular Let-7a and thus initiated efficient cascade amplification PDT against MCF-7 cells, which was further confirmed in the timelapse movies via sequential image acquisition as well (Movie S1 to S4). In Vivo Fluorescence Imaging of Let-7a miRNA in MCF7 Tumor-Bearing Mice. To demonstrate the capacity of Ymotif/FA@HyNPs for real-time monitoring of Let-7a levels in vivo, 100.0 L of 200.0 M Y-motif/FA@HyNPs were directly injected into MCF-7 tumors in living mice. Afterward, the whole-body fluorescence images were acquired by an IVIS Lumina XR III in vivo Imaging System. As illustrated in Figure 7a & 7b, the fluorescence of TMPyP-Zn-QDs was observed in Y-motif/FA@HyNPstreated MCF-7 tumor, and gradually increased with the post-injection time increase. The maximum fluorescence intensity was acquired at 3.0 h post-injection, and

Let-7a-triggered activation of Y-motif/FA@HyNPs in living MCF-7 cells. To this end, MCF-7 cells were pretreated with iodoacetic acid (IAA, 100.0 mM) which could prevent glycolysis and thus decrease the level of intracellular ATP,44, 45 and then treated with 2.0 M Ymotif/FA@HyNPs for 2.0 h. The corresponding result indicated that the intracellular fluorescence was obviously weaken when compared with that of the MCF-7 cells treated with alone Y-motif/FA@HyNPs, confirming that endogenous ATP could assist and boost intracellular Let7a-triggered activation of Y-motif/FA@HyNPs. In view of the facts above, Y-motif/FA@HyNPs using intracellular ATP as power could be specifically used for the dual-color fluorescence cascade amplification imaging of endogenous Let-7a at a low level in lysosome of folate receptoroverexpressed MCF-7 cells.

Figure 6. Fluorescence images of MCF-7 cells treated with 2.0 M Y-motif/FA@HyNPs and 20.0 M DCFH-DA in the absence and presence of light only, light with 2.0 mM VC, 200.0 nM Let-7a inhibitor, and 100.0 mM IAA, respectively. Scale bars: 20 m. Red arrows indicated the cell collapse and blebs, implying the apoptosis degree of MCF-7 cells.

Intracellular Let-7a miRNA-Triggered Activation of Cascade Amplification to Achieve Efficient PDT against Tumor Cells. Having confirmed that intracellular ATP-powered Y-motif/FA@HyNPs could be specifically applied to observe cascade amplification imaging of intracellular Let-7a in living MCF-7 cells, we next explored the capacity of endogenous Let-7a to activate Ymotif/FA@HyNPs for controllable generating 1O2 in living MCF-7 cells upon a white light irradiation (LED, 20.0 mW/cm2, 400 nm long pass filter). 2′,7′-dichlorofluorescin diacetate (DCFH-DA) that displayed nonfluorescence, but could be rapidly oxidized to emissive dichlorofluorescein (DCF) by 1O2, was used as the indicator to reflect 1O2 level in MCF-7 cells.9 As demonstrated in Figure 6, without irradiation, MCF-7 cells that incubated with 2.0 M Ymotif/FA@HyNPs for 2.0 h only displayed the dual activation fluorescence of R6G and TMPyP-Zn-QDs in Ymotif/FA@HyNPs, revealing little 1O2 generation. However, irradiation of MCF-7 cells that incubated with 2.0 M Y-motif/FA@HyNPs for 2.0 h not only exhibited dual activation fluorescence of R6G and TMPyP-Zn-QDs in Y-motif/FA@HyNPs, but also showed strong DCF fluorescence. Once addition of Vitamin C (VC, a known 1O scavenger), the intracellular DCF fluorescence could 2 be effectively eliminated. These results proved that a large amount of 1O2 could be produced by Y-motif/FA@HyNPs 1O 2

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Analytical Chemistry injection of either 100.0 L PBS (pH = 7.4, 1X) or 100.0 L of 200.0 M Y-motif/FA@HyNPs. At 3.0 h postinjection, half of the mice were treated with light irradiation (Xenon lamp, 120.0 mW/cm2, 400 nm long pass filter) for three consecutive exposures of 10.0 min each, with a 10-min interval, while the other half mice were maintained in dark. To confirm that efficient PDT of Ymotif/FA@HyNPs in vivo was initiated by endogenous Let-7a, 100.0 L of 20.0 mM Let-7a inhibitor was intratumorally injected into MCF-7 tumors bearing nude mice for 12.0 h, and then 100.0 L of 200.0 M Ymotif/FA@HyNPs was also intratumorally injected into these mice for another 3.0 h, following irradiation (Xenon lamp, 120.0 mW/cm2, 400 nm long pass filter) for three consecutive exposures of 10.0 min each, with a 10-min interval. After that, the tumor size and body weight of each mouse were monitored to assess the PDT efficacy over a period of 14 days. It was found that mice neither treated with alone PBS, PBS plus light irradiation, nor alone Ymotif/FA@HyNPs could inhibited the tumor growth, nevertheless the mice treated with Y-motif/FA@HyNPs plus light irradiation displayed an obvious decrease of tumor volume. More significantly, light irradiation of mice treated with Let-7a inhibitor plus Y-motif/FA@HyNPs showed a similar tumor growth trend when compared with that of the alone PBS (Figure S27a). Furthermore, the body weight of each group mice showed an insignificant change before and after 14 days treatment (Figure S27b). These results exhibited that Y-motif/FA@HyNPs in vivo possessed nontoxicity in dark, but excellent PDT efficacy to MCF-7 tumors upon light irradiation, owing to intracellular cancer-relevant Let-7a miRNA-activatable recycling amplification. To further visualize the PDT efficacy of Ymotif/FA@HyNPs in vivo, we performed hematoxylineosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining on histological sections resected from the different treatment groups at 24.0 h post irradiation. The results showed large areas of apoptosis and necrosis of tumor cells in mice treated with Y-motif/FA@HyNPs and light irradiation, which was significantly more than that in other four experimental groups. Notably, apoptotic and necrotic tumor cells were almost indiscernible in the group of mice treated with Let7a inhibitor and Y-motif/FA@HyNPs upon light irradiation, which was close to the results of mice treated with either PBS, PBS with irradiation, or Ymotif/FA@HyNPs alone, more intuitively revealing endogenous Let-7a-activatable PDT activity of Ymotif/FA@HyNPs for desirable antitumor efficacy in vivo (Figure S27c).

maintained for over 18.0 h. After 108.0 h post-injection, most nanoparticles were eliminated out from MCF-7 tumors. At this moment, the tumor volume was increased ~3.0-fold referred to 0 h post-injection, indicating an excellent biocompatibility of Y-motif/FA@HyNPs in vivo (Figure 7c). In addition, we also found that the fluorescence intensity in MCF-7 tumor treated with Ymotif/FA@HyNPs alone was ~2.0-fold stronger than that of Let-7a inhibitor plus Y-motif/FA@HyNPs at 3.0 h postinjection, revealing that Y-motif/FA@HyNPs could efficiently enter into MCF-7 cells and be activated by endogenous Let-7a in vivo (Figure 7d & 7e), which was also verified by fluorescence imaging of MCF-7 tumor tissue slices ex vivo at 3.0 h post-injection. It was found that MCF-7 tumor tissue slices injected with Ymotif/FA@HyNPs showed both bright TMPyP-Zn-QDs and R6G fluorescence signals, while negligible fluorescence signals were found in the tissue slices that injected with Let-7a inhibitor and Y-motif/FA@HyNPs (Figure 7f).

Figure 7. Fluorescence imaging of Let-7a in vivo. (a) In vivo time-dependent whole-body fluorescence images of MCF-7 tumor-bearing living mice after intratumoral injection of Ymotif/FA@HyNPs. Red arrows pointed out the tumor site in mice. (b) Quantification analysis of the relative fluorescence intensity in the tumor site as shown in (a). (c) Quantification analysis of the relative tumor volume before and after injection of Y-motif/FA@HyNPs for 0 and 108.0 h. (d) In vivo whole-body fluorescence images of MCF-7 tumorbearing living mice injected with alone Y-motif/FA@HyNPs and Let-7a inhibitor plus Y-motif/FA@HyNPs at 3.0 h postinjection. (e) Quantification analysis of the relative fluorescence intensity in the tumor site as shown in (d). (f) Ex vivo fluorescence images of MCF-7 tumor-tissue slices obtained from living mice after 3.0 h intratumoral injections of Y-motif/FA@HyNPs or Let-7a inhibitor plus Ymotif/FA@HyNPs and stained with DAPI. Blue fluorescence was observed from 420 nm - 460 nm with the excitation of DAPI channel, green fluorescence was observed from 570 nm - 600 nm with the excitation of R6G channel, and red fluorescence was observed from 600 nm - 650 nm with the excitation of TMPyP-Zn-QDs channel. Scale bar: 20 μm. All data were mean ± SD (n = 5).

CONCLUSIONS In summary, we programmed a molecule machine of Ymotif DNA structure on hybrid micellar nanoparticles for constructing a tumor-targeting and trace under-expressed intracellular miRNA-activatable nanophotosensitizer Ymotif/FA@HyNPs, which was based on ATP-powered cascade strand-displacement reaction strategy. We demonstrated that Y-motif/FA@HyNPs was capable of targeting to tumor cells through FA receptor-mediated endocytosis and specifically bound with cancer-relevant miRNA to initiate ATP-powered cascade strand-

Evaluation of Endogenous Let-7a miRNA-Activatable PDT Efficacy In Vivo Antitumor. With the guidance of fluorescence imaging results, we finally estimated the in vivo PDT efficacy of Y-motif/FA@HyNPs to MCF-7 tumors bearing nude mice that received intratumoral 7



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displacement reaction, leading to efficient activation of dual color fluorescence and cascade amplification PDT capacity. Y-motif/FA@HyNPs was successfully applied for sensitive enough fluorescence imaging of endogenous miRNA both in living tumor cells and tumors in vivo. With the guidance of fluorescence imaging, selective and efficient cascade amplification PDT of tumors in vivo was observed, resulting in almost complete ablation of tumors upon light irradiation. These results showed great prospects in utilizing a smart powered and target-triggered recycling cascade strand-displacement reaction strategy to design activatable PSs for extremely low abundant intracellular miRNA imaging and efficient PDT of tumors in vivo, thus opening up an avenue to design personalized PSs for early precise diagnosis and therapy of cancer.

AUTHOR INFORMATION Supporting Information Supplementary Figures and nano-characterization. The Supporting Information is available free of charge on the ACS Publications website.

Corresponding Authors * E-mail: [email protected] (Y. Ye). * E-mail: [email protected] (J.-J. Xu).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21804029, 31671951, 21535003), Natural Science Foundation of Anhui Province, China (1908085QB67), and China Postdoctoral Science Foundation (2019M651770).

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