Mitochondrial-targeted multifunctional mesoporous Au@Pt nanoparticles for dual-mode photodynamic and photothermal therapy of cancers.

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Nanoscale View Article Online

DOI: 10.1039/C7NR04881E

Mitochondrial Reactive Oxygen Species Burst for Cancer Therapy

Yang Song‡ a, Qiurong Shi‡ a, Chengzhou Zhua, Yanan Luoa,b, Qian Lua, He Lia, Ranfeng Yea, Dan Dua,b*, and Yuehe Lin*a a

School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164 b

Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, P.R. China

Keywords: near-infrared spectroscopy, reactive oxygen species, mesoporous Pt shell, core-shell nanoparticles, PDT/PTT

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Published on 12 September 2017. Downloaded by Fudan University on 15/09/2017 06:08:17.

Triggered by Near-Infrared Light

Nanoscale

Page 2 of 35 View Article Online

DOI: 10.1039/C7NR04881E

Abstract In the conventional non-invasive cancer treatments, such as photodynamic therapy (PDT) and photothermal therapy (PTT), light irradiation is focused precisely on cancer cells to induce

overconsumption of oxygen and the restricted diffusion distance of ROS limit the therapeutic effects on hypoxic tumors. We developed a platform for the rapid uptake of Au@Pt nanoparticles

(NPs)

labeled

with

a

cell-targeting

ligand

(folic

acid)

and

a

mitochondria-targeting group (triphenylphosphine (TPP)) into mitochondria in cancer cells. Mitochondria were targeted via the subsequent loading of these Au@Pt NPs with a photosensitizer (Ce6), which led to significant improvement of PDT efficacy due to enhanced cellular uptake, an effective mitochondrial ROS burst and a rapid intelligent oxygen release. Meanwhile, Au@Pt NPs converse laser radiation into heat, resulting in thermally induced cell damage. This nanosystem could be adopted to be a dual-model phototherapeutic agent for enhanced cancer therapy and molecular targets associated with disease progression. We achieved a mitochondria-targeting multifunctional theranostic strategy (a combination of PDT and PTT) to improve theranostic efficiency substantially.

2



apoptosis by generating reactive oxygen species (ROS) or localized heating. However, the

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1. Introduction Photodynamic therapy (PDT) has been used as a noninvasive and safe method for tumor therapy. Compared to occur with conventional cancer treatments, such as chemotherapy and radiotherapy, PDT provides treatment with minimum invation, as well as negligible drug

target cancer tissue locally with light irradiation. The excited photosensitizer interacts with the surrounding molecules, converting oxygen into reactive oxygen species (ROS). ROS can induce serious oxidative damage to tumor tissue and ultimately cause the apoptosis of cancer cells.5, 6 However, because of their intrinsic disadvantages, the clinical utilization of PDT drugs for cancer treatment remains challenging. First, the extremely short lifespan of ROS limits their diffusion process and therefore directly restricts the damage to biomolecules.7-9 Second, photosensitizers located within cytoplasm are significantly detrimental to PDT therapeutic effect since the mitochondria are the primary organelles for cellular ROS generation. Mitochondrial dysfunction is directly related to the ROS level being out of balance.10 Moreover, mitochondria control the intrinsic pathway of apoptosis that results in cancer cell death in tumor therapy.11-14 Therefore, a targeted mitochondrial photosensitizer could generate a high yield of ROS to induce cancer cell apoptosis, which would further improve PDT therapeutic efficiency and reduce system toxicity and undesired side effects. However, the effeciency of uptaking of a (typically hydrophobic) photosensitizer into mitochondria remains one of the challenges encountered in PDT.15 Third, tumor hypoxia could also lower the therapeutic efficiency of PDT by significantly depriving the environmental oxygen, hindering photosensitizers from mediating their cytotoxic effect.16-18 Besides, further oxygen depletion during PDT would potentiate tumor hypoxia. Thus, the development of an alternative system that combines oxygen evolution and the PDT process would be highly desirable for vanquishing tumor hypoxia in the application of PDT.

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resistance and side effects.1-4 In a photodynamic system, a photosensitizer is used to treat the

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Photothermal therapy (PTT) is a hyperthermia method, in which irreversible cell damage in the tumor area is caused by the conversion of energy absorbed from photons to heat.19 Several types

of

photoabsorbing

agents,

including

carbon

nanomaterials,20-23

gold

(Au)

to their high near-infrared surface plasma resonance (SPR) properties. The integratation of noninvasive PTT and PDT into nanosystem have attracted great attentions because of the enhanced therapeutic efficiency.27, 28 However, the development of this PDT/PTT multifunctional nanosystem, which can both retain the desirable intrinsic properties of PTT and self-oxygen evolution in cancer cells in situ, is still a challenge. In this paper, we developed an oxygen-based PDT/PTT platform that targets mitochondria using mesoporous core-shell structured (MCSS) Au@Pt NPs. Taking advantage of a combined therapeutic approach, the developed platform is a nanosystem that can achieve oxygen self-sufficiency, selective uptake, high molecular loading capacity, providing a powerfull tool for cancer ablation with minimized side effects. MCSS Au@Pt NPs were synthesized using a one-pot template-directed method and subsequently passivated with PEG to increase their biocompatibility as well as their physiological stability. Because of the short half-life of ROS (microseconds) and the weak stability of the photosensitizer, an effective therapeutic outcome is greatly expected where the PDT agent can be deliveried into the cells. Due to its high affinity toward cancerous cells, folic acid (FA) is the ideal endocytic ligand for folate receptors that are overexpressed in various human cancer cells. The nanosystem was modified with FA for targeted delivery to FA-positive cancer cells. We also assessed the possibility of drug delivery into the mitochondria. Triphenylphosphine (TPP), a mitochondria-targeting group, was modified on the surface of the NPs to target mitochondria selectively for a mitochondrial ROS burst (Scheme 1)29. This could result in a mitochondria-mediated intrinsic apoptotic pathway that is associated with cascade reactions in apoptosis. In addition, the excellent catalytic activity of Pt NPs was expected to boost the peroxidase-like catalysis of 4



nanomaterials24, 25 and palladium (Pd) nanosheets,26 have been developed for tumor PTT due

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H2O2 for in situ generation of massive amounts of oxygen, leading to a significant improvement of PDT efficacy (Scheme 2). The feasibility of enhancing the capacity of PDT by cellular oxygen production and the effiency of killing cancer cells using this system were

the nanosystem in cancer therapy and shed light on a new avenue to enhance the efficacy of PDT. Moreover, because of the broad optical absorption windows, this nanosystem is a



confirmed using an in vitro cancer cell model. The results suggest a potential application of

promising candidate for PTT therapy.

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2. Results and discussion

2.1. Preparation of APPTFC Nanosystem In this work, MCSS Au@Pt NPs were synthesized via a one-pot reduction method based on

PtCl42-([PtCl42-/Pt] (0.76 V vs. SHE)) at 60 °C.30 First, Au3+ is reduced to form an Au core, and then Pt2+ is reduced to form a Pt shell.31 In this work, Brij 58, a nonionic surfactant, was employed as a soft template to generate the mesoporous Pt shells.31-33 Figure 1A, S1 and 1D shows the transmission electron microscope (TEM) image of the as-obtained MCSS Au@Pt NPs. The TEM image shows an obviously core-shell structure with mesoporous shells. The average size of the Au core was about 15 ± 2 nm, and the average thickness of the Pt shell was about 6.5 ± 2 nm. The crystalline structure of the as-synthesized Au@Pt NPs was also verified by X-ray diffraction (XRD) (Figure 1B). The diffraction peaks of the Au@Pt NPs were indexed as the (111), (200), (220), (311), (222) planes, which belong to the typical face-centered cubic (fcc) lattice of Au and Pt. The peak positions of Au and Pt were in accordance with JADE PDF#04-0783 and PDF#65-2868, respectively.34, 35 The atomic ratio of Pt and Au was about 2.5 (70.95% Pt and 29.05% Au), determined by X-ray energy dispersive spectroscopy (EDS) (Figure 1C). The atomic ratio was in agreement with the feeding ratio of the Pt and Au precursors, indicating a 100% yield for the one-pot reduction method. The lattice spacing of 0.232 nm marked in the HRTEM images in

Figure 1E is

indexed to the (111) planes of Pt. A high-angle annular dark field/scanning transmission electron

microscope/energy-dispersive

spectrometer

(HAADF-STEM-EDS)

also

demonstrated that the NPs had a core-shell structure (Figure 1F) which was composed of elements Au (red, orange) and Pt (green, yellow). The element Au mainly formed the core, while Pt formed the shell.

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the different redox potentials of AuCl4- ([AuCl4-/Au] (0.93 V vs. SHE)) and

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Generally, nanomaterials are unstable under physiological conditions.36 They aggregate in PBS solution because of the electron screen effect. Surface modification is required before any subsequent biomedical applications. Polyethylene glycol (PEG), a polymer of ethylene

NPs for biomedical applications. It helps NPs escape from the reticuloendothelial system (RES).37-39,40 It is fairly well-known that amine groups strongly bind with Pt NPs due to the amine group can form dative covalent bond to the Pt surface via lone pair of electrons.41-43 As expected, MCCS Au@Pt NPs co-modified with PEG diamine and FA-PEG-amine, denoted as APPF, exhibited a well-dispersed state in PBS even at concentrations up to 1 mg mL-1 and could be easily stored in various physiological solutions for at least one month (Figure S2). TEM images of APPF indicated a negligible aggregation of NPs (Figure 1A). Ultraviolet-visible-NIR (UV-vis-NIR) absorbance spectra of surface-passivated APPF showed unchangeable UV to NIR absorbance, which is consistent with the as-prepared Au@Pt NPs (Figure 2a). In addition, the photothermal conversion efficiency of APPF upon 808 nm laser irradiation (1.2 W cm-2) was tested for various concentrations of NPs. Photothermal irrational curves measured with a thermo-couple detector showed a strong concentration-dependent temperature increase for APPF, with the highest temperature of ~ 65 °C at the concentration of 50 µg mL-1 (Figure 2b, 2c). To promote the therapeutic efficiency, our nanosystems were co-introduced with TPP and FA, denoted as APPTFC. Dynamic light scattering (DLS) analysis found no aggregation of the APPTFC. The size of the NPs increased to 29 ± 5 nm due to the uniform loading of TPP and Ce6 onto the surface. In addition, UV-vis-NIR spectra and fluorescence emission spectra were used to confirm the successful functionalization of APPTFC. Compared with APPTF, APPTFC displayed new absorption bands located at 406 nm and 675 nm, which correspond to the characteristic absorption peaks of Ce6 loaded on the surface of NPs (Figure 2e). About 10% of drug loading is achieved in this experiment by virtue of the high specific surface area of NPs. Moreover, due to the strongly additive effect 7



oxide, is commonly introduced to improve the physiological stability and biocompatibility of

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of the absorbance of APP from 300 nm to 900 nm, the absorbance intensity of APPTFC was obviously higher than that of free Ce6 in PBS, suggesting strong interactions between NPs and aromatic drug molecules (Figure 2e). As illustrated in Figure S3, Au@Pt NPs was

conjugation with PEG . When further introducing Ce6 and TPP with –COOH, the zeta potential decreased to be -34.6 mV. In addition, the Ce6 loading was also confirmed by Fourier transform infrared (FTIR) spectra. As shown in Figure 2f, the sharp stretching vibration of the carbonyl-amide was observed at 1638 cm-1, which was produced by amidation between carboxylic group from Ce6/TPP and amine group from PEG. The peak emerged at 3300 cm-1 was due to the stretch vibration of –NH2, indicating the amino-PEG attached on the Au@Pt NPs surface. According to the Au@Pt NPs-H2O2 peroxidase-like reaction equation (Figure S4a), Au@Pt NPs can serve as a catalyst to promote the disproportionation reaction of H2O2 and produce oxygen and water.44-46 To investigate the peroxidase-like activity of Au@Pt NPs, 3,3',5,5'-tetramethylbenzidine (TMB) as a peroxidase substrate would be oxidized in the presence of H2O2. As shown in Figure S4b, oxygen serves as the electron donor in charge-transfer complexes derived from the one-electron oxidation of TMB under an acidic condition, resulting in a solution with a deep blue color. The color intensity was concentration-dependent (Figure S5 a-b). Even at low concentration levels, the pure-clear blue color could indicate that Au@Pt NPs have intrinsic peroxidase-like activity. The relationship between the peroxidase-like activity of Au@Pt NPs and H2O2 concentration was further tested. As exhibited in Figure S6, from 5x10-6 to 10 mM H2O2 concentration was in a linear relationship to absorption intensity. In addition, increasing bubbles were clearly observed with an increasing concentration of H2O2 (0-50 mM), indicating the generation of much more dissolved oxygen (Figure S7 a-b). The generation of the active ROS via photoinduced

energy

transfer

from 8

Ce6

was

evaluated

using



endowed with a negative charge, while the zeta potential of APP has a significant change after

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9,10-anthracenediyl-bis-(methylene) diamalonic acid (ABDA) as the indicator. This triggered a reaction with ROS and resulted in the absorption diminishing at 380 nm (Figure S8).

As

shown in Figure S8a, less ROS were produced because of the strong quenching ability of

H2O2 indicates enhanced ROS generation due to oxygen production through the peroxidase reaction between H2O2 and Au@Pt NPs (Figure S8c). In addition, the sharp decline in ABDA absorbance was only observed at 660 nm and for a combined laser. There was no obvious perturbation in ABDA absorbance under 808 nm laser irradiation (Figure S8b). Hence, only 660 nm laser irradiation can effectively induce ROS generation from APPTFC. To confirm the generation of ROS from APPTFC, a comparison between APPTFC and free Ce6 was conducted under 660 nm and 808 nm laser irradiation. As indicated in Figure S9, both APPTFC and free Ce6 produced ROS in solution upon 660 nm laser irradiation, while APPTFC and free Ce6 hardly generated ROS upon 808 nm laser irradiation. Because of the intermolecular energy transfer from Ce6 to NPs, APPTFC slightly affected the generation of ROS insignificantly compared to free Ce6. It should be noted that Ce6 alone showed increasing ROS production because of the quenching of Ce6 on APPTF (Figure S9a). ROS production from APPTFC increased obviously upon 660 nm laser irradiation after the addition of H2O2, because of the oxygen-enhanced ROS burst. In contrast, it did not occur without laser irradiation. Also, the APPTFC in 10% fetal bovine serum (FBS) exhibited faster and higher ROS generation than free Ce6 in 10% FBS because free Ce6 interacts easily with FBS and thus loses its photodynamic activity (Figure S9b),47, 48 but here the APPTFC

could be maintained the photodynamic activity of Ce6.

2.2. Cellular Internalization Pathway To promote therapeutic efficiency, TPP and FA were co-introduced in our system to target mitochondria selectively. In order to investigate the efficacy of APPTFC in targeted delivery 9



Au@Pt NPs. The significantly decreased ABDA absorption in the acidic solution containing

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and PDT, we performed confocal laser scanning microscopy (CLSM) to detect the internalization of the photosensitizer in human breast cancer MCF-7 cells. As shown in Figure 3A, most of the APPTFC accumulated around the cell membrane within 30 min. In

distributed through the cytosol. The fast cellular uptake rate is presumably caused by the significant targeting ability of FA. To evaluate the target recognition capability of the APPTFC nanosystem further, A549 cells (FR-negative) incubated with APPTFC were assessed in a control experiment. As shown in Figure S10, APPTFC distributed in the cytomembrane and cytoplasm of an MCF-7 cell exhibited strong red fluorescence. On the other hand,

APPTFC in A549 cells exhibited dim red fluorescence after 12 h of incubation.

The relative fluorescence intensity profiles revealed that the fluorescence intensity of APPTFC incubated with MCF-7 was approximately 4-fold higher than that of A549 cells (Figure S11). In addition, the MCF-7 cells incubated with APPTFC clearly displayed prominent concentration-dependent fluorescence intensity in the cytoplasm (Figure 3B), which was higher than that of the cells treated only with free Ce6. The red fluorescence intensity of the cells incubated with a high concentration of free Ce6 (10 µg mL-1) was significantly lower than that of the cells incubated with a low concentration of the APPTFC nanosystem. Thus, the APPTFC nanosystem has the capacity to enhance the cellular delivery efficiency of Ce6. The flow cytometer results for the fluorescence intensity of the cells incubated with the APPTFC nanosystem containing 5 µg mL-1 of Ce6 are almost equal to those for cells treated with 15 µg mL-1 of free Ce6, which are in good agreement with the results of CLSM analysis (Figure 3C). These results imply that APPTFC can be internalized into cancer cell through endocytosis much faster than free Ce6. This may be due to their effective cellular uptake via folate receptor mediated endocytosis.49 Efficient cellular internalization is of critical significance to ensuring their combined photothermal and photodynamic efficacy. To clarify the cellular internalization pathways, the cancer cells were 10



the following 2 hours, the APPTFC continuously crossed the cell membrane and was

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pre-treated with a variety of endocytosis inhibitors, such as ethylisopropylamiloride (EIPA), chlorpromazine, dynasore, and filipin. As shown in Figure 3D and Figure S12, the filipin- or EIPA-treated cells showed similar fluorescence with that of the control, indicating that the

caveolae. However, the cells treated with other inhibitors show significantly decreased fluorescence, suggesting that the internalization of the APPTFC nanosystem in cancer cells is clathrin-mediated. These results corroborate that the targeting affinity and efficacy based on FA facilitate the internalization of the APPTFC nanosystem and induce preferential uptake via mediated endocytosis. Since the passive targeting of Ce6 is inadequate for in vivo photodynamic treatment through systemic administration, modification with ligands of the surface of the APPTFC nanosystem is critical to increase the local concentration of the photosensitizer in the tumor area, and thus to reduce side effects and improve therapeutic efficacy. Next, CLSM was performed for a convenient assessment of cellular uptake and the subcellular distribution of the nanosystem in MCF-7 cells. APPTFC was previously shown to be taken up by cells via an endocytosis-dependent pathway. The efficient endosome escape of the nanosystem was further confirmed with CLSM. As shown in Figure 4A and Figure 4B, a 3D reconstructional Z-stack CLSM of MCF-7 cells showed most of the nanosystem accumulated around the cytoplasm (incubated for 12 h), with less evidence of its being trapped inside endolysomeal vesicles (colored in yellow) (Pearson’s correlation coefficient ～0.20). The cross-sectional CLSM in Figure 4C for MCF-7 cells incubated with the nanosystem also verifies that the low degree of co-localization of APPTFC with the cellular acidic vesicles, which were quantified by the Pearson’s correlation coefficient representing the effective endosome escape of the system. This endosome escape efficacy can be attributed to the high buffering capacity caused by the amino residual on the surface, which has the function of a proton sponge.50 As shown in Figure 5A and S14, after incubated with 11



endocytosis pathway of the APPTFC nanosystem is not mediated by macropinocytosis or

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APPTFC for 4 h, the nanosystem was concentrated at endosomes/lysosomes (colored in green). Afterward, MCF-7 cells were incubated with fresh culture medium for an additional 1 to 4 h. A yellow fluorescence signal with a colocalization ratio decreasing continuously from

escape. The endocytosis process of APPTFC is an active and energy-dependent process via actin filaments. As shown in Figure 6A-C, MCF-7 cells incubated with the nanosystem generally also showed colocalization with cytoskeleton actin (Pearson’s correlation coefficient, ρ=0.27) and accumulation in the perinuclear region in the cells after 8 h incubation, suggesting that the nanosystem stayed out of the actin and had less colocalization with actin after endosome escape.

However, as shown in Figure 6D, the MCF-7 cells

incubated with the nanosystem for 4 h exhibited a higher colocalization with cytoskeleton actin (Pearson’s correlation coefficient, ρ=0.42), which indicated a high association between the nanosystem and the actin meshwork. We have demonstrated that APPTFC uptake into cancer cell via an endocytic pathway and exhibited interactions with the actin meshwork when are present on the endosomes. This suggests that the movement of the nanosystem on actin occurred in the early stage of endocytosis and mainly in the cell periphery. Internalized APPTFC actively moved and fused with static early endosomes. This observation was further verified by quantifying the fluorescence intensity of the line scanning profiles (Figure S13). The cross-sectional confocal images of MCF-7 cells incubated with the nanosystem, shown in Figure 6, also verify that the nanosystem had negligible colocalization with actin, partially confirming the effectiveness of the mitochondrial targeting. In this work, to identify the targeting role of TPP in selectively internalizing the APPTFC nanosystem into the mitochondria of cancer cells, localization imaging of MCF-7 human breast cancer cells was performed using MitoTracker Green (MTG) to confirm the intracellular distribution of the nanosystem. As Figure 5B indicates, the fluorescence of the APPTFC nanosystem effectively overlapped that of MTG, which is evidenced by the clear 12



62.7% to 31.5% was observed in overlay images, indicating effective endosome/lysosome

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yellow fluorescence signal. A quantitative colocalization analysis (Pearson’s correlation coefficient ～0.58) and 3D reconstruction of Z-stack CLSM of the successfully internalized nanosystem with MTG (green stain) clearly showed the superiority of our novel nanosystem

endolysosomal trapping (Figure 5C). However, as shown in Figure S15, the MCF-7 cell

showed less degree of co-localization of Au@Pt@PEG-FA (APPFC) with the mitochondria (Pearson’s correlation coefficient ～0.19), which is contrary to the data shown in Figure 5B-C. These results showed that the TPP-functionalized APPTFC nanosystem could specifically target the mitochondria of cancer cells.

2.3. Evaluation of ROS Generation in vitro The hypoxia is one of the major hallmarks of solid tumors, which usually only preserves the oxygen concentration as low as 4% and thus seriously limits the PDT effect. It has been reported that hypoxic cells in tumors generally produce a high concentration of endogenous acid H2O2, which could be catalyzed by nanosystem to generate oxygen in vitro. Dysfunction of mammalian mitochondria is corrected by governing ROS production. Besides, mitochondria can precisely regulate the intrinsic apoptotic cell death pathway. Hence, the targeted disruption of mitochondria could improve oxidative stress, resulting in the apoptosis of cancer cells. To further study the mechanism, through which APPTFC nanosystem based ROS bursts in mitochondria initiate cell apoptosis, an investigation of subcellular organelle rupture and the chemistry of damaged cells was conducted. Commercial singlet oxygen sensor green (SOSG) kits, an indicator for ROS, were employed to monitor the ROS burst in vitro conveniently. As shown in Figure 7A, the untreated cells exhibited negligible SOSG fluorescence, indicating that few ROS were generated during PDT treatment (Figure 7A-a). Meanwhile, neither 660 nm laser irradiation after incubation with the APPTFC nanosystem 13



over the current reported NPs for intracellular mitochondria targeting with minimal

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(Figure 7A-b) nor the presence of the APPTFC nanosystem without laser irradiation (Figure 7A-c) induced any additional ROS generation. This was proven by the negligible green fluorescence signal in these experiments. In contrast, cancer cells treated with the APPTFC

(Figure 7A-f), which was significantly stronger than that of free Ce6 treated cancer cells (Figure 7A-d) or APPTFC nanosystem treated cancer cells upon 808 nm laser irradiation (Figure 7A-e). This stronger fluorescence intensity is attributed to the higher uptake ability and the superior stability of the APPTFC nanosystem under physiological conditions. The PDT effects were significantly improved owing to the relatively high level of oxygen generation (Figure S16). In contrast, there was no ROS generation upon 808 nm laser irradiation, indicating that an 808 nm laser is less efficient. These results indicate that the APPTFC nanosystem has a promising ability to generate an ROS burst in vitro upon irradiation with a 660 nm laser and thus enhance the therapeutic efficiency of photodynamic therapy. Encouraged by the promising PDT efficacy in vitro, we furthermore monitored changes in ROS in cancer cells incubated with the APPTFC nanosystem for a range of irradiation times (0, 30, 60, 90, 120, and 150 s) with incubation of 12 h. As shown in Figure 7B and Figure S17, no obvious fluorescence signal was observed when the irradiation time was less than 90 s, whereas a bright green fluorescence signal was obtained after irradiation for longer times (90 s, 120 s, 150 s). These results clearly confirm that the generation of ROS in mitochondria can be triggered by the APPTFC nanosystem when the appropriate irradiation threshold time is reached; this was referred to them as the “mitochondrial threshold” in previous reports.51 Moreover, considering that apoptosis resulting from PDT is a programmed process involving a time-consuming pathway, long-term monitoring of the mitochondrial ROS burst would be beneficial for evaluating photoactivated cytotoxicity triggered by the process. The time dependence of the ROS response was evaluated as variation in fluorescence over time using 14



nanosystem exhibited strong green fluorescence intensity upon 660 nm laser irradiation

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CLSM imaging. After incubation with the APPTFC nanosystem, cancer cells were irradiated with a 660 nm laser for 150 s to trigger ROS generation. Fluorescent imaging was carried out using CLSM. As depicted in Figure 7C, the time-dependent increase of green fluorescence

therapeutic modality for overcoming the limitations of PDT in promoting cancer cell apoptosis. Moreover, the significant change in cell morphology and strong green fluorescence signal were obtained after 14 h of incubation, which confirms photoactivated cell apoptosis. To shed light on the apoptotic mechanism in our present study, the commercial fluorescent dye rhodamine 123 was employed as a convenient marker to validate mitochondrial membrane potential (MMP) loss induced by the mitochondrial ROS burst. As depicted in Figures S18 and S19, the MMP loss was observed as a decrease of green fluorescence intensity (rhodamine 123) after irradiation for more than 90 s and excitation by a 488 nm laser. In contrast, for cancer cells treated with ROS quenchers, less MMP loss was observed, as reflected by the negligible change in green fluorescence intensity. These results are attributed to increased activity in the inner membrane anion channel and promotion of the opening of mitochondrial permeability transition pores via the oxidation of glutathione during the process of ROS generation. When mitochondria are seriously damaged, a cascade process is initiated, resulting in the release of certain biomolecules into the cytosol. These molecules form a complex that activates cytoplasmic protease caspase-3, which induces the apoptotic process. In our study, the apoptosis of MCF-7 cells through mitochondrial signaling pathways was examined as the activity of caspase-3. The activity of caspase-3 was monitored using a caspase protein In-cell ELISA kit to stain the cell with NIR fluorescence. As shown in Figure 7D, increased activity of caspase-3 was observed when MCF-7 cells were pulsed with the APPTFC nanosystem and irradiated for 150 s. Less caspase-3 was expressed for cells without any treatment, as evidenced by the relatively dim red fluorescence of the cancer cells.

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All



intensity revealed that a mitochondrial ROS burst, as an ablation strategy, is an automatically

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these results support the mitochondria-targeting nanosensitizer mediating an ROS burst for cancer cell apoptosis via an intrinsic mitochondrial pathway.

To identify the phototherapeutic efficiency of the APPTFC nanosystem, quantitative MTT cell viability assays were conducted for the nanosystem with and without laser irradiation. Figures 8A and 8B exhibit the cell viabilities of the APPTF/APPTFC nanosystem. The APPTF/APPTFC nanosystem exhibited minimal cytotoxicity on MCF-7 and bEnd.3 cells compared to its effect on free Ce6. Both MCF-7 and bEnd.3 cells retained ~90% of their viability, indicating excellent biocompatibility. MCF-7 cells were incubated with free Ce6 and the APPTFC nanosystem, followed by treatment with 660 nm laser irradiation (50 mW cm2) (Figure 8C). With the same concentration of Ce6 loading, the nanosystem exhibited the efficiency of PDT that is superior to that of free Ce6. Notably, even for MCF-7 cells treated with the nanosystem, cell viability was still more than 87% when irradiation was less than 60 s, whereas cell viability was significantly decreased to 35.2% with long irradiation times. This result clearly confirms that both the APPTFC nanosystem and an appropriate irradiation time are necessary for highly effective PDT. Moreover, the significant difference in viability between cells treated with the APPTFC nanosystem and cells treated with free Ce6 is consistent with the preferential internalization of the nanosystem into MCF-7 cells, leading to a considerably improved therapeutic efficacy of photodynamic action. An APPTFC nanosystem without modified TPP groups was also prepared for comparison to verify the significance of the ROS burst induced by the photosensitizer targeting the mitochondria to the induction of cell apoptosis. After MCF-7 cells were incubated with an APPTFC nanosystem without TPP groups under the conditions described above, the cell viability was more than 56% even for irradiation times up to 150 s (Figure S20), demonstrating that the ROS burst due to targeting mitochondria is pivotal for inducing cell apoptosis. In addition, when MCF-7 16



2.4. Therapeutic Effect of the APPTFC Nanosystem

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cells were incubated with the nanosystem and irradiated with an 808 nm laser (1.2 W cm2), the cell viability decreased to 46.2 % after 150 s, which is significantly lower than that of the free Ce6 plus PDT. Moreover, upon combination with NIR laser irradiation, the viability of

demonstrates that this nanosystem can enhance Ce6 uptake and maintain remarkable PDT/PTT efficacy.

3. Conclusion In summary, detailed experiments were carried out to design a mitochondria-targeting nanosensitizer based on Au@Pt-PEG-Ce6 NPs which could be used for not only PDT but also PTT for cancer cells in vitro. We preliminarily hypothesized that the photosensitizer would specifically target mitochondria to trigger a mitochondrial ROS burst and initiate a series of reactions, which will result in mitochondrial collapse and cell apoptosis. Meanwhile, the peroxidase activity of Au@Pt NPs toward H2O2 would generate enough oxygen to overcome the hypoxia induced by oxygen loss during the PDT process. CLSM imaging indicated that the nanosensitizer targeted mitochondria and induced a mitochondrial ROS burst in vitro. MTT assay confirmed the designed nanosensitizer could effectively induce cell apoptosis (~10%). We hope that this novel PDT approach can provide new insights for cancer therapy.

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MCF-7 cancer cells treated with the nanosystem was dramatically decreased to 13%, which

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Supporting Information The Supporting Information is available free of charge on the RSC website. Experimental details, CLSM of endocytosis, peroxidase-like performance of nanosystem.

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected]; [email protected] ‡ These authors contributed equally to this work.

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Acknowledgements This work was supported by start-up fund of Washington State University. We thank the financial support from National Natural Science Foundation of China (21575047).

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Scheme 1. Schematic illustration of mechanism in which near-infrared light triggers a mitrochondrial ROS burst, enhancing the availability of H2O2 for synergetic PDT/PTT effects.

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Scheme 2. Schematic illustration of the structure of nanosystem and ROS generation.

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Figure 1. (A, D) TEM, (E) HRTEM images, (B) XRD and (C) EDS spectra of the as-obtained MCSS Au@Pt NPs. (F) HAADF-STEM-EDS mapping images.

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Figure 2. Photothermal profile of Au@Pt NPs. (A) UV-visible spectra of Au@Pt NPs and APPF. (B) Temperature increase of water and APPF dispersions for various concentrations as a function of irradiation time. (C) Plot of temperature change over a period of 10 min versus APPF concentration. (D) Size distribution of Au@Pt NPs. (E) UV-visible spectra of free Ce6, APPF, and AFFTFC in PBS. (F) FTIR of Au@Pt NPs and APPTFC.

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Figure 3. (A) CLSM of MCF-7 cells treated with APPTFC. Scale bar: 7.5 µM. (B) CLSM images of MCF-7 cells incubated with various concentrations of free Ce6 and APPTFC (Ce6 amount: 5 µg mL-1 or 10 µg mL-1) for 4 h. Scale bar: 50 µM. (C) Flow cytometer analysis of MCF-7 cells treated with APPTFC (Ce6 amount: 5 µg mL-1) and various concentrations of free Ce6. (C) CLSM imaging of mitochondria targeting of APPTFC in cancer cells. MCF-7 cells were incubated with APPTFC before CLSM imaging. (D) CLSM images of untreated MCF-7 cells (control) and MCF-7 cells treated with filipin (an inhibitor of caveolae-mediated uptake, 5 µM), ethylisopropylamiloride (EIPA, an inhibitor of macropinocytosis, 50 µM), dynasore (an inhibitor of dynamin-mediated uptake, 100 µM), and chlorpromazine (an 23

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inhibitor of clathrin-mediated uptake, 10 µM) before being incubated with APPTFC (Ce6 amount: 10 µg mL-1). Scale bar: 50 µM.

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Figure 4. (A) CLSM of live MCF-7 cells treated with APPTFC (Ce6 amount: 2 µg mL-1, in purple). The cells were stained with LysoTracker (yellow) and DAPI (red). (B) A 3D projection showing Z-stack images of MCF-7 cells treated with APPTFC (Ce6 amount: 2 µg mL-1), followed by staining with either LysoTracker (yellow) or DAPI (red). (C) Relative CLSM of multiple cross sections exhibiting various locations of APPTFC within the cancer cells. Scale bar: 7.5 µM.

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Figure 5. (A) CLSM of MCF-7 cells incubated with APPTFC (Ce6 amount: 10 µg mL-1) and further incubated for various periods of time. The lysosomes were stained with LysoTracker. Scale bar: 25 µM. (B) Mitochondria targeting of APPTFC under CLSM: CLSM of APPTFC, mitochondria stained with MitoTracker Green (MTG) , and the overlay channel of APPTFC and MTG. Scale bar: 25 µM. (C) Three-dimensional projections show Z-stack images of MCF-7 cells in Figure 5B. The MCF-7 cells were treated with MitoTracker Green. Scale bar: 10 µM.

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Figure 6. (A) Three-dimensional projection showing Z-stack images at 45° of MCF-7 cells treated with APPTFC (Ce6 amount: 2 µg mL-1) for 8 h, followed by staining with cytoskeleton actin (green) and DAPI (blue). (B) Confocal images of overlay channel for (A). (C) CLSM of stained cytoskeleton actin, DAPI-stained nucleus and APPTFC. (D) Confocal images of multiple cross sections of MCF-7 cells treated with APPTFC (Ce6 amount: 2 µg mL-1) for 4 h, exhibiting various locations of the APPTFC within the cancer cells. Scale bar: 10 µM. 27




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Figure 7. (A) CLSM images of MCF-7 cells subjected to a variety of treatments; here, the green color represents ROS indicator SOSG: (a) untreated MCF-7 cancer cells; (b) MCF-7 cells irradiated with a 660 nm laser; (c) cells incubated with APPTFC without any laser irradiation; (d) MCF-7 cells incubated with free Ce6, then irradiated with a 808 nm laser; (e) MCF-7 cells incubated with APPTFC, then irradiated with a 808nm laser; and finally (f) MCF-7 cells incubated with APPTFC, then irradiated with combined 660 nm/808 nm laser irradiation. (The power intensity of the 660 nm laser is 50 mW cm-2, and the power intensity of the 808 nm laser is 1.2 W cm-2) Scale bar: 100 µM. (B) CLSM fluorescence microscopy image of a real-time intercellular ROS burst in MCF-7 cells. After being incubated with APPTFC (Ce6 amount: 10 µg mL-1) and irradiated for various lengths of time, CLSM images were captured at 12 h. Scale bar: 250 µM. (C) CLSM images of real-time intercellular ROS burst in MCF-7 cells taken at 2 h intervals for 14 h. The increasing intensity of the green fluorescence indicates a domino effect of the ROS burst. Scale bar: 250 µM. (D) Immunofluorescence staining images of caspase-3: (a) untreated MCF-7 cells and (b) MCF-7 cells incubated with APPTFC (Ce6 amount: 10 µg mL-1) and irradiated with an NIR laser for 90 s. CLSM images were taken 12 h after irradiation. Scale bar: 50 µM.

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Figure 8. (A) Results of MTT tests for determining the viability of MCF-7 cancer cells. MCF-7 cells were treated with various concentrations of APPF, APPTFC, and Ce6 without laser irradiation. (B) Cell viability of normal cells (bEnd.3 cells). MCF-7 cells were treated with various concentrations of APPF, APPTFC, and Ce6 without laser irradiation. (C) Results of MTT tests for determination of viability of MCF-7 cancer cells under PDT treatment, PTT treatment or combined treatment. (D) Immunofluorescence staining images of caspase-3: (a) MCF-7 cells without any treatment and (b) MCF-7 cells incubated with APPTFC nanosystem (Ce6 amount: 10 µg mL-1) and irradiated with an NIR laser for 90 s. CLSM images were taken 12 h after irradiation.

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