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Feb 2, 2017 - compared with PTT or PDT treatment alone. In this work ...... incubation for 2, 4, 8, or 12 h in the dark, cells were washed with PBS th...
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Graphene Oxide Decorated with Ru(II)−Polyethylene Glycol Complex for Lysosome-Targeted Imaging and Photodynamic/Photothermal Therapy Dong-Yang Zhang, Yue Zheng, Cai-Ping Tan,* Jing-Hua Sun, Wei Zhang, Liang-Nian Ji, and Zong-Wan Mao* MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, PR China S Supporting Information *

ABSTRACT: The combination of photothermal therapy (PTT) and photodynamic therapy (PDT) can kill cancer cells more efficiently as compared with PTT or PDT treatment alone. In this work, we use nanohybrid rGO-Ru-PEG composed of reduced nanographene oxide (rGO) sheet and a phosphorescent polyethylene glycol modified Ru(II) complex (Ru-PEG) for combined PTT and PDT of cancer. Photosensitizer and imaging agent RuPEG is decorated onto delivery and PTT agent rGO via π−π stacking and hydrophobic interactions. The chemical structure and morphology have been characterized by various methods. The release of Ru-PEG from rGO surface is pH-dependent, and irradiation can increase the release rate considerably. The combined effects of PDT and PTT have been evaluated by cytotoxicity assay under serial irradiation at 808 nm (PTT) and 450 nm (PDT). Mechanism investigation shows that the nanohybrid can induce apoptosis through generation of reactive oxygen species (ROS) and cathepsin-initiated apoptotic signaling pathways under light excitation. rGO-Ru-PEG can be applied to in vivo photothermal imaging, and high treatment efficacy was achieved for in vivo antitumor experiments when irradiated with an 808 nm laser and a 450 nm laser. Our work provides an effective strategy for the construction of multifunctional imaging and phototherapeutic nanohybrids for the treatment of cancer. KEYWORDS: graphene oxide, Ru(II) complex, photodynamic therapy, photothermal therapy, lysosome, apoptosis

1. INTRODUCTION Conventional cancer therapy has many limitations and often fails to eradicate tumor completely. Combined therapy that can lead to synergistic or combined effect of different therapeutic modalities is a promising approach to enhance anticancer efficacy and reduce systemic toxicity as well as side effects.1−4 Photodynamic therapy (PDT) utilizing light to produce reactive oxygen species (ROS) and photothermal therapy (PTT) using light to generate heat are two typical phototherapeutic approaches for killing of cancer cells.5 Combining these two phototherapy techniques in one system may improve the cancer therapeutic efficacy through a synergistic effect.6,7 Photodynamic therapy (PDT) is a noninvasive medical technology to treat cancer.8−11 The use of Ru(II) complexes, especially Ru(II)−polypyridyl complexes, for PDT has gained great interests in recent years.12−15 Compared with the traditional photosensitizers (PSs), Ru(II)−polypyridyl complexes have adjustable spectroscopic and photophysical properties and their structures can be easily modified. Additionally, Ru(II)−polypyridyl complexes have many advantages as imaging/sensing agents, e.g., visible-light excitation and emission based on metal-to-ligand charge transfer (MLCT), high chemical and photochemical stabilities, relatively high quantum yields, and large Stokes shifts (usually greater than 150 nm).16,17 However, © 2017 American Chemical Society

in most cases, Ru(II)−polypyridyl complexes enter cells by passive diffusion, and high lipophilicity, which results in poor aqueous solubility, is necessary for optimized cellular uptake.18,19 Both limited water solubility and poor tumor targeting capability of Ru(II)−polypyridyl complexes hinder their broader clinical applications as PSs. To address these problems, one possible solution is to modify Ru(II)−polypyridyl complexes with polyethylene glycol (PEG) to increase their water solubility and bioavialability.20,21 Conjugation or absorption of PSs with nanoparticles can also optimize the intracellular uptake levels by altering the penetration mechanisms, and improve the tumor targeting capability by enhanced permeability and retention (EPR) effects.22 Recently, PTT-mediated by nanomaterials has attracted great attention. Upon near-infrared (NIR) illumination, a number of nanomaterials, such as Au nanomaterials, carbon nanomaterials, Pd nanosheets, copper sulfide, and W18O49 nanoparticles have shown the capabilities to destroy cancer cells by photothermal heating.23−25 Loading of PSs in nanomaterials for combined PTT and PDT can extend circulation time within the body and Received: October 28, 2016 Accepted: February 2, 2017 Published: February 2, 2017 6761

DOI: 10.1021/acsami.6b13808 ACS Appl. Mater. Interfaces 2017, 9, 6761−6771

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Figure 1. (A) Schematic illustration of the formation of rGO-Ru-PEG. (B) AFM images of rGO-Ru-PEG dispersed in H2O. Scale bar: 250 nm. (C) XPS spectrum of rGO-Ru-PEG. (D) UV/vis absorbance spectra of Ru1, Ru-PEG, rGO, and rGO-Ru-PEG in H2O. (E) Fluorescence spectra of Ru1 (5 μM), Ru-PEG (20 μg/mL), and rGO-Ru-PEG (5 μM based on the concentration of Ru-PEG) in H2O.

2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of rGO-Ru-PEG. rGO and [Ru(DIP)2(H2dcbpy)][Cl]2 (Ru1; DIP = 4,7diphenyl-1,10-phenanthroline; H2dcbpy = 2,2′-bipyridine-4,4′dicarboxylic acid) were synthesized following literature procedures with slight modifications.38,39 The ESI-MS and 1H NMR spectra of Ru1 are shown in Figures S1 and S2. Ru-PEG was obtained by a simple condensation reaction of Ru1 with NH2-PEG using N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. The 1H NMR spectrum of Ru-PEG is shown (Figure S3). rGO-Ru-PEG was readily formed by sonication of Ru-PEG with rGO, and the product was purified by ultrafiltration centrifuge. Ru-PEG was loaded onto rGO by π−π interaction between Ru-PEG and rGO as well as intermolecular hydrophobic interaction between Ru-PEG molecules, which were also reported to be the main driving forces of the loading of Ru(II) complexes on other nanosystems.40,41 Successful loading of Ru-PEG on rGO was evidenced by atomic force microscopy (AFM). The thickness of rGO-RuPEG is about 25 nm, which is much higher than that of Ru-PEG (0.5 nm) or rGO (2 nm) (Figures 1A and S4A,B). X-ray photoelectron spectroscopy (XPS) measurements were performed to probe the composition of rGO-Ru-PEG. The survey spectrum in Figure 1B indicates the presence of ruthenium. FTIR spectrum of rGO-Ru-PEG shows the typical symmetric CH2 stretch of the methylene groups in rGO at 2890 cm−1 (peak 1), and the attachment of Ru-PEG to rGO is indicated by the peak at 1113 cm−1 (peak 2) assigned to the vibrational band of C−O bonds in the PEG moiety (Figure S5). The absorption spectrum of rGO-Ru-PEG clearly shows the characteristic absorption band of Ru-PEG with a maximum at approximately 460 nm, which indicates the successful loading of Ru-PEG onto rGO (Figure 1C). The UV/vis peak at 460 nm was then used to

achieve the EPR effect at the tumor site. Unfortunately, the loading amount of PS in most of the nanocarriers is typically very low.26,27 Graphene, a single-layer or few-layered two-dimensional (2D) sp2-bonded carbon sheet, has attracted remarkable attention in biological applications including nanomedicine.28−31 Due to its excellent drug loading capability and minimal cytotoxicity, graphene is an ideal carrier for high drug loading through physical interaction or chemical conjugation.32,33 Among graphene-based nanomaterials, reduced GO sheet (rGO) is one of the most effective PTT agents that can absorb NIR light and induce a temperature increase in the local environment, causing irreversible cell damage.34,35 Lysosomes contain a large variety of hydrolytic enzymes, and they are capable of degrading almost all kinds of biomolecules. Disruption of the lysosomal integrity can cause lysosomal membrane permeabilization (LMP) to initiate cell death.36 Lysosomes are emerging as attractive anticancer targets as they are involved in various aspects of cancer cell immortalization.37 Additionally, inducing photodamage in lysosomes is also proved to be a feasible stratagy to demolish cancer cells.13 In this work, we designed nanohybrid rGO-Ru-PEG constructed by rGO loaded with a PEG-modified Ru(II) complex (Ru-PEG) for lysosome-targeted phosphorescent imaging and combined PTT−PDT therapy (Figure 1A). The structure and composition of rGO-Ru-PEG were characterized by various methods. The drug loading capacity and stability were studied. The efficiency of combined PTT−PDT therapy was evaluated by in vitro cytotoxicity assay. The anticancer mechanisms including induction of apoptosis, elevation of cellular ROS, and damage to lysosomal membrane were investigated. 6762

DOI: 10.1021/acsami.6b13808 ACS Appl. Mater. Interfaces 2017, 9, 6761−6771

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2.3. Photothermal Properties. To investigate their photothermal therapeutic potential, rGO and rGO-Ru-PEG (2.5, 5, and 10 μg/mL) in water were irradiated with an 808 nm laser (1 W/cm2) for different durations (0−10 min). A thermometer sonde was used to record the temperature variation every 10 s. Pure water shows no obvious changes in temperature before and after irradiation. An obvious increase in temperature is observed for both rGO and rGO-Ru-PEG in time- and concentrationdependent manners (Figures 3 and S7). After the water solutions

determine the concentrations of Ru-PEG in rGO-Ru-PEG samples after subtraction of the absorbance contributed by rGO. The loading capacity (the weight ratio of Ru-PEG/rGO in rGORu-PEG) is calculated to be approximately 128%. Fluorescence spectra show that the fluorescence of Ru-PEG is partially quenched as compared with Ru1, which may be attributed to the aggregation of Ru-PEG (Figure 1D). Diminished emission intensity is also observed for rGO-Ru-PEG as compared with that of Ru-PEG, which may be caused by the photoinduced electron transfer between the chromophore (Ru-PEG) and graphene.6,42−44 2.2. Release of Ru-PEG from rGO-Ru-PEG. rGO-Ru-PEG is well dispersed in deionized water, and the suspension is very stable with no sign of aggregation and precipitation observed for 48 h (Figure S6). In contrast, rGO aggregates into small visible particles under the same conditions. Similar observations are also obtained in PBS and cell culture medium. The result indicates that the absorption of Ru-PEG can increase the aqueous dispersibility of rGO. In vitro release of Ru-PEG from rGO-RuPEG was investigated in phosphate-buffered saline (PBS) by UV/vis spectroscopy. The results show that the release of RuPEG is pH-dependent. rGO-Ru-PEG is rather stable in the solution at physiological pH (Figure 2A). The release of Ru-PEG

Figure 3. Temperature change curves of water and rGO-Ru-PEG solutions at different concentrations exposed to the 808 nm laser (1 W/ cm2) for different time intervals.

containing rGO-Ru-PEG are irradiated for 10 min, the temperature of the solutions increases by 16, 26, and 33 °C for 2.5, 5, and 10 μg/mL, respectively. Under the same conditions, the temperature of the rGO solutions increases by 9.4, 14.7, and 22.2 °C for 2.5, 5, and 10 μg/mL, respectively. The results indicate that both rGO and rGO-Ru-PEG can effectively convert NIR light to heat, and the photothermal effect of rGO-Ru-PEG is higher than that observed for rGO. 2.4. Generation of 1O2. 1O2 is considered to be the main cytotoxic species in PDT. The abilities of rGO-Ru-PEG and RuPEG to generate 1O2 under irradiation at 450 nm were detected by the bleaching of p-nitrosodimethylaniline (RNO; Figure 4).45

Figure 2. Release of Ru-PEG from rGO-Ru-PEG: (A) pH-dependent in vitro release of Ru-PEG from rGO-Ru-PEG. (B) Absorption spectra of rGO-Ru-PEG (6 μM based on the concentration of Ru-PEG) before and after 808 nm laser irradiation (0.5 W/cm2, 5 min).

Figure 4. Photooxidation of RNO by Ru-PEG, rGO, and rGO-Ru-PEG under light irradiation. In the presence of Ru-PEG (10 μM), rGO (40 μg/mL), and rGO-Ru-PEG (10 μM based on the concentration of RuPEG), changes in the absorption spectra of RNO at 440 nm upon irradiation at 450 nm in aerated PBS were monitored. [Ru(bpy)3]Cl2 was used as the standard.

from rGO is accelerated in the acidic solution (pH 5) mimicking the lysosomal/endosomal environments, which may be attributed to the protonation/deprotonation process of carboxyl groups on rGO. After rGO-Ru-PEG is incubated in PBS at room temperature for 12 h, 14 and 24% of Ru-PEG is released from rGO under physiological and acidic conditions, respectively. Interestingly, Ru-PEG can be released from rGO via photothermal heating triggered by an 808 nm laser (Figure 2B). After irradiation with the 808 nm laser at a power density of 0.5 W/cm2 for 5 min, almost all of Ru-PEG is released from rGO as calculated by the absorption intensity at 460 nm.

The absorbance of RNO at 440 nm can be diminished in the presence of 1O2. By using [Ru(bpy)3]Cl2 (bpy = 2,2′-bipyridine) as the reference,46 the quantum yields of Ru-PEG and rGO-RuPEG to produce 1O2 (ΦΔ) are determined to be 0.31 and 0.06, respectively. The capability of rGO-Ru-PEG to produce 1O2 is lower than that of Ru-PEG, which may be due to the quenching effect of rGO. It has been shown that PSs loaded in nanomaterial 6763

DOI: 10.1021/acsami.6b13808 ACS Appl. Mater. Interfaces 2017, 9, 6761−6771

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ACS Applied Materials & Interfaces produce less 1O2 than free PSs as the nanomaterial can act as efficient quencher, and the ability of PSs to produce 1O2 can be restored upon release from surface of the nanomaterials.6,7,42 2.5. Cellular Uptake Properties. The cellular uptake of RuPEG and rGO-Ru-PEG in human lung cancer A549 cells were first investigated by confocal laser microscopy utilizing the intrinsic fluorescence of the Ru(II) complex. A549 cells were incubated with Ru-PEG or rGO-Ru-PEG for different time intervals. A time-dependent increase in the emission intensity indicates that both rGO-Ru-PEG (Figure 5A) and Ru-PEG

with MTDR are 0.32 and 0.39, respectively. The results indicate that Ru-PEG and rGO-Ru-PEG possess lysosomal specificity. The extent of cellular uptake of Ru-PEG and rGO-Ru-PEG is also quantitatively determined by ICP-MS. After a treatment for 24 h, the intracellular ruthenium content is 9.46 ± 0.43 ng/106 cells and 15.40 ± 0.69 ng/106 cells for Ru-PEG (2.5 μM) and rGO-Ru-PEG (2.5 μM based on the concentration of Ru-PEG), respectively. The result indicates the nanocarrier rGO can increase the cellular uptake efficacy of Ru-PEG. 2.6. In Vitro Cytotoxicity. The in vitro PTT and PDT efficacy of rGO, Ru-PEG, and rGO-Ru-PEG was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A moderate PTT effect is observed for rGO when A549 cells are irradiated with the 808 nm laser (Figure 6A).

Figure 5. (A) Representative confocal microscopic images showing the cellular uptake of rGO-Ru-PEG. A549 cells were incubated with rGORu-PEG (10 μM based on the concentration of Ru-PEG) for different time intervals. (B) Upper row: Confocal microscopy images of A549 cells colabeled with rGO-Ru-PEG (10 μM, 3.5 h) and MTDR (150 nM, 0.5 h); bottom row: Confocal microscopy images of A549 cells colabeled with rGO-Ru-PEG (10 μM, 3.5 h) and LTDR (50 nM, 0.5 h). rGO-Ru-PEG was excited at 405 nm; MTDR and LTDR were excited at 633 nm. The phosphorescence/fluorescence was collected at 640 ± 20, 665 ± 20, and 668 ± 20 nm for rGO-Ru-PEG, MTDR, and LTDR, respectively. Scale bars: 10 μm.

(Figure S8) can penetrate into A549 cells and are mainly retained within the cytoplasm. Colocalization experiments of rGO-RuPEG (Figure 5B) or Ru-PEG (Figure S9A) with LysoTracker Deep Red (LTDR) demonstrate that they can specifically target lysosomes. The colocalization coefficients of rGO-Ru-PEG and Ru-PEG with LTDR are 0.79 and 0.78, respectively. Meanwhile, minimal colocalization of rGO-Ru-PEG (Figure 5B) or Ru-PEG (Figure S9B) with Mito Tracker Deep Red (MTDR) is observed. The colocalization coefficients of rGO-Ru-PEG and Ru-PEG

Figure 6. (A) Relative viability of A549 cells incubated with various concentrations of rGO and irradiated with 808 or 450 nm laser. (B) Relative viability of A549 cells incubated with various concentrations of Ru-PEG and irradiated with 808 or 450 nm laser. (C) Relative viability of A549 cells incubated with various concentrations of rGO-Ru-PEG and irradiated with 808 or 450 nm laser or treated with an 808 nm laser and a 450 nm laser serially. The control groups were kept in the dark. (808 nm: 0.5 W/cm2, 5 min; 450 nm: 20 mW/cm2, 2 min). 6764

DOI: 10.1021/acsami.6b13808 ACS Appl. Mater. Interfaces 2017, 9, 6761−6771

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Figure 7. (A) Hoechst 33342 stained A549 cells after the cells were treated with rGO-Ru-PEG and irradiated with either the 808 nm laser (0.5 W/cm2) or the 450 nm laser (20 mW/cm2) alone or with both lasers. (B) Representative confocal microscopic images of A549 cells after the cells were treated with rGO-Ru-PEG (Ru-PEG: 5 μM) and irradiated with either the 808 nm laser (0.5 W/cm2) or the 450 nm laser (20 mW/cm2) or both lasers. (C) Histograms of annexin V-FITC stained A549 cells after the cells were treated with different concentrations of rGO-Ru-PEG and irradiated by either the 808 nm laser or the 450 nm laser or both lasers. The control groups were kept in the dark. Scale bars: 10 μm. (D) Detection of caspase 3/7 activity in A549 cells after treated with rGO-Ru-PEG (5 μM based on the concentrations of Ru-PEG) in the absence or presence of light. (E) Measurement of cellular ATP content. (a) Cells kept in the dark. (b) Cells irradiated with an 808 nm laser and a 450 nm laser serially. (c) Cells treated with rGO-Ru-PEG and kept in the dark. (d) Cells treated with rGO-Ru-PEG and irradiated with an 808 nm laser. (e) Cells treated with rGO-Ru-PEG and irradiated with a 450 nm laser. (f) Cells treated with rGO-Ru-PEG and irradiated with an 808 nm laser and a 450 nm laser serially. (808 nm laser: 0.5 W/cm2, 5 min; 450 nm laser: 20 mW/cm2, 2 min)

the increased Ru cellular uptake of rGO-Ru-PEG as compared with that of Ru-PEG. Moreover, combined PTT−PDT treatment causes significantly higher cells death than does PTT

Upon irradiation with the 450 nm laser, the PDT effect in RuPEG-treated cells (Figure 6B) is lower than that observed in rGO-Ru-PEG-treated cells (Figure 6C), which may be caused by 6765

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Figure 8. Confocal microscopic images (top) and flow cytometric quantification (bottom) of cellular ROS levels detected by DCFH-DA staining. A549 cells were incubated with Ru-PEG and rGO-Ru-PEG at the indicated concentrations and irradiated with a 450 nm laser (20 mw/cm2, 2 min). Scale bar: 10 μm. MFI: mean fluorescence intensities.

percentages of cells in the apoptotic phase (annexin V-positive) are 1.0 ± 0.1, 20.6 ± 2.2, 31.1 ± 3.5, and 37.5 ± 4.0% for control, PTT, PDT, and PTT−PDT combined treatments, respectively. The activation of caspases has been identified as one of the key events in apoptosis.52 As compared with the control cells in the dark, negligible increase in caspase 3/7 activity was detected in cells treated with light or rGO-Ru-PEG alone. The combination of rGO-Ru-PEG (5 μM based on the concentrations of RuPEG), PTT and PDT caused an approximately 2.3-fold increase in caspase 3/7 activity (Figure 7D). Apoptosis also can cause a decrease in ATP levels. The ATP levels in cells after combined PTT−PDT treatment was reduced to 64%, which is much lower than those obtained in cells with either PTT or PDT alone (Figure 7E). These results collectively indicate that combined PTT−PDT treatment of rGO-Ru-PEG mainly induce apoptotic cell death.53,54 2.8. Elevation of ROS. ROS, particularly 1O2, are the most important mediators of cell death induced by PDT.55 A 2′,7′dichlorofluorescin diacetate (DCFH-DA) assay was used to measure ROS in A549 cells produced by PDT treatment of rGORu-PEG. Once in the cells, DCFH-DA is hydrolyzed by esterase enzymes to DCF, which is vulnerable to ROS and can be oxidized to highly fluorescent 2,7-dichlorofluorescein (DCF).56 A549 cells were incubated with rGO-Ru-PEG (2.5 and 5 μM based on the concentrations of Ru-PEG) and stained with DCFH-DA. Confocal microscopy images show that after A549 cells were treated with rGO-Ru-PEG for 24 h and irradiated with 450 nm laser (20 W/cm2) a significant increase in DCF fluorescence was observed (Figure 8). Similar results were also obtained by flow cytometric analysis (Figure 8). This finding suggests that rGORu-PEG can efficiently produce ROS in A549 cells upon PDT treatment. 2.9. Lyososomal Damage. Lysosomal membrane permeabilization can cause the release of lysosomal proteases, e.g., cathepsin B, from lysosomes to cytosol to promote apoptosis.57 As rGO-Ru-PEG can localize to lysosomes, the release of cathepsin B from lysosomes caused by PTT−PDT combined treatment was detected using the fluorogenic substrate Magic Red MR-(RR)2 assay.58 The control cells display red dotlike fluorescence mostly localized in the lysosomes, while cells with PTT, PDT and PTT−PDT combined treatments show different levels of diffused red fluorescence. The release of cathepsin B from lysosomes into the cytosol is more obvious for PTT−PDT combined treatments (Figure 9). The results indicated that the rGO-Ru-PEG can induce apoptosis through lysosomal damage upon PTT−PDT treatment.

or PDT treatment alone (Figure 6C). After treatment with rGORu-PEG at 6.25 μM (based on the concentration of Ru-PEG), the cell viabilities for dark control, PTT treatment, PDT treatment, and combined PTT−PDT treatment are 17.6 ± 3.6, 51.8 ± 1.0, and 8.3 ± 0.5%, respectively. The results indicate that a synergistic effect can be achieved by the combined PTT−PDT treatment for rGO-Ru-PEG. It should be pointed out that no obvious increase in cytotoxicity is observed for Ru-PEG irradiated with the 808 nm laser or rGO irradiated with the 450 nm laser (Figure 6A,B). 2.7. Induction of Apoptosis. Apoptosis is reported to be the primary pathway for cell death execution induced by PDT as well as PTT.47,48 We then investigated whether the combined PTT−PDT effects of rGO-Ru-PEG occurred through the apoptotic pathway. Apoptosis is characterized by a series of defined biochemical and morphological events, such as activation of caspase family proteases, loss of cell membrane asymmetry accompanied by phosphatidylserine translocation from the inner plasma membrane to the outer cell surface, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation.49,50 First, the changes in cell morphology of rGO-Ru-PEG-treated A549 cells induced by PTT−PDT combined treatment were examined by 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)2,5′-bi-1H-benzimidazole trihydrochloride (Hoechst 33342) staining. As shown in Figure 7A, control cells show normal overall morphology and a homogeneous nuclear staining pattern. After PTT−PDT combined treatment, A549 cells show typical apoptotic changes, including cell shrinkage, membrane bubbling, bright staining, condensed chromatin, fragmented nuclei, and apoptotic bodies. Compared with PTT or PDT treatment alone, the combination of PTT and PDT can markedly increase the proportion of cells with abnormal morphology. Then, annexin V labeling was used to detect phosphatidylserine externalization, a hallmark of the early phase of apoptosis.51 rGO-Ru-PEG-treated cells with PTT and PDT treatment show apparent features of apoptosis, as evidenced by the appearance of green fluorescence in the cell membrane (Figure 7B). Flow cytometric analysis shows that both PTT and PDT treatment of rGO-Ru-PEG-treated A549 cells leads to an apparent dosedependent increase in the percentage of A549 cells that are annexin V positive. As expected, an enhancement in apoptosisinducing capability is observed for combined PTT−PDT treatment as compared with either treatment alone. As shown in Figure 7C, after A549 cells are treated with rGO-Ru-PEG (5 μM based on the concentration of Ru-PEG) for 24 h, the 6766

DOI: 10.1021/acsami.6b13808 ACS Appl. Mater. Interfaces 2017, 9, 6761−6771

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Figure 9. Observation of cathepsin B release from lysosomes to the cytosol induced in A549 cells incubated with rGO-Ru-PEG (5 μM based on the concentrations of Ru-PEG) upon PTT, PDT, or PTT− PDT combined treatment in A549 cells. (i) Cells kept in the dark. (ii) Cells irradiated with an 808 nm laser and a 450 nm laser serially. (iii) Cells treated with rGO-Ru-PEG and kept in the dark. (iv) Cells treated with rGO-Ru-PEG and irradiated with an 808 nm laser. (v) Cells treated with rGO-Ru-PEG and irradiated with a 450 nm laser. (vi) Cells treated with rGO-Ru-PEG and irradiated with an 808 nm laser and a 450 nm laser serially. (808 nm laser: 0.5 W/cm2, 5 min; 450 nm laser: 20 mW/ cm2, 2 min) Scale bar: 5 μm.

2.10. In Vivo Imaging and Antitumor Evaluation. For in vivo imaging of tumors by rGO-Ru-PEG, an infrared thermal mapping apparatus was used to record the temperature change under NIR laser irradiation. As shown in Figure 10A, after 5 min irradiation, a moderate increase in temperature (37.8 °C) is observed in tumors treated with PBS. In tumor treated with rGO-Ru-PEG (100 μL, rGO = 1 mg/mL, Ru-PEG = 250 μM), the temperature increases rapidly to 58.7 °C. The result indicates that photothermal imaging by rGO-Ru-PEG can provide an effective tool for monitoring the outcomes of the treatment. For in vivo treatment evaluation, mice bearing A549 tumors with initial volumes of 100−150 mm3 were chosen and randomly divided into four groups. The corresponding treatments were conducted 4 h after injection. The control group was irradiated by 808 and 450 nm lasers. The other three groups were injected with rGO-Ru-PEG (100 μL, rGO = 1 mg/mL, Ru-PEG = 250 μM). For the PTT treatment, the mice were irradiated with an 808 nm laser at a power density of 0.5 W/cm2 for 5 min. The mice for the PDT treated group were irradiated with a 450 nm laser at a power density of 50 mW/cm2 for 2 min. For the combined PTT−PDT treatment, the mice were irradiated with 808 and 450 nm lasers at the same power densities serially. Tumor volumes and body weights were monitored every 3 days. For tumors with PTT treatment alone, their growth is largely inhibited in the first 6 days, after which rapid regrowth occurs. The tumor growth in the PDT treated group is also only partially inhibited. In marked contrast, the combined PTT−PDT treatment can greatly inhibit the growth of tumors (Figure 10B,C). Moreover, neither obvious body weight loss nor noticeable abnormality is observed for all the tested groups (Figure S10). These results indicate that rGO-Ru-PEG has great potential for efficient imaging guided PTT−PDT combined antitumor treatment.

Figure 10. (A) IR thermal images of A549 tumor-bearing mice exposed to 808 nm laser for 5 min (0.5 W/cm2). (B) Tumor growth curves of different groups of A549 tumor-bearing mice (5 mice per group). Error bars were based on standard error of mean. (C) Photos of mice after various treatments taken at day 15. Tumor sites are marked with red dashed circles.

thermal-responsive releasing properties. As expected, rGO-RuPEG shows photothermal activities upon 808 nm irradiation and 1 O2-producing capability upon 450 nm irradiation. rGO-RuPEG can be efficiently uptaked by A549 cancer cells and can specifically localized to lysosomes. In vitro cell viability assays show that rGO-Ru-PEG exhibits higher anticancer efficacy upon combined PTT−PDT treatment as compared with either PTT or PDT treatment alone. Further mechanistic studies show that rGO-Ru-PEG can induce apoptosis through ROS generation and lysosomal damage upon combined PTT−PDT treatment. Moreover, rGO-Ru-PEG can be applied for in vivo photothermal imaging. In vivo tumor ablation was achieved with excellent PTT−PDT combined treatment efficacy. Our study demonstrates the potential of rGO-Ru-PEG for multifunctional imaging and combinational PTT−PDT treatment of cancer, which merits further investigations.

4. EXPERIMENTAL SECTION 4.1. Materials. Ruthenium chloride hydrate and H2dcbpy were purchased from J&K Chemical (China). Single-layer grapheme oxide sheets were purchased from Nanjing XF NANO Materials Tech Co., Ltd. (China). O-(2-Aminoethyl)-o′-methylpolyethylene glycol (H2NPEG, MW = 2000), EDC, NHS), RNO, and imidazole were purchased from Aladdin Reagent (China). The dialysis membrane was sourced from Rancho Dominguez (USA). MTT, DCFH-DA, Hoechst 33342, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and antibiotics (penicillin/streptomycin) were obtained

3. CONCLUSIONS We present nanohybrid rGO-Ru-PEG constructed by rGO loaded with a PEG-modified ruthenium complex (Ru-PEG). Interestingly, rGO-Ru-PEG shows pH-sensitive and photo6767

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ACS Applied Materials & Interfaces

ultrafiltration centrifuge unit (MW 10 kDa). The product was washed with water until the filtrate became free of yellow color. The residue was dispersed in 5 mL of water. 4.4. AFM Measurement. The aqueous solutions of Ru-PEG (25 μM), rGO (50 μg/mL) and rGO-Ru-PEG (25 μM based on the concentration of Ru-PEG) were dropped onto newly clipped mica and air-dried. The samples were analyzed using a Bruker Multimode 8 AFM under ScanAsyst mode in air at room temperature. 4.5. XPS Measurement. The aqueous solution of rGO-Ru-PEG (25 μM based on the concentration of Ru-PEG) was dropped onto new glass slide and air-dried. The samples were analyzed using a VG Thermo ESCALAB 250 spectrometer. 4.6. Photophysical Properties and Stability of rGO-Ru-PEG. The concentration of rGO was determined by absorbance at 245 nm according to a standard curve of rGO. The concentration of Ru-PEG was quantified by the strong absorption peak at 460 nm, and the absorption of rGO at the same wavelength was subtracted. Fluorescence spectra of rGO-Ru-PEG and Ru-PEG were measured upon 460 nm excitation. To measure the release of Ru-PEG from the nanohybrid, rGO-Ru-PEG was incubated in PBS (pH 5 or 7.4) for different time intervals (0, 4, 8, and 12 h). The amount of Ru-PEG retained on rGORu-PEG was calculated by absorption at 460 nm after the removal of the detached Ru-PEG by centrifugal filtration. 4.7. Photothermal Activity of rGO-Ru-PEG. rGO and rGO-RuPEG (2.5, 5, or 10 μg/mL) in water were placed in a quartz cuvette of 4 mm path length and irradiated with an 808 nm laser (1 W/cm2, 5 min). Light-induced temperature changes in the solutions were collected using a thermal temperature probe. Millipore water was used as the control group. 4.8. Photothermal-Triggered Release of Ru-PEG. The solution of rGO-Ru-PEG (Ru-PEG, 50 μM) was irradiated with an 808 nm laser at an output power of 0.5 W/cm2 for 5 min. After centrifugation, the concentration of Ru-PEG in supernatants was measured by absorption at 460 nm. 4.9. Detection of Singlet Oxygen (1O2). The generation of 1O2 was determined following the literature procedure using RNO as the 1 O2 indicator.45 Briefly, air-equilibrated solutions containing the tested samples, RNO (25 μM), and imidazole (500 μM) were prepared in the dark and irradiated with a 450 nm LED light array. The quantum yields for 1O2 production (ΦΔ) of rGO-Ru-PEG, Ru-PEG, and rGO under irradiation in solution were calculated using [Ru(bpy)3]Cl2 as the standard (ΦΔ = 0.18 in H2O).46 4.10. Cell Lines and Culture Conditions. A549 cells were obtained from Experimental Animal Center of Sun Yat-Sen University (Guangzhou, China). Cells were maintained in RPMI 1640 (Roswell Park Memorial Institute 1640, Gibco BRL) medium, which contained 10% FBS (fetal bovine serum, Gibco BRL), 100 μg/mL streptomycin (Gibco BRL), and 100 U/mL penicillin (Gibco BRL). The cells were cultured in a humidified incubator, which provided an atmosphere of 5% CO2 and 95% air at a constant temperature of 37 °C. 4.11. Cellular Uptake. 4.11.1. Confocal Microscopy. A549 cells (1 × 105 cells) were seeded in 35 mm culture dishes (Corning) for 24 h. Culture medium containing Ru-PEG (10 μM) or rGO-Ru-PEG (10 μM based on the concentration of Ru-PEG) was added. After incubation for 2, 4, 8, or 12 h in the dark, cells were washed with PBS three times and visualized by confocal microscopy immediately upon excitation at 405 nm. 4.11.2. Colocalization Assay. A549 cells were coincubated with RuPEG (10 μM) or rGO-Ru-PEG (10 μM based on the concentration of Ru-PEG) and LTDR (150 nM) or MTDR (150 nM) at 37 °C for 30 min. Cells were washed three times with PBS and visualized by confocal microscopy immediately. The wavelength for the excitation of Ru-PEG is 405 nm. The excitation wavelength of MTDR and LTDR is 633 nm. Emission was collected at 640 ± 20 nm (Ru-PEG), 665 ± 20 nm (MTDR) and 668 ± 20 nm (LTDR). 4.11.3. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Measurement. A549 cells were seeded in 10 cm tissue culture dishes and incubated for 24 h. The medium was removed and replaced with fresh medium containing Ru-PEG (2.5 μM) or equivalent rGO-RuPEG. After incubation for 24 h, the cells were washed with PBS,

from Sigma-Aldrich (USA). MTDR and LTDR were purchased from Thermo Fisher Scientific (USA). CellTiter-Glo luminescent assay kit and Caspase-Glo 3/7 kit was purchased from Promega (USA). Magic Red MR-(RR)2 was purchased from Immunochemistry Tech (USA). All the other chemicals were of analytical grade. Deionized water, purified by a Milli-Q water purification system (Millipore, USA) to a minimum resistivity of 18.2 MΩ cm, was used in all experiments. 4.2. General Instruments. Microanalysis of elements (C, H, and N) was carried out using an Elemental Vario EL CHNS analyzer (Germany). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 400 MHz spectrometer (Germany) at room temperature. Shifts were referenced relative to the internal solvent signals. Electrospray ionization mass spectrometry (ESI-MS) was recorded on a Thermo Finnigan LCQ DECA XP spectrometer (USA). The quoted m/z values represented the major peaks in the isotopic distribution. UV/vis spectra were recorded on a Varian Cary 300 spectrophotometer (USA). The fluorescence emission spectra were obtained using a Shimadzu RF-5301PC spectrofluorophotometer. Fourier transform infrared (FT-IR) spectrometry was performed on a Bruker Vector-22 infrared spectrometer (Germany). Cell imaging experiments were carried out on a confocal microscope (Zeiss LSM-710, ZEISS, Germany). Flow cytometric analysis was done using a BD FACS Calibur flow cytometer (Becton Dickinson, USA). AFM images were obtained in air at room temperature with a scanning probe microscope (Dimension Fastscan, Bruker, Germany) and an SPI3800N control station (Seiko Instruments) operated in the tapping mode. Probes made of a single silicon crystal with the cantilever length of 129 mm and the spring constant of 33−62 N/m (OMCL-AC160TS-W2, Olympus) were used for imaging. The composition of the samples was investigated via an XPS (ESCALab250, Thermo VG) with 200 W Al KR radiation in twin anode. All core level XPS spectra were calibrated using C 1s photoelectron peak at 284.6 eV as the reference. 4.3. Preparation of rGO-Ru-PEG Nanohybrids. 4.3.1. Synthesis of [Ru(DIP)2(H2dcbpy)][Cl]2 (Ru1). Ru1 was synthesized according to published methods with slight modifications.38 A mixture of [Ru(DIP)2Cl2]·2H2O (0.5 mmol, 1 equiv), H2dcbpy (0.55 mmol, 1.1 equiv), and NaOAc (3.95 mmol, 7.9 equiv) in CH3OH/H2O (4:1, v/v) was heated to reflux for 24 h under nitrogen atmosphere in the dark. The solution was cooled to room temperature, and dilute hydrochloric acid was added to adjust the pH to 1 with vigorous stirring. The solution was concentrated by evaporation, and then NaCl (200 mg) was added. The solution was stored at 5 °C overnight. The precipitate was filtered and washed with CH2Cl2. The crude product was recrystallized from a mixture of methanol and diethyl ether to give the pure complex as a dark red powder. Yield: 357 mg (62%). 1H NMR (400 MHz, methanol-d4) δ 9.28 (s, 2H), 8.42 (d, 2H), 8.32 (m, 4H), 8.24 (d, 2H), 8.19 (d, 2H), 7.95 (d, 2H), 7.89 (d, 2H), 7.63 (m, 20H). ESI-MS (CH3CN) m/z 504.9 [M − 2Cl]2+. Elemental analysis: calcd (%) for C60H49Cl2N6O8.5Ru: C, 62.02; H, 4.25; N, 7.23; found: C, 62.01; H, 4.19; N, 7.21. 4.3.2. Synthesis of Ru-PEG. A mixture of the Ru1 (0.04 mmol, 1 equiv), EDC (60 mg), and NHS (60 mg) dissolved in DMF (10 mL) was stirred at 25 °C for 30 min. Then, H2N-PEG (0.08 mmol, 2 equiv) was added, and the mixture was vigorously stirred for 24 h at room temperature. Free PEG and Ru1 were removed by dialysis (dialysis bag MW cutoff 2000). The solution was freeze-dried to obtain a red solid powder. Yield: 20 mg (10%). 1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 2H), 8.34 (d, 2H), 8.23 (t, 8H), 7.92 (d, 4H), 7.74 (d, 4H), 7.64 (m, 20H), 3.49 (s, 360H), 3.22 (s, 6H). 4.3.3. Synthesis of Reduced Graphene Oxide (rGO). rGO was synthesized following a literature procedure with slight modifications.39 NaOH (0.05 M) and graphene oxide (1 mg/mL) dispersed in water were subjected to ultrasonication for 4 h in an ice bath. The solution was filtered through a filter (0.2 μm) to exclude large GO sheets. The filtrate (5 mL), hydrazine hydrate (5 μL, 80 wt %), and ammonium hydroxide (80 μL, 25 wt %) were mixed in water (15 mL). The solution was sonicated for 2 min and then heated at 95 °C for 2 h with vigorous stirring. 4.3.4. Preparation of rGO-Ru-PEG. rGO (2 mL, 1 mg/mL) and RuPEG (1 mL, 2.5 mg/mL) were mixed in H2O (5 mL) and incubated at 25 °C overnight. Unabsorbed excess Ru-PEG was removed with a single 6768

DOI: 10.1021/acsami.6b13808 ACS Appl. Mater. Interfaces 2017, 9, 6761−6771

Research Article

ACS Applied Materials & Interfaces

serially irradiated with an 808 nm laser (0.5 W/cm2) and a 450 nm laser (20 mW/cm2). Relative luminescent units (RLU) were detected with a microplate reader (Infinite F200, Tecan, Switzerland). The results are averaged among 3 replicates and have been normalized by the values obtained on untreated control cells. Error bars represent the standard deviation. 4.16. Detection of Intracellular ROS. 4.16.1. Confocal Microscopy. A549 cells were seeded into 35 mm culture dishes and incubated for 24 h. The cells were treated with medium containing Ru-PEG (1.25 and 2.5 μM) or rGO-Ru-PEG (1.25 and 2.5 μM based on the concentration of Ru-PEG) at 37 °C for 24 h in the dark. The cells were then washed twice with serum-free medium and incubated with DCFHDA (10 μM) for 15 min at 37 °C in the dark. The samples were washed twice with serum-free medium and then irradiated with a 450 nm laser (20 mW/cm2, 2 min). The samples were then analyzed immediately by confocal microscopy. Emission was collected at 530 ± 20 nm upon excitation at 488 nm. 4.16.2. Flow Cytometry. After treatment with Ru-PEG (1.25 and 2.5 μM) or rGO-Ru-PEG (1.25 and 2.5 μM based on the concentration of Ru-PEG) for 24 h, the cells were irradiated with a 450 nm laser (20 mW/cm2, 2 min). The cells were then harvested and incubated with DCFH-DA (10 μM) in serum-free medium for 15 min at 37 °C in the dark. After being washed twice with serum-free DMEM, the samples were analyzed by flow cytometry with excitation at 488 nm and emission at 530 ± 15 nm. MFI was analyzed using FlowJo 7.6 software (Tree Star, USA). 4.17. Detection of Cathepsin B Release. Cathepsin B activity was detected using the fluorogenic susbtrate Magic Red MR-(RR)2 according to the manufacturer’s instructions. Briefly, A549 cells seeded into 35 mm dishes (Corning) were treated with rGO-Ru-PEG (5 μM based on the concentrations of Ru-PEG) for 24 h and then irradiated with 808 nm laser (0.5 W/cm2, 5 min) or a 450 nm laser (20 mW/cm2, 2 min). For the combined PTT−PDT treatment, cells were serially irradiated with an 808 nm laser (0.5 W/cm2, 2 min) and a 450 nm laser (20 mW/cm2, 5 min). The cells were washed twice with PBS and then incubated with Magic Red MR-(RR)2 at 37 °C for 1 h. After being washed twice with PBS, the cells were visualized by confocal microscopy. Emission was collected at 630 ± 20 nm upon excitation at 543 nm. 4.18. In Vivo Imaging and Photothermal/Photodynamic Therapy. BALB/c-(nu/nu) female nude mice aged 4−5 weeks were purchased and bred in the Center of Experiment Animals at Sun Yat-Sen University. All experimental protocols were approved by the Sun YatSen University Animal Care and Use Committee. A549 xenografts were established by inoculating 2 × 106 cells via subcutaneous injection (s.c.) into BALB/c-(nu/nu) female nude mice. When the tumor volume reached 100−150 mm3, the nude mice were randomly allocated into four groups (5 mice per group) before the experiments. Thermal imaging was recorded by a thermal camera (MAG30, Magnity Electronics, Thermal Imaging Expert) when the tumors were exposed to an 808 nm laser with a power density at 0.5 W/cm2 for 5 min. The PTT/PDT process was conducted as follows: (1) group 1 (laser only): Mice were only irradiated by 808 and 450 nm laser as a control. (2) group 2 (rGO-Ru-PEG + 808 nm laser): mice were intratumorally injected with rGO-Ru-PEG (100 μL/20 g body weight of 1 mg/mL solution, 100 μg of rGO-Ru-PEG/20 g body weight), and then irradiated with an 808 nm NIR laser (0.5 W/cm2, 5 min). (3) group 3 (rGO-Ru-PEG + 450 nm laser): Mice were intratumorally injected with the rGO-Ru-PEG (100 μL/20 g body weight of 1 mg/mL solution, 100 μg of rGO-Ru-PEG/20 g body weight), and then irradiated with a 450 nm NIR laser (0.5 W/cm2, 5 min). (4) group 4 (rGO-Ru-PEG + 808 and 450 nm laser): Mice were intratumorally injected with rGO-RuPEG (100 μL/20 g body weight of 1 mg/mL solution, 100 μg of rGORu-PEG/20 g body weight), and then serially irradiated with an 808 nm laser (0.5 W/cm2, 5 min) and a 450 nm laser (20 mW/cm2, 2 min). After irradiation (day 0), the tumor sizes were measured using a caliper every 3 days. The tumor volumes were calculated based on the following formula:

trypsinized, and collected. The cells were counted and digested with HNO3 (65%, 0.2 mL) at room temperature for 24 h. The solution was then diluted to a final volume of 10 mL with Milli-Q water. The concentration of ruthenium was measured using the XSERIES 2 ICPMS (Thermo Scientific, USA). 4.12. In Vitro Cytotoxicity Assay. The cells were seeded in 96-well plates at 1 × 104/well and cultured for 24 h. Then, the medium was replaced with medium containing different concentrations of Ru-PEG, rGO or rGO-Ru-PEG. After 24 h, the media was removed, and fresh media was added. Then, PTT groups were irradiated with an 808 nm NIR laser (0.5 W/cm2, 5 min), whereas PDT groups were irradiated with a 450 nm laser (20 mW/cm2, 2 min). For the combined PTT− PDT treatment, cells were serially irradiated with an 808 nm laser (0.5 W/cm2) and a 450 nm laser (20 mW/cm2). After 20 h, 20 μL of MTT (5 mg/mL) solution was added to each well. The plates were incubated in the dark for an additional 4 h. The media was carefully removed. DMSO was added (150 μL per well), and the plate was incubated at room temprature for 10 min with shaking. The absorbance at 595 nm was measured using a microplate reader (Infinite F200, Tecan, Switzerland). The cells treated under identical conditions in dark were kept as control groups. The percentage of viability was calculated as the following formula: (% viable cells) = (OD of treated sample/OD of untreated sample) × 100%. 4.13. Hoechst 33342 Staining. A549 cells were seeded into 35 mm dishes (Corning) and incubated for 24 h. The cells were treated with rGO-Ru-PEG (2.5 μM based on the concentration of Ru-PEG) for 24 h, after which the samples were irradiated with an 808 nm laser (0.5 W/ cm2, 5 min) or a 450 nm laser (20 mW/cm2, 2 min). For the combined PTT−PDT treatment, cells were serially irradiated with an 808 nm laser (0.5 W/cm2, 2 min) and a 450 nm laser (20 mW/cm2, 5 min). After incubated for 12 h, the cells were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. Then, the cells were labeled with Hoechst 33342 (5 μg/mL in PBS) for 5 min and wash twice with PBS. The cells were imaged immediately with a confocal laser-scanning microscope with excitation at 405 nm and emission at 460 ± 20 nm. 4.14. Annexin V-FITC Staining. 4.14.1. Confocal Microscopy. A549 cells were seeded into 35 mm dishes (Corning) and then treated with rGO-Ru-PEG (2.5 μM based on the concentration of Ru-PEG) for 24 h, after which the samples were irradiated with an 808 nm laser (0.5 W/cm2, 5 min) or a 450 nm laser (20 mW/cm2, 2 min). For the combined PTT−PDT treatment, cells were serially irradiated with an 808 nm laser (0.5 W/cm2, 2 min) and a 450 nm laser (20 mW/cm2, 5 min). After incubation for 12 h, the cells were stained using the annexin V-FITC apoptosis detection kit (Sigma-Aldrich, USA) according to the manufacturer’s recommendations. The samples were then analyzed immediately by confocal microscopy with excitation at 488 nm and emission at 530 ± 20 nm. 4.14.2. Flow Cytometry. A549 cells were cultured in 6-well tissue culture plates for 24 h and then treated with rGO-Ru-PEG (2.5 μM based on the concentration of Ru-PEG) for 24 h. Then, the samples were irradiated with an 808 nm laser (0.5 W/cm2, 5 min) or a 450 nm laser (20 mW/cm2, 2 min). For the combined PTT−PDT treatment, cells were serially irradiated with an 808 nm laser (0.5 W/cm2, 2 min) and a 450 nm laser (20 mW/cm2, 5 min). After incubation for 12 h, the cells were harvested and stained using the annexin V apoptosis detection kit according to the manufacturer’s recommendations. The samples were measured by flow cytometry with excitation at 488 nm and emission at 530 ± 20 nm. Data were analyzed by FlowJo software (Tree Star, USA). A total of 10 000 events were acquired for each sample. 4.15. Measurement of Intracellular ATP Levels and Caspase 3/7 Activity Assays. Measurement of adenosine triphosphate (ATP) content was carried out using the CellTiter-Glo kit (Promega), and caspase 3/7 activity was determined using Caspase-Glo 3/7 kit (Promega) according to the manufacturer’s instructions. Briefly, the cells were seeded in 96-well plates, cultured for 24 h, and then treated with rGO-Ru-PEG (5 μM based on the concentrations of Ru-PEG) for 24 h. PTT groups were irradiated with an 808 nm NIR laser (0.5 W/cm2, 5 min), whereas PDT groups were irradiated with a 450 nm laser (20 mW/cm2, 2 min). For the combined PTT−PDT treatment, cells were

tumor volume (V ) = (tumor length) × (tumor width)2 /2 6769

DOI: 10.1021/acsami.6b13808 ACS Appl. Mater. Interfaces 2017, 9, 6761−6771

Research Article

ACS Applied Materials & Interfaces The relative tumor volumes were calculated as V/V0, where V0 was the initial tumor volume at day 0. 4.19. Statistical Analysis. All biological experiments were performed at least twice with triplicates in each experiment. Representative results were depicted in this report and data were presented as means ± standard deviations (SD).

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(8) 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. (9) Lucky, S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015, 115 (4), 1990−2042. (10) Nam, J. S.; Kang, M. G.; Kang, J.; Park, S. Y.; Lee, S. J. C.; Kim, H. T.; Seo, J. K.; Kwon, O. H.; Lim, M. H.; Rhee, H. W.; Kwon, T. H. Endoplasmic Reticulum-Localized Iridium(III) Complexes as Efficient Photodynamic Therapy Agents via Protein Modifications. J. Am. Chem. Soc. 2016, 138 (34), 10968−10977. (11) Park, J.; Jiang, Q.; Feng, D. W.; Mao, L. Q.; Zhou, H. C. SizeControlled Synthesis of Porphyrinic Metal-Organic Framework and Functionalization for Targeted Photodynamic Therapy. J. Am. Chem. Soc. 2016, 138 (10), 3518−3525. (12) Frei, A.; Rubbiani, R.; Tubafard, S.; Blacque, O.; Anstaett, P.; Felgentrager, A.; Maisch, T.; Spiccia, L.; Gasser, G. Synthesis, Characterization, and Biological Evaluation of New Ru(II) Polypyridyl Photosensitizers for Photodynamic Therapy. J. Med. Chem. 2014, 57 (17), 7280−7292. (13) Huang, H. Y.; Yu, B. L.; Zhang, P. Y.; Huang, J. J.; Chen, Y.; Gasser, G.; Ji, L. N.; Chao, H. Highly Charged Ruthenium(II) Polypyridyl Complexes as Lysosome-Localized Photosensitizers for Two-Photon Photodynamic Therapy. Angew. Chem., Int. Ed. 2015, 54 (47), 14049−14052. (14) Mari, C.; Pierroz, V.; Ferrari, S.; Gasser, G. Combination of Ru(II) Complexes and Light: New Frontiers in Cancer Therapy. Chem. Sci. 2015, 6 (5), 2660−2686. (15) Padilla, R.; Rodriguez-Corrales, J. A.; Donohoe, L. E.; Winkel, B. S. J.; Brewer, K. J. A New Class of Ru(II) Polyazine Agents with Potential for Photodynamic Therapy. Chem. Commun. 2016, 52 (13), 2705−2708. (16) Gill, M. R.; Thomas, J. A. Ruthenium(II) Polypyridyl Complexes and DNA-From Structural Probes to Cellular Imaging and Therapeutics. Chem. Soc. Rev. 2012, 41 (8), 3179−3192. (17) Gill, M. R.; Garcia-Lara, J.; Foster, S. J.; Smythe, C.; Battaglia, G.; Thomas, J. A. A Ruthenium(II) Polypyridyl Complex for Direct Imaging of DNA Structure in Living Cells. Nat. Chem. 2009, 1 (8), 662−667. (18) Puckett, C. A.; Barton, J. K. Methods to Explore Cellular Uptake of Ruthenium Complexes. J. Am. Chem. Soc. 2007, 129 (1), 46−47. (19) Puckett, C. A.; Barton, J. K. Mechanism of Cellular Uptake of A Ruthenium Polypyridyl Complex. Biochemistry 2008, 47 (45), 11711− 11716. (20) Li, S. P.; Liu, H. W.; Zhang, K. Y.; Lo, K. K. Modification of Luminescent Iridium(III) Polypyridine Complexes with Discrete Poly(ethylene glycol) (PEG) Pendants: Synthesis, Emissive Behavior, Intracellular Uptake, and PEGylation Properties. Chem. - Eur. J. 2010, 16 (28), 8329−8339. (21) Li, S. P.; Lau, C. T.; Louie, M. W.; Lam, Y. W.; Cheng, S. H.; Lo, K. K. Mitochondria-Targeting Cyclometalated Iridium(III)-PEG Complexes with Tunable Photodynamic Activity. Biomaterials 2013, 34 (30), 7519−7532. (22) Lee, D. E.; Koo, H.; Sun, I. C.; Ryu, J. H.; Kim, K.; Kwon, I. C. Multifunctional Nanoparticles for Multimodal Imaging and Theragnosis. Chem. Soc. Rev. 2012, 41 (7), 2656−2672. (23) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114 (21), 10869−10939. (24) Shi, S.; Huang, Y.; Chen, X.; Weng, J.; Zheng, N. Optimization of Surface Coating On Small Pd Nanosheets for in Vivo Near-Infrared Photothermal Therapy of Tumor. ACS Appl. Mater. Interfaces 2015, 7 (26), 14369−14375. (25) Chen, Z.; Wang, Q.; Wang, H.; Zhang, L.; Song, G.; Song, L.; Hu, J.; Wang, H.; Liu, J.; Zhu, M.; Zhao, D. Ultrathin PEGylated W18O49 Nanowires as a New 980 nm-Laser-Driven Photothermal Agent for Efficient Ablation of Cancer Cells in Vivo. Adv. Mater. 2013, 25 (14), 2095−2100. (26) Teng, I. T.; Chang, Y. J.; Wang, L. S.; Lu, H. Y.; Wu, L. C.; Yang, C. M.; Chiu, C. C.; Yang, C. H.; Hsu, S. L.; Ho, J. A. Phospholipid-

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b13808. ESI-MS and 1H NMR spectra; AFM image of rGO and Ru-PEG; FTIR spectra of rGO, Ru-PEG and rGO-RuPEG; stabilities of rGO and rGO-Ru-PEG; photothermal activities of rGO; cellular uptake of Ru-PEG and subcellular localization of Ru-PEG; body weights of mice with various treatments (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: (+86)20-8411-2245. *E-mail: [email protected]. ORCID

Zong-Wan Mao: 0000-0001-7131-1154 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Science Foundation of China (Nos. 21231007, 21572282 and 21571196), the 973 program (Nos. 2014CB845604 and 2015CB856301), the Guangdong Natural Science Foundation (2015A030306023), and the Fundamental Research Funds for the Central Universities.

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REFERENCES

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DOI: 10.1021/acsami.6b13808 ACS Appl. Mater. Interfaces 2017, 9, 6761−6771

Research Article

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DOI: 10.1021/acsami.6b13808 ACS Appl. Mater. Interfaces 2017, 9, 6761−6771