A Near-Infrared Triggered Nanophotosensitizer Inducing Domino

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A Near-Infrared Triggered Nanophotosensitizer Inducing Domino Effect on Mitochondrial Reactive Oxygen Species Burst for Cancer Therapy Zhengze Yu, Qiaoqiao Sun, Wei Pan, Na Li, and Bo Tang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b04501 • Publication Date (Web): 12 Oct 2015 Downloaded from http://pubs.acs.org on October 12, 2015

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A Near-Infrared Triggered Nanophotosensitizer Inducing Domino Effect on Mitochondrial Reactive Oxygen Species Burst for Cancer Therapy Zhengze Yu, Qiaoqiao Sun, Wei Pan, Na Li,* and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, P. R. China). KEYWORDS: near-infrared, nanophotosensitizer, reactive oxygen species, domino effect, cancer therapy

ABSTRACT: Photodynamic therapy (PDT) is a well-established modality for cancer therapy, which locally kills cancer cells when light irradiates a photosensitizer. However, conventional PDT is often limited by the extremely short lifespan and severely limited diffusion distance of reactive oxygen species (ROS) generated by photosensitizer, as well as the penetration depth of visible light activation. Here, we develop a near-infrared (NIR) triggered nanophotosensitizer based on mitochondria targeted titanium dioxide-coated upconversion nanoparticles for PDT against cancer. When irradiated by NIR laser, the nanophotosensitizer could produce ROS in

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mitochondria, which induced the domino effect on ROS burst. The overproduced ROS accumulated in mitochondria, resulting in mitochondrial collapse and irreversible cell apoptosis. Confocal fluorescence imaging indicated that the mitochondrial targeting and real-time imaging of ROS burst could be achieved in living cells. The complete removal of tumor in vivo confirmed the excellent therapeutic effect of the nanophotosensitizer.

Photodynamic therapy (PDT) has emerged as one of significant therapies in the treatment of cancer.1-4 Compared to the conventional therapeutics, PDT possesses several advantages owing to its noninvasive nature, negligible drug resistance, and low systemic toxicity.5-8 Most modern PDT applications involve three key components: a photosensitizer, a light source and tissue oxygen.9 Upon irradiation, the excited photosensitizer transfers energy to the surrounding O2 to generate reactive oxygen species (ROS), which can be exploited to destroy cancer cells for cancer therapy.10-12 However, the produced ROS exhibits the extremely short lifespan and severely limited diffusion distance, so the damage of ROS to biomolecules is strongly restricted to the immediate vicinity of ROS generation.13-15 Recently, most of the photosensitizers were performed in cytoplasm to generate ROS, which greatly limits the therapeutic effect of PDT.16-18 Mitochondria are the primary source of cellular ROS generation (approximately up to 90%),19, 20 and mitochondrial dysfunctions are closely correlated with the disruption in the balance of mitochondrial ROS.21-24 In addition, mitochondria are decisive regulators of the intrinsic pathway of apoptosis, which is regarded as the major mode of cell death in cancer therapy.25-27 Therefore, a mitochondria-targeted photosensitizer can perturb ROS homeostasis and further induce cell apoptosis, which is beneficial to improve the effect of PDT treatment.


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Furthermore, another obstacle of conventional PDT is the limited penetration depth of visible light activation.28, 29 The upconversion nanoparticles (UCNPs) can convert a near-infrared (NIR) excitation into ultraviolet or visible emission through the lanthanide doping.30-34 Along with the improved tissue penetration depth and reduced autofluorescence background in biological samples, the UCNPs have attracted considerable interest.35-37 Titanium dioxide (TiO2) is considered to be an ideal candidate due to high stability, nontoxicity and high efficiency.38 TiO2 with band gap energies of 3.2 eV requires activation under UV exposure conditions. However, UV is cytotoxic to living cells and has low tissue penetration capabilities, thus preventing its use in deep tissues in a clinical setting.39 When modified on the surface of UCNPs, TiO2 can be photoactivated by the NIR excitation to generate harmful radicals.40-42 Herein, we present a novel strategy to construct an NIR-responsive nanophotosensitizer for PDT based on mitochondria targeted TiO2-coated UCNPs. The Tm3+-doped UCNPs can emit UV light with 980 nm laser excitation and activate TiO2 to produce a flux of ROS, especially superoxide anion radicals(O2·-). The triphenylphosphine (TPP), a mitochondria-targeted group, was then anchored on the surface of TiO2 to selectively trigger the localized ROS burst in the mitochondria, resulting in initiation of mitochondria-mediated intrinsic apoptotic pathway, which was associated with cascade reactions, such as the activation of an inner membrane anion channel (IMAC), the opening of mitochondrial permeability transition pores, the decrease in mitochondrial membrane potential (∆Ψm), the release of cytochrome C to cytoplasm.25-27 The released cytochrome C will activate the caspase-3 and caspase-7 to induce apoptosis. These caspases can, in return, disturb the mitochondrial electron transport chain, inducie the domino effect on ROS burst and cause irreversible cell death.43 The structure of the nanophotosensitizer (UCNPs@TiO2-TPP) and details of this approach are illustrated in Scheme 1.


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RESULTS AND DISCUSSION Synthesis and characterization of the nanophotosensitizer. NaYF4:Yb3+,Tm3+ nanocrystals (β-phase) were firstly prepared via a solvothermal method with some modifications.44 As given in Figure 1a-c, high resolution transmission electron microscopy (HRTEM) showed that the oleic acid (OA)-capped UCNPs possessed uniform morphology with sizes around 25 nm and the pattern was hexagonal (β-) phase. After treated with hydrochloric acid, the OA free UCNPs were obtained with good monodispersity in aqueous solution. Meanwhile, the size did not show an obvious change. Then a layer of TiO2 was coated on the surface of UCNPs (denoted as UCNPs@TiO2) through a versatile kinetics-controlled method.45 The TiO2 shell of UCNPs@TiO2 is estimated to have a homogeneous thickness of about 3 nm. The EDX spectra showed a Kα energy peak of titanium element appears at 4.51 Kev for UCNPs@TiO2 compared with UCNPs (Figure S1a, b).46 X-ray photoelectron spectroscopy (XPS) was also employed to confirm the TiO2 coating. As shown in Figure S1c, the XPS pattern of titanium (Ti) was observed only for UCNPs@TiO2 and not for UCNPs. The results indicated that the UCNPs were successfully modified with TiO2. Moreover, the appearance of the strong absorbance in the UV region of UCNPs@TiO2 further confirmed the coating of TiO2 (Figure 2a). In order to further modify TPP groups on the surface of UCNPs@TiO2, the surface of TiO2 was functionalized with amino groups. Zeta potential experiments further verified the successful treatment, i.e. -20.2±0.3 mV (before functionalization) and +18.9±0.7 mV (after functionalization). And the content of amino groups was calculated to be 6.87 µmol/mg UCNPs@TiO2 by TGA analysis (Figure S2a). Next, the ability of the nanophotosensitizer for the UV emission of UCNPs to activate the TiO2 to generate O2•- was evaluated. The upconversion emission spectra of UCNPs and UCNPs@TiO2 were shown in Figure 2b. The emission peaks at 347 nm and 362 nm were assigned to 1I6 - 3F4 and 1D2 - 3H6 transitions of Tm3+ ions doped in NaYF4, respectively. Except emissions in UV


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region, the two blue emission peaks at 452 nm, 476 nm were contributed to the 1D2 - 3F4 and 1G4 - 3H6 transitions of Tm3+ ions. As can be seen from Figure 2a, b, the absorption band of UCNPs@TiO2 overlaps with the UV emissions of the UCNPs, thereby enabling the generation of FRET. Compared with UCNPs, the upconversion emission peaks for UCNPs@TiO2 at 347 nm and 362 nm decreases sharply, which indicated the efficient energy transfer from UCNPs to TiO2 (Figure 2c). IR806 or hydroethidine (HE) probe on the surface of the nanophotosensitizer were employed to label the nanoparticle or determine the O2•- produced by TiO2. The content of IR806 and HE was calculated to be 0.81 and 2.35 µmol/mg UCNPs@TiO2 using fluorescence spectra, respectively (Figure S2b, c). And TEM images showed that almost no obvious morphology change and crosslinking were found after the modifications (Figure S3). As shown in Figure 2d, the fluorescence intensity at 610 nm of HE probe obviously increased compared to that before irradiation when the nanophotosensitizer was irradiated continuously with 980 nm laser for 15 min. The results indicated that the TiO2 shell of the nanophotosensitizer can effectively absorb the UV emission of UCNPs to generate O2•-. Co-localization and cellular internalization pathways. To evaluate the capability of the nanophotosensitizer to selectively target in mitochondria, co-localization imaging experiments in human breast cancer cell line (MCF-7) were performed. Mito-Tracker Green (MTG), a commercial mitochondrial dye, was employed to label mitochondria. First of all, the concentration of TPP group was optimized through the co-localization ability of the nanophotosensitizer. A series of nanoparticles (UCNPs@TiO2-TPP-IR806) (Figure 3a) with different concentrations of TPP were prepared (calculated to be 0.94, 1.95, 2.94, 3.85, 4.87, 5.89 µmol/mg UCNPs@TiO2 using UV-Vis spectra) (Figure S2d). MCF-7 cells were incubated with UCNPs@TiO2-TPP-IR806 for 12 h, and mitochondria were labeled with MTG before the


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imaging experiment. As shown in Figure S4, the co-localization effect increased until the TPP concentration reached 3.85 µmol/mg, so it was chosen in this study. Figure 3b indicated that the fluorescence of nanophotosensitizer overlapped well with that of MTG (Pearson’s correlation coefficient, ρ =0.653), which was evidenced by the clear yellow signals. The above observations are further verified by quantifying fluorescence intensity of the line scanning profiles (Figure 3b). Bio-TEM images of MCF-7 cells incubated with nanophotosensitizer were taken to demonstrate the spatial localization of the nanoparticles. As shown in Figure S5, the nanophotosensitizer were indeed located inside mitochondria. The results confirmed that the nanophotosensitizer could specifically localize in mitochondria of living cells. The intracellular trafficking profile was further evaluated by confocal laser scanning microscopy. MCF-7 cells were incubated with UCNPs@TiO2-TPP-IR806. As shown in Figure S6, when the incubatation time was 2 h, most of the nanoparticles were in endo/lysosome (Pearson’s correlation coefficient, ρ =0.653). After MCF-7 cells were incubated with UCNPs@TiO2-TPP-IR806 for 4 h and the excess nanophotosensitizer was removed, the cells were incubated with fresh culture media for an additional 1, 2, 4 and 8 h. A continuously increased Pearson's correlation coefficient was obtained in the overlay channel. The increased colocalization ratio of nanophotosensitizer within mitochondria from 34.8% to 62.4% showed the transfer to mitochondria within 8 h in living cells (Figure S7). The efficient endosomal escape of the designed nanophotosensitizer can be attributed to their high buffering capacity caused by the untreated amino groups on the surface, which was caused as “proton sponges”.47, 48 To confirm the uptake of nanoparticles in MCF-7 cells, inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed. The analysis showed that the nanoparticles


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were taken up into the cells and the content of Ti in each group was almost the same. The similar result was obtained by quantifying the fluorescence of IR806 in each group (Figure S8). The cellular uptake and internalization pathways were also investigated by applying various endocytosis inhibitors including ethylisopropylamiloride (EIPA, inhibitor of macropinocytosis, 50 µM),49 dynasore (inhibitor of both endocytotic pathways, 100 µM),49 chlorpromazine (inhibitor of clathrin-mediated endocytsis 10 µM),50 and filipin (inhibitor of caveolae-mediated endocytosis, 5 µM).50 As shown in Figure 3c and Figure S9, the fluorescence intensity did not show an obvious change for EIPA-treated cells, indicating that the pathway of endocytosis of the nanophotosensitizer is not mediated by macropinocytosis. However, the fluorescence intensity of cells treated with the other three inhibitors significantly decreased. The results suggested that the nanophotosensitizer might be mainly internalized via caveolae-mediated and clathrin-mediated endocytic pathways, which generally plays a key role for the internalization of nanoparticles into cells.51 Real-time monitoring O2•- burst in living cells. The ability of the nanophotosensitizer to trigger ROS burst in mitochondria and induce cell apoptosis was then evaluated. In order to monitor the ROS change in real-time, hydroethidine (HE), a probe response to O2•-, was employed to anchor on the surface of the nanophotosensitizer (UCNPs@TiO2-TPP-HE) (Figure 3a). After incubated with UCNPs@TiO2-TPP-HE, 6 groups of MCF-7 cells were irradiated for different periods of time (0, 30, 60, 90, 120 and 150 s), respectively. As shown in Figure 4a and Figure S10, no obvious fluorescence signal was observed when the irradiation time was less than 90 s, while bright green fluorescence signals were obtained for longer irradiation time (90, 120, 150 s). The results suggested that the nanophotosensitizer could trigger O2•- burst in


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mitochondria as expected when the irradiation time reached the threshold time, which was consistent with the concept of “mitochondrial criticality”.52, 53 The unique O2•--responsive fluorescence of HE on the nanophotosensitizer made it possible to monitor the mitochondrial O2•- change in real-time, which was beneficial to evaluate the photoactivated cytotoxicity arising from triggered apoptosis process. To verify this capability, the fluorescent changes of UCNPs@TiO2-TPP-HE in MCF-7 cells were tracked in real-time by confocal fluorescence imaging. After incubated with the UCNPs@TiO2-TPP-HE, the cells were irradiated for 90 s to induce apoptosis and performed on the CLSM immediately. The confocal images were captured for 12 h at 1 h interval at the same region. As shown in Figure 4b, a timedependent increase of fluorescence intensity suggested that ROS were continuously produced without further irradiation. The results confirmed that the domino effect of ROS burst indeed happened. After 12 h, very strong fluorescence signal and obvious change of cell morphology were observed, which further confirmed the NIR triggered nanophotosensitizer could induced cell apoptosis (Figure S11). The therapeutic effect of the nanophotosensitizer in living cells. An MTT (3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromide) assay was then employed to evaluate the ability of the nanophotosensitizer to induce cell apoptosis. The absorbance of MTT at 490 nm is dependent on the degree of activation of the cells. Then the cell viability was expressed by the ratio of absorbance of the treated cells (incubated with the nanophotosensitizer or irradiated with NIR laser) to that of the untreated cells. After incubated with UCNPs@TiO2-TPP for 12 h, the MCF-7 cells were irradiated for different periods of time. Figure 5a showed that the cell viability was more than 90% when the irradiation time was less than 60 s, while the cell viability was less than 10% after administration of longer irradiation time (> 90 s). The results indicated when the


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irradiation time reached the critical toxic time, that is, the initial concentration of O2•- was large enough to induce the domino effect on ROS burst in mitochondria, which could induce cell apoptosis. To evidence the pivotal role of the mitochondria targeting for the nanophotosensitizer in inducing domino effect on ROS burst to kill cells, the nanophotosensitizer without TPP groups was also prepared as comparison. After MCF-7 cells incubated with UCNPs@TiO2-NH2 without TPP groups under the same condition as mentioned above, the cell viability was more than 70% even though the irradiation time up to 150 s (Figure 5b). It demonstrated that the initiation of mitochondrial domino effect on O2•- burst is the key factor for cell apoptosis. As a control, the viability of cells treated with only nanophotosensitizer or only NIR laser irradiation were also investigated. The results indicated that more than 90% cells were still alive in both the samples, suggesting that the nanophotosensitizer possessed good biocompatibility and the irradiation of NIR light showed negligible side effects (Figure S12). We further investigated whether mitochondrial antioxidant (MitoQ10) had effect on the apoptosis. As shown in Figure S13a, cell viability was still less than 15% in the presence of MitoQ10, which indicated that it could not reverse cell death. Determination of mitochondrial membrane potential. Previous report showed the loss of mitochondrial membrane potential (∆ψm) was an early event in mitochondria triggered apoptosis.54 The change of ∆ψm was monitored using rhodamine 123 staining. MCF-7 cells were incubated with UCNPs@TiO2-TPP and irradiated for different time as above. Confocal fluorescence imaging indicated that the fluorescence of rhodamine 123 sharply reduced when the cells irradiated more than 90 s from confocal images and the intensity quantification, suggesting the decrease of ∆ψm (Figure S14). This is because the ROS burst activates an inner membrane anion channel (IMAC) and promotes the opening of mitochondrial permeability transition pore


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via oxidation the matrix glutathione.25,55 Cyclosporine A (CsA), a desensitizer of the mitochondrial permeability transition pore (PTP) was employed to clarify the apoptosis mechanism of mitochondrial membrane potential depolarization. MTT results showed that the cell viability was less than 20% in the presence of CsA, which confirmed that the opening of MPTP was not the only reason for ∆ψm reduction (Figure S13b). Cell death pathways. Cell death pathways induced by the nanophotosensitizer were examined by exploring ann exin V-fluorescein isothiocyanate (annexin V-FITC) and propidium iodide (PI) staining. Apoptosis initially induces phosphatidylserine exposure outside the cell membrane without permeabilization. This process enables annexin V-FITC to bind to phosphatidylserine, but PI is unable to enter into cells owing to the integrity of the cell membrane at the initial stage of apoptosis. When the membrane is disrupted upon onset of necrosis, annexin V-FITC and PI interact with the surface and DNA inside the cell, respectively. As shown in Figure 6, neither annexin V-FITC nor PI-stained cells are detected before NIR light irradiation. After treated with NIR light for 4 h, only annexin V-FITC stained cells are detected, indicating the inversion of phosphatidylserine and no permeabilization of the cell membrane, which confirmed the early apoptosis stage. As controls, neither annexin V-FITC nor PI-stained cells were observed when cells were treated with UCNPs@TiO2-NH2 upon irradiation for 90 s or with only irradiation for 90 s. When the incubation time extended to 12 h after irradiation, both annexin V-FITC and PI stained cells were observed, indicating that the membrane is disrupted at late apoptosis stage. (Figure S15) These results suggested that O2•- induced cell death by the nanophotosensitizer occurred predominantly through mitochondria. Caspase 3 activation. Apoptosis of MCF-7 cells through the mitochondrial signaling pathways was also evidenced by the activity of caspase-3 using the method of


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immunefluorescent staining. As shown in Figure 7, increased activity of caspase-3 was detected when MCF-7 cells were loaded the nanophotosensitizer and irradiated for 90 s. The therapeutic effects of the nanophotosensitizer in vivo. We next assessed the ability of the nanophotosensitizer for PDT against cancer in a mouse model. A xenograft mouse model (the mice were treated with MCF-7 cells) was then applied to evaluate the therapeutic effect of the nanophotosensitizer in tumor tissue. Figure 8a illustrates the schematic diagram of the NIR light triggered PDT for cancer therapy. MCF-7 cells are first xenografted to the flank of the mice. The nanophotosensitizer dispersed in PBS buffer (1 mg/ml, 50 µL), was then directly injected into the tumor for each mouse. The NIR laser with a parameter of 3 W·cm-2 was conducted at the tumor region for 90 s. As can be seen in Figure 8b, the tumor was completely removed after 14 days when treated with the nanophotosensitizer upon NIR laser irradiation for 90 s. The change in the tumor volume was monitored over a period of 14 days without extra irradiation. In the control group of mice treated with PBS buffer, the tumor size was found to increase about 5-fold over this period (black line in Figure 8c). Notably, the tumors of mice for the experimental group with nanophotosensitizer mediated PDT were eliminated by day 6 (blue line in Figure 8c). Another 3 groups of mice were treated with NIR laser only, nanophotosensitizer only and UCNPs@TiO2-NH2 with irradiation for 90 s, respectively. The changes of the tumor size were monitored in the same way and a similar trend was observed as the control group. As can be seen in Figure 8b, c, the tumor grew rapidly as same as the control group. Body weight is an important parameter to evaluate the systemic toxicity of the material to the body. As shown in Figure 8d, the body weight of all groups does not decrease with the time prolonged in 14 days, implying that the treatments did not show obvious toxicity. In addition, the treatment efficacy in term of tumor cell death was also evaluated by H&E staining on tissue sections from the


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different treatment groups at 12 h after treatment. The tumors treated with nanophotosensitizer upon irradiation exhibited a wide range of tissue damage in histological sections, while most tumor cells showed no obvious change in the control groups (Figure 9e, f). The histological effect of nanophotosensitizer on five major organs (liver, lung, spleen, kidney, and heart) of healthy mice was monitored at 7 days after intratumor injection and no histopathological abnormalities were found (Figure S16). These results indicated that the nanophotosensitizer is highly effective for cancer therapy using PDT and has little side effects to normal issue via intratumor administration. CONCLUSION In conclusion, we have demonstrated a near-infrared triggered nanophotosensitizer based on mitochondria targeted titanium dioxide-coated upconversion nanoparticles, which can be used for photodynamic therapy against cancer in living cells and in vivo. To the best of our knowledge, this is the first time that cancer therapy could be achieved using a photosensitizer to induce mitochondrial ROS burst. The nanophotosensitizer makes use of the advantages of UCNPs, such as remarkable light penetration depth, large anti-Stokes shifts and high photochemical stability. The coated TiO2 can be photoactivated by the emission of UCNPs with an NIR laser, which could produce large amounts of ROS. The triphenylphosphine modification can guide the nanophotosensitizer to specifically target mitochondria. When irradiated with a 980 nm NIR laser, the nanophotosensitizer can selectively trigger the mitochondrial ROS burst and initiate a series of cascade reaction, leading to mitochondrial collapse and irreversible cell apoptosis. Confocal fluorescence imaging indicated that the nanophotosensitizer could target mitochondria and induce domino effect on ROS burst above the critical irradiation time in living cells. MTT assay confirmed the designed nanophotosensitizer could effectively destroy cancer cells with less


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than 10% of the cell viability. Moreover, it revealed mitochondria targeting of the nanophotosensitizer played a pivotal role in inducing the domino effect on ROS burst and cell apoptosis. In vivo study demonstrated that the tumor could be completely removed owing to the excellent therapeutic effect of the nanophotosensitizer. We anticipate that this novel approach can provide new insights for cancer therapy.


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Scheme 1. Schematic illustration of (a) the structure of the naophotosensitizer (TPP anchored UCNPs@TiO2

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Figure 1. Characterization of the nanophotosensitizer. High resolution transmission electron microscopy images of OA-capped NaYF4:Yb3+,Tm3+ (a); OA free NaYF4:Yb3+,Tm3+ (b); UCNPs@TiO2 (c). Scale bars are 25 nm.


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12000

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Figure 2. Characterization of the FRET and O2•- production. (a) Absorption spectra of the pure UCNPs and UCNPs@TiO2. (b) Emission spectra of pure UCNPs and UCNPs@TiO2 under 980 nm laser. (c) Amplified images of black box shown in b. (d) Fluorescence spectra of nanophotosensitizer UCNPs@TiO2-TPP-HE before and after irradiation with 980 nm laser for 15 min. λex/λem=488/610 nm.


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Figure 3. Mitochondrial targeting and cellular uptake pathways. (a) Schematic illustration of the structure of UCNPs@TiO2-TPP-IR806 and UCNPs@TiO2-TPP-HE. Structure formulae of IR806 and HE are showed on the bottom. (b) Mitochondrial targeting of nanophotosensitizer under confocal imaging. MCF-7 cells were incubated with UCNPs@TiO2-TPP-IR806 for 12 h before

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emission=750-800 nm), Mito-Tracker Green (MTG) stained mitochondria (excitation=488 nm, emission=500-550 nm), the overlay channel of nanophotosensitizer and mitochondria(top); The quantification of fluorescent intensity of the line scanning profiles in the corresponding confocal images in b(bottom). (c) Confocal imaging of MCF-7 cells treated without (control) with dynasore (inhibitor of dynamin-mediated uptake, 100 µM), chlorpromazine (inhibitor of clathrinmediated uptake, 10 µM), ethylisopropylamiloride (EIPA, inhibitor of macropinocytosis, 50 µM)


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and filipin (inhibtor of caveolaemediated uptake, 5 µM) before incubated with UCNPs@TiO2TPP-IR806 (0.1 mg/mL).

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Figure 4. Real-time imaging of intracellular O2•- burst. (a) Confocal imaging of O2·- in MCF-7 cells. After incubated with UCNPs@TiO2-TPP-HE (0.1 mg/mL) and irradiated for different time, the confocal images were captured at 12 h. (b) Real-time monitoring the fluorescence of O2·- for 12 h at 1 h interval. The increasing green pixels indicated the domino effect on O2·-. The power of the irradiation was 3W•cm-2.


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(a)

(b) 100 Cell viability/ %

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80 60 40 20 0 control 30s 60s 90s 120s 150s

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Figure 5. Cell viability. Cell viability of MCF-7 cells incubated with the nanophotosensitizer (0.1 mg/mL) (a) and UCNPs@TiO2-NH2 (0.1 mg/mL) (b) upon irradiation for different period of time. The power of the irradiation was 3W·cm-2. Cell viability was measured 24 h after the irradiation.


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DAPI

Annexin V

PI

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Figure 6. Cell apoptosis pathway. Confocal images of DAPI and Annexin V-FITC/PI stained MCF-7 cells with different treatments: (a) the nanophotosensitizer (0.1 mg/mL) with laser irradiation for 90 s; (b) UCNPs@TiO2-NH2 (0.1 mg/mL) with laser irradiation for 90 s; (c) laser irradiation for 90 s only; (d) control group without treatment. The power of the irradiation was 3W•cm-2. Confocal images were captured 4 h after irradiation.


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hochest

caspase 3

merge

bright field

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Figure 7. Immunofluorescent staining images of caspase 3. (a) MCF-7 cells without any treatment. (b) MCF-7 cells incubated with the nanophotosensitizer (0.1mg/ mL) and irradiated with NIR laser for 90 s (3W·cm-2). Confocal images were taken 12 h after irradiation.

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Figure 8. In vivo application of nanophotosensitizer in a mouse model. (a) Schematic illustration of the PDT treatment set-up. (b) Photographs of the mice taken before treatment (0 day) and at 14 days with different treatments: i, PBS only; ii, PBS with laser irradiation for 90 s; iii, nanophotosensitizer only; iv, UCNPs@TiO2-NH2 with irradiation for 90 s; v, nanophotosensitizer with irradiation for 90 s. A dosage of nanophotosensitizers in PBS (1 mg/mL, 50 µL) was administrated intratumorlly for all mice (n ≥ 5). Tumor growth curves (c) and mice body weight curves (d) of different groups of tumor-bearing mice after PDT. They were measured at 2 days interval for 14 days. The power of the irradiation was 3W·cm-2.

(a)

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Figure 9. H&E staining of tumor slides. The tumors were treated differently: (a) PBS only; (b) PBS with laser irradiation for 90 s; (c) nanophotosensitizer only; (d) UCNPs@TiO2-NH2 with irradiation for 90 s; (e) nanophotosensitizer with irradiation for 90 s. (f) Amplified images of the black box shown in e. The power of the irradiation was 3W·cm-2.


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MATERIALS AND METHODS Materials and Regions. Hydroethidine (HE), 1-Octadecene (ODE), Rare earth oxides Yttrium(III) oxide (Y2O3), Ytterbium(III) oxide (Yb2O3), Thulium(III) oxide (Tm2O3) and 3(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Company; Oleic acid (OA), (4-Carboxybutyl)triphenylphosphonium bromide, (3aminopropyl)triethoxysilane

(APTES),

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Alfa Aesar Chemical Ltd (tianjin, China); Tetrabutyl titanate (TBOT) was purchased from China National Pharmaceutical Group Corporation (Shanghai, China). Annexin V-FITC/propidium iodide (PI) Cell Apoptosis Kit and Cyclosporine A (CsA) were obtained from Sangon Biotechnology Co., Ltd (Shanghai, China). Mito-Tracker Green was purchased from Molecular Probes (Invitrogen, USA); MitoQ was purchased from Vosun chemical Co., Ltd (Suzhou, China). Oleic acid and 1Octadecene were of technical grade and the others were of analytical grade. All the chemicals were used without further purification. The human breast cancer cell line (MCF-7) was purchased from KeyGEN biotechnology Company (Nanjing, China). Synthesis of NaYF4:20%Yb3+,0.2%Tm3+ nanocrystals. The β-phase NaYF4:Yb3+,Tm3+ nanocrystals were prepared via a solvothermal method with some modifications.44 To obtain rare earth chlorides, 1 mmol rare earth oxides Y2O3, Yb2O3, and Tm2O3 with a stoichiometric ratio of 79.8:20:0.2 were dissolved in hydrochloric acid, and then the solution was stirred and heated to evaporate the water completely. In the typical synthesis procedure, YCl3 (0.798 mmol), YbCl3 (0.20 mmol), and TmCl3 (0.002 mmol) were dispersed in oleic acid (OA, 8 mL) and 1octadecene (ODE, 18 mL), and then the mixture was heated to 160 °C for 30 min. After a homogeneous solution was formed, the mixture was cooled to room temperature, then followed by adding NaOH (0.1 g, 2.5 mmol) and NH4F (0.148 g, 4 mmol) in 10 mL methanol solution


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under vigorously stirring for 30 min. The temperature was heated to 100 °C to evaporate methanol, then was raised to 295 °C in an argon atmosphere for 90 min and finally cooled down to room temperature naturally. The resulting NaYF4:Yb,Tm nanoparticles were precipitated by adding ethanol and then centrifuged and washed with ethanol and cyclohexane for several times. The precipitates were redispersed in 10 mL cyclohexane solution. Synthesis of UCNPs@TiO2 core-shell nanoparticles. Before the synthesis, the OA ligand on the NaYF4:Yb,Tm surface was removed.44 0.5 mL of as-synthesized Oleate-capped UCNPs was dispersed in a 10 mL aqueous solution, and the pH was adjusted to 2 by adding a solution of HCl 0.5 M. The reaction was performed with vigorous stirring for 4 h. The nanoparticles were centrifuged and washed with ethanol for three times. UCNPs@TiO2 was synthesized according to a versatile kinetics-controlled coating method with some modifications.45 The core particles were dispersed in 25 mL absolute ethanol and mixed with concentrated ammonia solution (75 µL, 28 wt%) under ultrasound for more than 15 min. Afterward, 120 µL of TBOT was added dropwise in 5 min, and the reaction was allowed to proceed for 24 h at 45 oC under continuous mechanical stirring. The resultant products were separated and collected, followed by washing with deionized water and ethanol for 3 times, respectively. Synthesis of UCNPs@TiO2-TPP and UCNPs@TiO2-TPP-IR806. UCNPs@TiO2-NH2 was synthesized firstly. As-prepared UCNPs@TiO2 (2 mg) was dispersed in a solution of ethanol (10 mL) and deionized water (100 µL) under stirring for 15 min. Then, 10 µL APTES (40 µmol) was added to the mixture and the reaction was processed for another 12 h. The precipitates were washed and redispersed in 4 mL MES buffer (10 mM, pH 6.0). The content of amino groups was measured by TGA analysis. UCNPs@TiO2-TPP-IR806 was obtained by coupling the carboxl groups of the (4-Carboxybutyl)triphenylphosphonium bromide and infrared dye IR806 and the


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amino group on the surface of UCNPs@TiO2-NH2 to form the amido bonds. EDC (20 µmol), NHS (20 µmol) and EDC (20, 40, 60, 80, 100, 120 µmol), NHS (20, 40, 60, 80, 100, 120 µmol) were added to IR806 (2 µmol) and (4-Carboxybutyl)triphenylphosphonium bromide (2, 4, 6, 8 ,10, 12µmol ) solution, respectively. The reaction was performed for 30 min at room temperature in the dark to activate carboxylate groups and then both of them were added to above UCNPs@TiO2-NH2 solution under gentle stirring for 12 h which resulted in the formation of the amido bonds. Following this, the precipitates were centrifuged (10000 rpm, 10 min) and washed with methanol and PBS buffer (10 mM, pH 7.4) for three times and finally redispersed in PBS buffer (1 mg/mL). The content of TPP groups and IR806 molecules was calculated according to the standard linear calibration curve of each group using subtraction through UVVis absorption spectra and fluorescence analysis, respectively. Synthesis of UCNPs@TiO2-TPP-HE. UCNPs@TiO2-NH2 and UCNPs@TiO2-TPP were prepared successively with the method mentioned above and further modified with carboxyl groups.56 UCNPs@TiO2-TPP were collected by centrifugation and redispersed in 4 mL DMSO containing triethylamine (1 mg) and succinic anhydride (1 mg). The mixture was allowed to stir at 40 °C for 48 h. The carboxylic acid-functionalized particles were centrifuged and washed for three times and finally redispersed in MES buffer (10 mM, pH 6.0). HE probe was anchored on the nanophotosensitizer by formation of amido bond.57 19.1 mg EDC (100 µmol) and 11.5 mg NHS (100 µmol) were added to the above solution with reaction for 30 min to activate carboxylate groups. Finally, HE probe (10 µmol) in methanol was mixed under gentle stirring for 12 h in the dark and the resulting core-shell nanoparticles were centrifuged and washed with deionized water and redispersed in PBS buffer (1 mg/ml). The content of HE molecules was


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calculated according to the standard linear calibration curve of each group using subtraction through fluorescence analysis. Cell culture. MCF-7 cells were cultured in Dulbecco’s modified Eagles medium (DMEM) with 10% fetal bovine serum and 100 U/ml 1% antibiotics penicillin/streptomycin and maintained at 37 °C in a 100% humidified atmosphere containing 5% CO2. Co-localization into mitochondria. MCF-7 cells were seeded in a confocal dish for 24 h. Then, UCNPs@TiO2-TPP-IR806 (0.1 mg/mL) was delivered into the cells in DMEM culture medium. After incubation for 12 h, Cells were then washed three times with PBS buffer to remove the nanoparticles that were not uptake into the cells. Then, 2 mL fresh DMEM culture medium was added and the cells were stained by Mito-Tracker Green (25 nM) at 37 °C for 15 min. The cells were then washed by PBS twice and immediately observed using confocal laser scanning microscopy (CLSM) and confocal images of cells fluorescence were captured with 488 nm excitation for Mito-Tracker Green (emission=500-550 nm) and 633 nm excitation for IR806 (emission=750-800 nm). The co-localization ratio of Mito-Tracker Green with IR806 of UCNPs@TiO2-TPP-IR806 was quantified using Image-Pro Plus Imaging software. Cellular uptake and internalization pathways. MCF-7 cells were cultured in 96-well microtiter plates and incubated at 37 °C in 5% CO2 for 24 h. Cells were incubated with different inhibitors including dynasore (inhibitor of dynamin-mediated uptake, 100 µM), chlorpromazine (inhibitor of clathrin-mediated uptake, 10 µM), ethylisopropylamiloride (EIPA, inhibitor of macropinocytosis, 50 µM) and filipin (inhibitor of caveolae-mediated uptake, 5 µM) in serum free DMEM medium for 30 min prior to incubation with the nanoparticles UCNPs@TiO2-TPPIR806 (0.1 mg/mL) for further 12 h. Subsequently, the medium was removed and the cells were


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washed 3 times using PBS. Confocal images were obtained by excitation of the samples at 633 nm. Intracellular trafficking UCNPs@TiO2-TPP-IR806. Six groups of MCF-7 cells were seeded in confocal dishes for 24 h. Then, UCNPs@TiO2-TPP-IR806 (0.1 mg/mL) was delivered into the cells in DMEM culture medium at 37 °C in 5% CO2. One group of cells were incubated for 2 h, washed three times and then stained with Lyso-Tracker DND-26 at 37 °C for 15 min. The cells were then washed by PBS twice and immediately observed using confocal laser scanning microscopy (CLSM) and confocal images of cells fluorescence were captured with 488 nm excitation for Lyso-Tracker DND-26 and 633 nm excitation for IR806. Another five groups of cells were incubated with UCNPs@TiO2-TPP-IR806 (0.1 mg/mL) for 4 h. Then the cells were washed with PBS buffer to remove the nanoparticles that were not uptake into the cells. Then, 2 mL fresh DMEM culture medium was added and the cells were further cultured for 0, 1, 2, 4 and 8 h. Subsequently, the cells were stained by Mito-Tracker Green (25 nM) at 37 °C for 15 min. The cells were then washed by PBS twice and immediately observed using confocal laser scanning microscopy (CLSM) and confocal images of cells fluorescence were captured with 488 nm excitation for Mito-Tracker Green and 633 nm excitation for IR806. The IR806 fluorescence intensity of the cells in each group was also quantified. The co-localization ratio of Lyso-Tracker DND-26 or Mito-Tracker Green with IR806 of UCNPs@TiO2-TPP-IR806 was quantified via Image-Pro Plus Imaging software. ICP-AES. Five groups of MCF-7 cells (seeded at 1×105/mL) were incubated with UCNPs@TiO2-TPP-IR806 (0.1 mg/mL) for 4 h. Then the cells were washed with PBS buffer to remove the nanoparticles that were not uptake into the cells. Then, 2 mL fresh DMEM culture medium was added and the cells were further cultured for 0, 1, 2, 4 and 8 h. At the end of


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incubation, the cells were washed with PBS buffer and collected in centrifuge tubes. Next, the cells were treated with nitric acid (2 mL), hydrofluoric acid (3 mL) and perchloric acid (0.5 mL), and then heated to dissolve TiO2. The samples were finally solved in 4 mL hydrochloric acid, and then analyzed for total Ti content by ICP-AES (Thermo, IRIS Advantage, 308.8 nm) and the measurement was repeated three times. In vitro detecting O2•- burst. 6 groups of MCF-7 cells were seeded in a confocal dish and incubated at 37 °C in 5% CO2 for 24 h. UCNPs@TiO2-TPP-HE (0.1 mg/mL) was delivered into the cells in DMEM culture medium for 12 h. Then, the cells were washed with PBS buffer to remove the nanoparticles outside the cells and fresh DMEM medium containing 10 % fetal bovine serum medium was added. 6 groups of the cells were irradiated with 980 nm laser for 0, 30, 60, 90, 120 and 150 s, respectively. Subsequently, the cells were examined with confocal laser scanning microscopy (CLSM) with 488 nm excitation. Next, the cells were cultured for another 12 h and were also examined with confocal laser scanning microscopy (CLSM) with the same parameters. Real-time monitoring O2•- experiment was also carried out. The cells were incubated with UCNPs@TiO2-TPP-HE (0.1 mg/mL) for 12 h. Then, the cells were washed with PBS buffer. After that, fresh DMEM medium containing 10% fetal bovine serum medium was added followed with irradiation with 980 nm laser for 90 s. Subsequently, the cells were examined with confocal laser scanning microscopy (CLSM) with 488 nm excitation for 12 h. Confocal images were obtained at 1 h interval. In vitro cytotoxicity. (1)To inspect the applicability of UCNPs@TiO2-TPP, 2 groups of MCF7 cells were cultured in 96-well microtiter plates and incubated at 37 °C in 5% CO2 for 24 h. UCNPs@TiO2-TPP (0.1 mg/mL) and UCNPs@TiO2-NH2 (0.1 mg/mL) were delivered into the


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cells in DMEM culture medium for 12 h followed by washing the cells with PBS buffer to remove the nanoparticles that were not uptake into the cells. Then, each plate of MCF-7 cells was divided into 6 groups and the laser irradiation of 980 nm (3W·cm-2) was performed on each group for 0, 30, 60, 90, 120 and 150 s, respectively. The cells were further cultured for 24 h. Next, 150 µL MTT solution (0.5 mg/mL) was added to each well. After 4 h, the remaining MTT solution was removed, and 150 µL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm with microplate reader (Synergy 2, Biotek, USA). (2) MCF-7 cells were cultured in 96-well microtiter plates and incubated at 37 °C in 5% CO2 for 24 h. Cells were divided into 3 groups. UCNPs@TiO2-TPP (0.1 mg/mL) without irradiation and only irradiation with 980 nm laser were performed, respectively. And another groups of cells as the control without any treatment. Next, 150 µL MTT solution (0.5 mg/mL) was added to each well. After 4 h, the remaining MTT solution was removed, and 150 µL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm with microplate reader (Synergy 2, Biotek, USA). (3) Two groups of MCF-7 cells loaded with the nanophotosensitizer (0.1mg/mL) were irradiated with the CsA (1 µM) and MitoQ10 (20 µM) for 5 min, respectively. Then the cells were irradiated with NIR laser for 90 s (3W·cm-2). After incubation for another 24 h, MTT assay was carried out as the same procedure above. Detection of mitochondrial membrane potential (∆Ψm). MCF-7 cells were cultured in 96well microtiter plates and incubated at 37 °C in 5% CO2 for 24 h. UCNPs@TiO2-TPP (0.1 mg/mL) was delivered into the cells in DMEM culture medium for 12 h. Then, the cells were irradiated for 90 s and further incubated for 4 h. Then, the cells were incubated with rhodamine


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123 (5 µg/mL) for 15 min in darkness at 37 °C. Confocal images of rhodamine 123-stained cells were obtained by excitation of the samples at 488 nm (emission=500-550 nm). Cell apoptosis. For monitoring cell death pathways, the Annexin V-FITC/PI Apoptosis Detection Kit is used. Here, MCF-7 cells were incubated with 0.1 mg/mL of UCNPs@TiO2-TPP at 37 °C in 5% CO2 for 12 hours. After that, the cells were washed thrice with DMEM medium and then subjected to the 980 nm NIR laser irradiation for 90 s. At 4 h post treatment, the cells were washed thrice with DMEM medium, twice with Millipore water and then changed with 0.2 mL Annexin-binding buffer containing 10 µL annexin V-FITC and PI. The cells were incubated for 15 min at room temperature. After the cellular incubation, annexin-binding buffer was added and mixed with samples gently, and then the samples were applied for imaging measurements. Cells were examined with confocal laser scanning microscopy (CLSM) with 405 nm excitation for DAPI, 488 nm excitation for FITC and 543 nm excitation for PI. As the control, cells treated with UCNPs@TiO2-NH2 (0.1 mg/mL) instead of UCNPs@TiO2-TPP, with only 980 nm laser irradiation for 90 s and without any treatment were examined the same as above. Another experiment of MCF-7 cells incubated with the nanophotosensitizer (0.1 mg/mL) was carried out at 12 h post irradiation (3W·cm-2) for 90 s as the same procedure above. Caspase 3 activation. Briefly, MCF-7 cells were incubated with 0.1 mg/mL of UCNPs@TiO2-TPP at 37 °C in 5% CO2 for 12 hours. Then, the cells were washed thrice with DMEM medium and then subjected to the 980 nm NIR laser irradiation for 90 s. After further incubation for 12 h, the cells were fixed with paraformaldehyde (4%) for 10 min, and then treated with primary antibody anti-caspase 3, enhanced secondary antibody for 1 h and 40 min at room temperature, respectively. At last, the cells were washed with PBS before CLSM


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experiment. (Excitation=633 nm, Emission=640-700 nm) Another group of cells without any treatment as the control was performed as the same procedure above. Animal tumor xenograft models. All animal experiments were carried out according to the Principles of Laboratory Animal Care (People's Republic of China) and the Guidelines of the Animal Investigation Committee, Biology Institute of Shandong Academy of Science, China. Female nude mice (4-6 week old, ~20 g) were housed under normal conditions with 12 h light and dark cycles and given access to food and water ad libitum. For xenografts established from cultured cells, MCF-7 cells were suspended and harvested after trypsinization and approximately 1×106 MCF-7 cells in 150 µL PBS were injected subcutaneously into the flank of the mice. The tumor volume (V) was determined by measuring length (L) and width (W), and calculated as V=L×W2/2. The relative tumor volumes were calculated for each mouse as V/V0 (V0 was the tumor volume when the treatment was initiated). The drug was administrated when the tumor grow to about 80-100 mm3. In vivo antitumor efficacy via injection. When the tumor volume reached to about 80-100 mm3 the tumor-bearing mice were weighed and randomly divided into different groups (n≥ 5). The mice were subjected to 5 different treatments: group1, PBS only; group 2, laser only; group 3, UCNPs@TiO2-TPP only; treatment group: group 4, UCNPs@TiO2-NH2 combined with laser irradiation; 5, UCNPs@TiO2-TPP combined with laser irradiation. 50 µL PBS, 50 µL 1.0 mg/ml UCNPs@TiO2-TPP or UCNPs@TiO2-NH2 in PBS were intratumorly injected into corresponding group. 8 h later, laser treatment was performed on groups 2, 3 and 4 by irradiating the tumor region with 980 nm laser at a power of 3 W·cm-2 for 90 s. The tumor size and the body weights of the mice were measured every other day for 14 days (day 0, 2, 4, 6, 8, 10, 12 and 14).


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ASSOCIATED CONTENT Supporting Information Instruments and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by 973 Program (2013CB933800), National Natural Science Foundation of China (21535004, 21227005, 21390411, 21422505, 21375081, 21505087), and Natural Science Foundation for Distinguished Young Scholars of Shandong Province (JQ201503). REFERENCES 1.

Chatterjee, D. K.; Fong, L. S.; Zhang, Y. Nanoparticles in photodynamic therapy: an emerging paradigm. Adv. Drug Delivery Rev. 2008, 60, 1627-1637.

2.

Lovell, J. F.; Liu, T. W. B.; Chen, J.; Zheng, G. Activatable Photosensitizers for Imaging and Therapy. Chem. Rev. 2010, 110, 2839-2857.

3.

Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869-10939.


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4.

Page 32 of 39

Ge, J.; Lan, M.; Zhou, B.; Liu, W.; Guo, L.; Wang, H.; Jia, Q.; Niu, G.; Huang, X.; Zhou, H. et. al. A Graphene Quantum Dot Photodynamic Therapy Agent with High Singlet Oxygen Generation. Nat. Commun. 2014, 5, 4596-4603.

5.

Olivo, M.; Bhuvaneswari, R.; Lucky, S. S.; Dendukuri, N.; Thong, P. S. Targeted Therapy of Cancer Using Photodynamic Therapy in Combination with Multi-faceted Anti-Tumor Modalities. Pharmaceuticals 2010, 3, 1507-1529.

6.

Dolmans, E. J. G. J.; Fukumura, D.; Jain, P. K. Photodynamic therapy for Cancer. Nat. Rev. Cancer 2003, 3, 380-387.

7.

Castano, A. P.; Mroz, P.; Hamblin, M. R. Photodynamic therapy and anti-tumour immunity. Nat. Rev. Cancer 2006, 6, 535-545.

8.

Moore, C. M.; Pendse, D.; Emberton, M. Photodynamic Therapy for Prostate Cancer-A Review of Current Status and Future Promise. Nat. Clin. Pract. Urol. 2009, 6, 18-30.

9.

Yuan, H.; Chong, H.; Wang, B.; Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Chemical Molecule-Induced Light-Activated System for Anticancer and Antifungal Activities. J. Am. Chem. Soc. 2012, 134, 13184-13187.

10. Niedre, M.; Patterson, M. S.; Wilson, B. C. Direct Near-infrared Luminescence Detection of Singlet Oxygen Generated by Photodynamic Therapy in Cells In Vitro and Tissues In Vivo. Photochem. Photobiol. 2002, 75, 382-391. 11. Kuimova, M. K.; Botchway, S. W.; Parker, A. W.; Balaz, M.; Collins, H. A.; Anderson, H. L.; Suhling, K.; Ogilby, P. R. Imaging Intracellular Viscosity of A Single Cell During Photoinduced Cell Death. Nat. Chem. 2009, 1, 69-73. 12. Ethirajan, M.; Chen, Y.; Joshi P.; Pandey, R. K. The Role of Porphyrin Chemistry in Tumor Imaging and Photodynamic Therapy. Chem. Soc. Rev. 2011, 40, 340-362.


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13. Lim, S. H.; Thivierge, C.; Nowak-Sliwinska, P.; Han, J.; van den Bergh, H.; Wagnieres, G.; Burgess, K.; Lee, H. B. In Vitro and In Vivo Photocytotoxicity of Boron Dipyrromethene Derivatives for Photodynamic Therapy. J. Med. Chem. 2010, 53, 2865-2874. 14. Hatz, S.; Lambert, J. D. C.; Ogilby, P. R. Measuring the Lifetime of Singlet Oxygen in A Single Cell: Addressing the Issue of Cell Viability. Photochem. Photobiol. Sci. 2007, 6, 1106-1116. 15. Rajaputra, P.; Nkepang, G.; Watley, R.; You, Y. Synthesis and In Vitro Biological Evaluation of Lipophilic Cation Conjugated Photosensitizers for Targeting Mitochondria. Bioorg. Med. Chem. 2013, 21, 379-387. 16. Lucky, S. S.; Idris, N. M.; Li, Z.; Huang, K.; Soo, K. C.; Zhang, Y. Titania Coated Upconversion Nanoparticles for Near-Infrared Light Triggered Photodynamic Therapy. ACS Nano, 2015, 9, 191-205. 17. Hou, Z.; Zhang, Y.; Deng, K.; Chen, Y.; Li, X.; Deng, X.; Cheng, Z.; Lian, H.; Li, C.; Lin, Jun. UV-Emitting Upconversion-Based TiO2 Photosensitizing Nanoplatform: Near-Infrared Light Mediated in Vivo Photodynamic Therapy via Mitochondria-Involved Apoptosis Pathway. ACS Nano, 2015, 9, 2584-2599. 18. Liu, K.; Liu, X.; Zeng, Q.; Zhang, Y.; Tu, L.; Liu, T.; Kong, X.; Wang, Y.; Cao, F.; Lambrechts, S. A. G. et. al. Covalently Assembled NIR Nanoplatform for Simultaneous Fluorescence Imaging and Photodynamic Therapy of Cancer Cells. ACS Nano, 2012, 6, 4054-4062. 19. Balaban, R. S.; Nemoto, S.; Finkel, T. Mitochondria, Oxidants, and Aging. Cell 2005, 120, 483-495.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 39

20. Hoye, A. T.; Davoren, J. E.; WIPF, P. Targeting Mitochondria. Acc. Chem. Res. 2008, 41, 87-97. 21. Kroemer, G.; Galluzzi, L.; Brenner, C. Mitochondrial Membrane Permeabilization in Cell Death. Physiol. Rev. 2007, 87, 99-163. 22. Wallace, D. C. Mitochondrial Diseases in Man and Mouse. Science 1999, 283, 1482-1488. 23. Lin, M. T.; Beal, M. F. Mitochondrial Dysfunction and Oxidative Stress in Neurodegenerative Diseases. Nature 2006, 443, 787-795. 24. Kroemer, G.; Pouyssegur, J. Tumor Cell Metabolism: Cancer's Achilles' Heel. Cancer Cell 2008, 13, 472-482. 25. Aon, M. A.; Cortassa, S.; Maack1, C.; O’Rourke, B. Sequential Opening of Mitochondrial Ion Channels as a Function of Glutathione Redox Thiol Status. J. Biol. Chem. 2007, 282, 21889-21900. 26. Green, D. R.; Reed, J. C. Mitochondria and Apoptosis. Science 1998, 281, 1309-1312. 27. Aronis, A.; Melendez, J. A.; Golan, O.; Shilo, S.; Dicter, N.; Tirosh, O. Potentiation of FasMediated Apoptosis by Attenuated Production of Mitochondria-Derived Reactive Oxygen Species. Cell Death Differ. 2003, 10, 335-344. 28. Kachynski, A. V.; Pliss, A.; Kuzmin, A. N.; Ohulchanskyy, T. Y.; Baev, A.; Qu, J.; Prasad, P. N. Photodynamic Therapy by In Situ Nonlinear Photon Conversion. Nat. Photonics 2014, 8, 455-461. 29. Cui, S.; Yin, D.; Chen, Y.; Di, Y.; Chen, H.; Ma, Y.; Achilefu, S.; Gu, Y. In Vivo Targeted Deep-Tissue Photodynamic Therapy Based on Near-Infrared Light Triggered Upconversion Nanoconstruct. ACS Nano 2013, 7, 676-688.


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Page 35 of 39

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30. Wang, F.; Han, Y.; Lim, C. S.; Lu, Y.; Wang, J.; Xu, J.; Chen, H.; Zhang, C.; Hong, M.; Liu, X. Simultaneous Phase and Size Control of Upconversion Nanocrystals Through Lanthanide Doping. Nature 2010, 463, 1061-1065. 31. Wang, F.; Deng, R.; Wang, J.; Wang, Q.; Han, Y.; Zhu, H.; Chen, X.; Liu, X. Tuning Upconversion through Energy Migration in Core–Shell Nanoparticles. Nat. Mater. 2011, 10, 968-973. 32. Xie, X.; Gao, N.; Deng, R.; Sun, Q.; Xu, Q.; Liu, X. Mechanistic Investigation of Photon Upconversion in Nd3+-Sensitized Core-Shell Nanoparticles. J. Am. Chem. Soc. 2013, 135, 12608-12611. 33. Zheng, W.; Huang, P.; Tu, D.; Ma, E.; Zhu, H.; Chen, X. Lanthanide-Doped Upconversion Nano-Bioprobes: Electronic Structures, Optical Properties, and Biodetection. Chem. Soc. Rev. 2015, 44, 1379-1415. 34. Wang, M.; Chen, Z.; Zheng, W.; Zhu, H.; Lu, S.; Ma, E.; Tu, D.; Zhou, S.; Huang, M.; Chen, X. Lanthanide-Doped Upconversion Nanoparticles Electrostatically Coupled with Photosensitizers for Near-Infrared-Triggered Photodynamic Therapy. Nanoscale, 2014, 6, 8274-8282. 35. Idris, N. M.; Gnanasammandhan, M. K.; Zhang, J.; Ho, P. C.; Mahendran, R.; Zhang, Y. In Vivo Photodynamic Therapy Using Upconversion Nanoparticles as Remote-Controlled Nanotransducers. Nat. Med. 2008, 18, 1580-1586. 36. Liu, J.; Liu, Y.; Liu, Q.; Li, C.; Sun, L.; Li, F. Iridium(III) Complex-Coated Nanosystem for Ratiometric Upconversion Luminescence Bioimaging of Cyanide Anions. J. Am. Chem. Soc. 2011, 133, 15276-15279.


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Page 36 of 39

37. Deng, R.; Xie, X.; Vendrell, M.; Chang, Y. T.; Liu, X. Intracellular Glutathione Detection Using MnO2-Nanosheet-Modified Upconversion Nanoparticles. J. Am. Chem. Soc. 2011, 133, 20168-20171. 38. Chen, X.; Mao, S. S. Titanium Dioxide Nanomaterials: Synthesis, Properties, Modifications, and Applications. Chem. Rev. 2007, 107, 2891-2959. 39. Vijayaraghavan, P.; Liu, C. H.; Vankayala, R.; Chiang, C. S.; Hwang, K. C. Designing Multi-Branched Gold Nanoechinus for NIR Light Activated Dual Modal Photodynamic and Photothermal Therapy in the Second Biological Window. Adv. Mater. 2014, 26, 6689-6695. 40. Qin, W.; Zhang, D.; Zhao, D.; Wang L.; Zheng, K. Near-Infrared Photocatalysis Based on YF3 : Yb3+,Tm3+/TiO2 Core/Shell Nanoparticles. Chem. Commun. 2010, 46, 2304-2306. 41. Li, Z.; Li, C.; Mei, Y.; Wang, L.; Dua, G.; Xiong, Y. Synthesis of Rhombic Hierarchical YF3 Nanocrystals and Their Use as Upconversion Photocatalysts after TiO2 Coating. Nanoscale 2013, 5, 3030-3036. 42. Su, W.; Zheng, M.; Li, L.; Wang, K.; Qiao, R.; Zhong, Y.; Hua, Y.; Li, Z. Directly Coat TiO2 on Hydrophobic NaYF4:Yb,Tm Nanoplates and Regulate Their Photocatalytic Activities with the Core Size. J. Mater. Chem. A 2014, 2, 13486-13491. 43. Dixon, S. J.; Stockwell, B. R. The role of Iron and Reactive Oxygen Species in Cell Death. Nat. Chem. Biol. 2014, 10, 9-17. 44. Zhao, L.; Peng, J.; Huang, Q.; Li, C.; Chen, M.; Sun, Y.; Lin, Q.; Zhu, L.; Li, F. NearInfrared Photoregulated Drug Release in Living Tumor Tissue via Yolk-Shell Upconversion Nanocages. Adv. Funct. Mater. 2014, 24, 363-371.


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45. Li, W.; Yang, J.; Wu, Z.; Wang, J.; Li, B.; Feng, S.; Deng, Y.; Zhang, F.; Zhao, D. A Versatile Kinetics-Controlled Coating Method to Construct Uniform Porous TiO2 Shells for Multifunctional Core-Shell Structures. J. Am. Chem. Soc. 2012, 134, 11864-11867. 46. Jeffryes, C.; Gutu, T.; Jiao, J.; Rorrer, G. L. Metabolic Insertion of Nanostructured TiO2 into the Patterned Biosilica of the Diatom Pinnularia sp. by a Two-Stage Bioreactor Cultivation Process. ACS Nano 2008, 2, 2103-2112. 47. Duan, H.; Nie, S. Cell-Penetrating Quantum Dots Based on Multivalent and EndosomeDisrupting Surface Coatings. J. Am. Chem. Soc., 2007, 129, 3333-3338. 48. Marrache, S.; Dhar, Shanta. Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics. Proc. Natl. Acad. Sci. U.S.A., 2012, 109, 16288-16293. 49. Gilleron, J.; Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C. et. al. Image-Based Analysis of Lipid Nanoparticle-Mediated siRNA Delivery, Intracellular Trafficking and Endosomal Escape. Nat. Biotechnol. 2013, 31, 638-646. 50. Chen, H.; Xiao, L.; Anraku, Y.; Mi, P.; Liu, X.; Cabral, H.; Inoue, A.; Nomoto, T.; Kishimura, A.; Nishiyama, N. et. al. Polyion Complex Vesicles for Photoinduced Intracellular Delivery of Amphiphilic Photosensitizer. J. Am. Chem. Soc. 2014, 136, 157163. 51. Huang, J. G.; Leshuk, T.; Gu, F. X. Emerging Nanomaterials for Targeting Subcellular Organelles. Nano Today 2011, 6, 478-492. 52. Aon, M. A.; Cortassa, S.; O’Rourke, B. Percolation and Criticality in a Mitochondrial Network. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4447-4452.


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Page 38 of 39

53. Aon, M. A.; Cortassa, S.; Aka, F. G.; Brown, D. A.; Zhou, L.; O’Rourke, B. From Mitochondrial Dynamics to Arrhythmias. Int. J. Biochem. Cell Biol. 2009, 41, 1940-1948. 54. Ly, J. D.; Grubb, D. R.; Lawen, A. The Mitochondrial Membrane Potential (∆ψm) in Apoptosis; An Update. Apoptosis 2003, 8, 115-128. 55. Lam, M.; Oleinick, N. L.; Nieminen, A. L. Photodynamic Therapy-Induced Apoptosis in Epidermoid Carcinoma Cells. Reactive Oxygen Species and Mitochondrial Inner Membrane Permeabilization. J. Biol. Chem. 2001, 276, 47379-47386. 56. Muhammad, F.; Guo, M.; Qi, W.; Sun, F.; Wang, A.; Guo, Y.; Zhu, G. pH-Triggered Controlled Drug Release from Mesoporous Silica Nanoparticles via Intracelluar Dissolution of ZnO Nanolids. J. Am. Chem. Soc. 2011, 133, 8778-8781. 57. Gao, X.; Ding, C.; Zhu, A.; Tian, Y. Carbon-Dot-Based Ratiometric Fluorescent Probe for Imaging and Biosensing of Superoxide Anion in Live Cells. Anal. Chem. 2014, 86, 70717078.


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A near-infrared (NIR) triggered nanophotosensitizer was developed based on mitochondria targeted titanium dioxide-coated upconversion nanoparticles for photodynamic therapy against cancer. When irradiated by NIR laser, the nanophotosensitizer could produce ROS in mitochondria, which induced the domino effect on ROS burst without further irradiation and resulted in mitochondrial collapse and irreversible cell apoptosis. A Near-Infrared Triggered Nanophotosensitizer Inducing Domino Effect on Mitochondrial Reactive Oxygen Species Burst for Cancer Therapy


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A Near-Infrared Triggered Nanophotosensitizer Inducing Domino

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