Fluorescent Phthalocyanine–Graphene Conjugate with Enhanced NIR

Article Cite This: ACS Appl. Nano Mater. 2018, 1, 2785−2795

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Fluorescent Phthalocyanine−Graphene Conjugate with Enhanced NIR Absorbance for Imaging and Multi-Modality Therapy Jiabao Pan,†,‡ Yang Yang,#,‡ Wenjuan Fang,† Wei Liu,*,† Kai Le,† Dongmei Xu,† and Xiangzhi Li*,# †

State Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, P. R. China School of Medicine, Shandong University, Jinan 250012, P.R. China

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ABSTRACT: There has been increasing interest in design theranostic agents for combining diagnosis and different treatment modalities, especially for development physiological stable materials to avoid instability and dissociation in biological environment. Herein, a covalently connected silicon phthalocyanine (SiPc) and graphene oxide (GO) conjugate SiPc-GO is designed and synthesized via conjugation reaction to render stability. This novel highly water-soluble material displays intrinsically fluorescence and synchronous photothermal-photodynamic therapy (PTT/PDT) effect, along with 3-fold higher near-infrared (NIR) absorbance comparing to pristine GO. In vitro cell studies show that SiPc-GO could cause intracellular fluorescence, photothermal effect and reactive oxygen species (ROS) generation synchronously, and effective photoablation of cancer cells could be triggered by both 671 and 808 nm lasers via synergistic PTT/PDT or NIR photothermal effects, respectively. In vivo systemic administration in MCF-7 xenograft mice shows that SiPc-GO could effectively accumulate in the tumor regions and induce the inhibition of tumor growth violently after laser irradiation. This work establish SiPc-GO as a multimodality nanosized photomedicine for cancer imaging, synergistic PTT/PDT, and NIR photothermal therapy. KEYWORDS: graphene oxide, phthalocyanine, photodynamic therapy, photothermal therapy, fluorescence

1. INTRODUCTION Currently, surgery, chemotherapy, and radiotherapy are still the mainstream of cancer therapeutic modalities. However, these approaches have many limitations such as invasive, toxic side effects, poor tumor-selectivity and limited efficacy. In contrast to these conventional methods, photodynamic therapy (PDT) has attracted increasingly interest in cancer treatment due to its noninvasive, reduced side effects and improved tumor targeting nature.1,2 Unlike ionizing radiation, this “friendly” therapy enables patients to be treated repetitively without dosage limit and harmful side effect.3 In PDT, singlet oxygen (1O2) is considered to be the main reactive oxygen species (ROS) produced upon triggering photosensitizer (PS) by light and then transferring the photon energy to surrounding oxygen molecules, which induces cellular and tissue damage.1,4 Therefore, photosensitizer, molecular oxygen, and light are identified as the three principal factors in PDT.5,6 Over the last decades, various classes of PS have been developed to improve their photophysical, photochemical and PDT efficacy.7−10 Among these PSs, phthalocyanine (Pc) is a kind of promising PS because of its high singlet oxygen generation and intense absorption in red and NIR region that is recognized as optimal therapeutic window.11 In addition, silicon phthalocyanine (SiPc) with two axial ligands on the opposite sides of the macrocycle is considered to be one of the most hopeful PS for PDT because of its better photoactivity and biocompatibility.12 © 2018 American Chemical Society

However, a typical feature of tumors is hypoxia, which would tremendously suppresses the efficiency of PDT, especially when oxygen is gradually exhausted as the treatment progresses.13,14 Hence, integrating PDT with other therapies to improve treatment efficacy is receiving much more attention. Photothermal therapy (PTT) is another type of phototherapy, which involves cell damage by heat generated from PTT agent via conversion of light absorption.15,16 So far, gold nanoparticles,17 self-assembled nanostructured dyes,18 and carbon nanomaterials19 have been identified as promising photothermal agents. Carbon nanomaterials, including fullerenes, carbon nanohorns,20 carbon nanotubes,21,22 and graphene,23−25 have attracted intensely attention as PTT agents, because of their good biocompatibility and photothermal conversion efficiency.26,27 Because of its abundant, low cost, and convenient preparation from graphite, graphene has the advantages as PTT agent comparing to other carbon-based nanomaterials.28,29 In particular, graphene oxide (GO), the water-soluble derivative of graphene, has attracted much more attention because of its water-solubility and plentiful surface functional groups (carboxylic, epoxide and hydroxyl groups), Received: March 21, 2018 Accepted: May 15, 2018 Published: May 15, 2018 2785

DOI: 10.1021/acsanm.8b00449 ACS Appl. Nano Mater. 2018, 1, 2785−2795

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ACS Applied Nano Materials

EDC (0.4 mM), NHS (0.4 mM), DIPEA (3 mM), and SiPc-NH2 (74.9 mg, 0.1 mM) were added and the mixture was stirred for 3 days at room temperature under a nitrogen atmosphere. The mixture was dialyzed (molecular weight cutoff: 3500) in DMF repetitively for 3 days until the dialyzate became colorless, and followed by dialysis using deionized water for 2 days. Then the final product was freezedried to give the conjugate of SiPc-GO (20.6 mg), which was stored below 4 °C for further use (mass fraction of SiPc in SiPc-GO was calculated at ∼66%, GO at ∼34%). 2.4. Characterizations and Instruments. Ultraviolet−visible (UV−vis) spectra were measured on a PerkinElmer Lambda-35 UV− vis spectrometer and the samples were dispersed in H2O or DMF. The Raman spectra were recorded on a Horbin PHS-3C Raman spectrometers. Steady-state fluorescence spectra were obtained on a Hitachi F-2700 spectrofluorimeter equipped with a 450 W Xe lamp. FT-IR spectra were acquired on a Nicolet NEXUS 670 Fourier transformation infrared (FTIR) spectrometer. Atomic force microscopy (AFM) images were carried out on Dimension Icon (Veeco Instruments Inc.) and by noncontact optical microscope (VHX1000C, Keyence). X-ray-photoelectron (XPS) were investigated on a K-Alpha X-ray Photoelectron Spectrometer System (ESCALAB 250, Thermo, USA), and C 1s (284.6 eV) was used to calibrate the peak positions for elemental analysis. The MTT assay was performed on a Bio-TEK ELX 800 microplate reader. 2.5. Singlet Oxygen Determination. 1,3-Diphenylisobenzofuran (DPBF) was used as a scavenger of the singlet oxygen (1O2). The DMF solution containing DPBF (4 mM) and SiPc-GO solution (20 μg mL−1) was irradiated by red light (λ > 610 nm, 0.2 mW/cm2). The absorbance of DPBF at 415 nm was recorded every 5 s along with the irradiation. 2.6. Photothermal Effect Measurement. SiPc-GO was dissolved in H2O with sonication to a final concentration of 20 μg mL−1, and equivalent amount of GO (6.8 μg mL−1) and SiPc-NH2 (13.2 μg mL−1) were diluted correspondingly. Then 1 mL of each solution was placed in a cuvette and irradiated by a 671/808 nm Laser for 5 min. The temperature change was recorded every 30 s using an infrared thermal imager (FLUKE Ti10, USA). 2.7. Cell Lines and Culture. Human breast cancer cell (MCF-7) and human cervical cancer cell (HeLa) were used for biological evaluation. The cells were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (100 units mL−1 , streptomycin 100 μg mL −1, respectively) at 37 °C under 5% CO2. 2.8. Intracellular Fluorescence Imaging. Cells were seeded on a 6-well plate and cultured overnight at 37 °C under 5% CO2. Then cells were incubated with SiPc-GO in medium (10 μg mL−1, 1.0 mL) for 24 h. After incubation, the cells were rinsed with PBS for three times and the fluorescence signals from the red channel were captured immediately with an Olympus FV 1200 confocal microscope. The control group was incubated with only medium and acquired under the same conditions. 2.9. Detection of Intracellular ROS. The chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was used as an indicator for the detection of intracellular ROS. Cells were seeded on coverslips and cultured overnight at 37 °C under 5% CO2. Thereafter, the cells were cultured with SiPc-GO (10 μg mL−1) in DMEM for 24 h. The control group was cultured in pure DMEM for 24 h. All cells were rinsed with PBS after incubation, then 100 μL of CM-H2DCFDA (10 μM) in PBS were added and cultured for another 15 min at 37 °C under 5% CO2. An LED array (λ ≈ 660 nm, energy destiny = 0.2 mW/cm2) was used to irradiate the cells for 5 min afterward. The fluorescence images were captured immediately after rinsed by PBS using an Olympus FV 1200 confocal microscope. 2.10. In Vitro Anticancer Efficacy. Cells were placed in 96-well plates (1 × 104 per well) and cultured overnight at 37 °C with 5% CO2. The cells were then incubated with different concentrations of SiPc-GO for 24 h. After incubation, the cells were rinsed with PBS twice and 100 μL of culture medium was added to each well before being illuminated. Then irradiation groups were exposed to 671 nm (2.5 W/cm2) or 808 nm (12.5 W/cm2) laser for 5 min, and cell

enabling it with desirable physicochemical and biological properties.30−32 Recently, combined use of PDT and PTT for cancer treatment is receiving more and more interest due to the enormous enhancement of therapeutic efficacy. For examples, Liang et al. developed SWNHs−TSCuPc nanohybrid for both PTT and PDT;33 Mao et al. reported a type of GO modified with Ru(II)−polyethylene glycol complex for combining of PDT and PTT therapy.34 However, so far these dual-modality agents prepared from PS and carbon nanomaterials were all fabricated via π−π stacking interaction or electrostatic attraction and van der Waals interaction,35−38 which are generally nonfluorescent and unstable/dissociated in physiological environment, resulting in reduced therapeutic efficiency and enhanced side effects. In the present work, a covalently connected silicon phthalocyanine (SiPc) and graphene oxide (GO) conjugate SiPc-GO was designed and synthesized. To the best of our knowledge, this is the first covalently bonded hybrid system between GO and Pc for integrating PDT/PTT, along with fluorescence imaging properties. This novel nanocomposite displays intrinsically synchronous fluorescence, photothermal and photodynamic properties upon exposure to red light, which enable SiPc-GO to be an applicable theranostic agent for fluorescence imaging and combined noninvasive photodynamic and photothermal therapy. Meanwhile, its highly enhanced absorption in NIR region (>800 nm) enables SiPc-GO to treat more deep-seated or large solid tumors using NIR laser-driven photothermal therapy. The biological evaluations both in vitro and in vivo indicate synergetic anticancer efficacy of PDT and PTT along with good fluorescence emitting could be achieved via laser irradiation of SiPc-GO, showing this as-prepared SiPcGO is an efficient and biocompatible multimodality anticancer agent for fluorescence imaging, synergistic photodynamicphotothermal therapy, and also NIR photothermal therapy.

2. MATERIALS AND EXPERIMENTS 2.1. Chemicals and Materials. Graphene oxide powder (50−100 nm) was purchased from Suzhou TANFENG graphene Tech Co., Ltd. Silicon phthalocyanine dichloride (SiPcCl2), 2-(2-aminoethoxy)ethanol, 1,3-diphenylisobenzofuran (DPBF), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), N,N-diisopropylethylamine (DIPEA), and polyoxyethylene castor oil (Cremophor EL, CEL) were purchased from Sigma-Aldrich. N,N-Dimethylformamide (DMF) was distilled from barium oxide (BaO) under reduced pressure. All other reagents are in the purity of analytical grade and were used without further purification. 2.2. Synthesis of SiPc-NH2. Silicon phthalocyanine dichloride (SiPcCl2) (0.33 mmol), 2-(2-aminoethoxy)ethanol (2.64 mmol), K2CO3 (2.64 mmol), and pyridine (2 mL) were dissolved in 30 mL of toluene. The reaction mixture was heated and refluxed at 130 °C for 12 h under nitrogen atmosphere. After evaporation the volatiles, the residue was dissolved in chloroform (100 mL) and filtered. The filtrate was washed three times with deionized water and then concentrated under reduced pressure. The crud product was recrystallized from chloroform/n-hexane to give SiPc-NH2 as a green solid (56.2%). 1H NMR (CD3OD, 300 MHz): δ −1.87 (4H, t, J = 3.4 Hz, CH2), 0.54 (4H, t, J = 3.6 Hz, CH2), 1.72 (4H, t, J = 3.6 Hz, CH2), 1.81 (4H, t, J = 3.8 Hz, CH2), 8.43−8.46 (8H, m, Pc-H), 9.68−9.70 (8H, m, PcH).13C NMR (CD3OD, 75 MHz): δ 150.9, 137.1, 132.8, 124.8, 73.6, 73.4, 73.2, 62.2. Anal. Calcd for C40H36N10O4Si: C 64.15, H 4.85, N 18.70%; found C 65.54, H 4.53, N 18.53%. MS (MALDI-TOF): m/z 749.004 (calcd. for [M + H]+ 749.269). 2.3. Synthesis of SiPc-GO. Graphene oxide (7 mg) was dispersed in DMF to a concentration of 0.5 mg/mL under sonication. Then 2786

DOI: 10.1021/acsanm.8b00449 ACS Appl. Nano Mater. 2018, 1, 2785−2795

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ACS Applied Nano Materials Scheme 1. Schematic Illustration of the Formation of SiPc-GO

EDC/NHS conjugation procedure (Scheme 1). The final product was purified by dialysis (molecular weight cutoff: 3500) in DMF and H2O successively to remove conjugation reagent (EDC/NHS) and excess of SiPc-NH2. The mass fraction of SiPc-NH2 in SiPc-GO was calculated at ∼66%, showing SiPcGO possesses a high Pc loading efficiency. Thus, this conjugation method is a very promising approach for integrating Pc and GO. The morphology of GO and as-prepared SiPc-GO were compared by AFM. As shown in Figure 1, the lateral dimension of GO ranges from 50 to 60 nm with a thickness, measured from the height profile of the AFM images, at ∼9 nm. Whereas, the mean size for SiPc-GO averages 20−30 nm in diameter with a mean thickness at 4 nm, revealing the grafting of SiPcNH2 onto GO breaks the GO sheets into much smaller pieces. The Fourier transform infrared (FT-IR) spectra were used to trace the covalent bond formation in SiPc-GO. As shown in FT-IR spectrum of SiPc-GO (Figure 2a), apart from the main characteristic absorption bands from SiPc-NH2 and GO, the CO absorption band at 1728 cm−1 (νC=O) attributed to the carboxylic acid groups from GO disappeared, followed by the appearance of a new band at 1663 cm−1 attributed to νC=O(NH) absorption in SiPc-GO, indicating the successful synthesis of SiPc-GO with amide linkage between SiPc and GO. The fluorescence and UV−vis spectra of GO, SiPc-NH2, and SiPcGO, with equivalent concentrations of GO and SiPc-NH2, were performed in DMF. As shown in Figure 2b, the absorption curve of SiPc-GO exhibits the characteristic Soret (λmax ∼ 350 nm) and Q-band (λmax ∼ 660 nm)40 peaks of SiPc-NH2 superimposing with the absorption of GO, suggesting the existence of both components of GO and silicon phthalocyanine. Compared to SiPc-NH2, a red-shift of B-band is observed for SiPc-GO. In addition, SiPc-GO exhibits stronger absorption in the NIR range with a ∼3-fold increase at wavelength longer than 800 nm compared to that of GO, resulting in effective

viability was assessed 24 h after irradiation using MTT assay. Dark groups were acquired under the same conditions without irradiation; the control group was cultured with pure medium. 2.11. In Vivo Therapeutic Efficacy. All experiments using mice described herein were approved by the Animal Care and Utilization Committee of Shandong University and performed in accordance with applicable guidelines and regulations. NOD-SCID IL-2 receptor gamma null (NSG) female nude mice of 4−5 weeks old bearing MCF-7 cancer xenografts in both flanks (tumor size ∼100 mm3) were randomized into two groups (n = 3) for control and evaluation of phototoxicity. SiPc-GO (3.8 mg/kg) in PBS was injected via the tail vein; control group was injected with PBS instead. At 24 h post injection, the mice were irradiated with a 671 nm laser (2.5 W/cm2, 5 min) and thermal imaging was recorded every 1 min with an infrared thermal imager along with irradiation time. After irradiation (day 0), the tumor size was measured every 3 days with a caliper and tumor volume was calculated according to eq 139

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

(1)

relative tumor volume calculated as V/V0, where V0 is the initial tumor volume on day 0.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of SiPc-GO. In modern oncology, there has been increasing interest in creating therapeutic agents for combining different treatment modalities to reinforce therapeutic effect. Among them, hybrid materials constructed by phthalocyanine and graphene have attracted remarkable attention because of their enormous potential for integrating PTT and PDT into a single system for cancer therapy. To improve their biological stability, herein, a covalently connected phthalocyanine and graphene oxide conjugate SiPc-GO was designed and synthesized. First, a novel axial −NH2 terminated silicon phthalocyanine SiPc-NH2 was synthesized via modification of SiPcCl2 with 2-(2aminoethoxy)ethanol. Subsequent SiPc-GO was constructed via reactions between excess SiPc-NH2 and GO by the standard 2787

DOI: 10.1021/acsanm.8b00449 ACS Appl. Nano Mater. 2018, 1, 2785−2795

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Figure 1. AFM imagines of GO (a, b) and SiPc-GO (c, d) and the height profile of GO (e) and SiPc-GO (f).

three peak components with binding energies 399.05, 400.60, and 402.30 eV, which are attributed to nitrogen functionalities in CN, C−N, and N−H, respectively (Figure 3c). After grafting SiPc-NH2 onto GO, one additional peak at 403.03 eV appears attributing to the N 1s species for amide bond (O C−N). In addition, all the bind energy of CN (398.30 eV), C−N (399.60), and N−H (400.69) decrease by 0.75, 1.0, and 1.61 eV, respectively, compared with that of free SiPc-NH2 (Figure 3d). Because of the binding energy is related to the electron density around the nucleus, these energy decrease indicate that an electron density shift should occurred from GO to SiPc. Unlike peripheral connected GO,41,42 herein, GO shows the electron-donating properties toward SiPc. We deem this electron-donating to be the result of the shielding effect of the macrocyclic diamagnetic ring-current of SiPc toward GO through axial ligands.12 This result can be also proved by the high resolution spectra of C 1s. The C 1s spectrum of GO can be fitted into four peaks, which contains C−C/CC at 284.60 eV, C−O at 286.63 eV, CO at 288.20 eV, and OC−O at 289.68 eV (Figure 3a). For comparison, the C 1s XPS spectrum of SiPc-GO contains two additional peaks at 285.92 and 290.53 eV (Figure 3 b), attributing to the C−N/CN species originating from the silicon phthalocyanine and newly formed OC−N, along with the disappearance of the peak for O C−O. Furthermore, the binding energy of C−O (287.08 eV)

photothermal heating of SiPc-GO in NIR region. With the same concentrations of SiPc, relative lower fluorescence intensity is observed for SiPc-GO comparing to that of free SiPc-NH2 upon excitation at 610 nm, indicating a slightly intracellular energy loss occurs within SiPc-GO. In contrast, GO shows no fluorescence emission under the same conditions (Figure 2c). Overall, the satisfactory fluorescence emission properties of SiPc-GO enable it to be a potential fluorescent imaging probe for cancers. The formation of SiPc-GO was further characterized by Raman spectrum and X-ray-photoelectron spectroscopy (XPS). Figure 2d shows the Raman spectra of pristine GO, free SiPcNH2, and SiPc-GO. The spectrum of SiPc-GO exhibits all the main typical bands for SiPc-NH2 along with the superimposing with two additional bands at ∼1337 and ∼1605 cm−1, which are corresponding to the D- and G-band of GO,33 respectively, confirming the existence of both components of SiPc-NH2 and GO. Moreover, the covalent attachment of SiPc-NH2 onto the GO was further evidenced by photoelectron information from XPS. As shown in Figure 2e, only two main characteristic peaks corresponding to the C 1s and O 1s species can be observed for GO. In contrast, the XPS spectrum of SiPc-GO shows the photoelectrons collected from Si 2p, C 1s, N 1s, and O 1s, which come from both silicon phthalocyanine and GO. The N 1s core-level spectrum of SiPc-NH2 can be curve-fitted into 2788

DOI: 10.1021/acsanm.8b00449 ACS Appl. Nano Mater. 2018, 1, 2785−2795

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Figure 2. (a) FT-IR spectra of SiPc-GO, GO, and SiPc-NH2. (b) UV/vis absorption curves and (c) fluorescent spectra of SiPc-GO (20 μg mL−1), GO (6.8 μg mL−1), and SiPc-NH2 (13.2 μg mL−1) in DMF. (d) Raman and (e) XPS spectra of SiPc-GO, GO, and SiPc-NH2.

(Figure 4a, b). The DPBF consumption rates in DMF and H2O are compared in Figure 4c. Although SiPc-GO is more aggregated in H2O, it could still produces 1O2 efficiently. The DPBF consumption ratios in DMF and H2O are 49.8% and 40% within 50 s of irradiation, showing its satisfactory 1O2 generation efficiency both in DMF and water. It worth to mention, herein, Cremophor EL (0.1%, v/v) was added to reduce the aggregation and chain reaction of DPBF in water during the photo-oxidative process by 1O2 (Figure S3). In addition, a homogeneous suspension in water could be obtained for SiPc-GO without sonication, however, clustering was found for GO under the same conditions (Figure 4d). SiPc-GO also displayed excellent dispersity in physiological solutions of PBS and DMEM as shown by dynamic light scattering (DLS) analysis (Figure 4e). The ξ potentials in PBS and pure H2O were measured to be −20.99 and −21.46 mV, respectively (Figure 4f). All these data show that SiPc-GO has good stabilities in physiological solutions. 3.3. Photothermal Properties of SiPc-GO. The photothermal responses of GO, SiPc-NH2, and SiPc-GO were

and CO (288.85 eV) increase by 0.45 and 0.65 eV, respectively, comparing with that of pristine GO. Such a binding energy increase again shows the decrease of electron density in GO, owing to its electron-donating properties toward silicon phthalocyanine. Furthermore, the C 1s binding energy of OC−N in SiPc-GO is another positive proof for the electron donating properties of GO. Theoretically, the C 1s binding energy of OC−N should be lower than that of O C−O, however, the C 1s energy of OC−N (290.53 eV) in SiPc-GO is 0.85 eV higher than that of OC−O (289.68 eV) in GO, which further confirms the electron-donating properties of GO. 3.2. Singlet Oxygen (1O2) Generation. The singlet oxygen (1O2) generation capability of SiPc-GO was evaluated by a steady-state method using 1,3-diphenylisobenzofuran (DPBF) as the quenching agent. The absorbance intensity of DPBF at 415 nm would be decreased gradually along with the generation of 1O2 by coexisting SiPc-GO under a red light (λ > 610 nm, 0.2 mW/cm2). For comparison, the 1O2 generation efficiency of SiPc-GO was detected in both DMF and water 2789

DOI: 10.1021/acsanm.8b00449 ACS Appl. Nano Mater. 2018, 1, 2785−2795

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Figure 3. High-resolution spectra of C 1s XPS of GO (a) and SiPc-GO (b) and N 1s XPS spectra of SiPc-NH2 (c) and SiPc-GO (d).

Figure 4. UV−vis absorption spectra for the determination of the 1O2 of SiPc-GO in (a) DMF and (b) H2O containing Cremophor EL (0.1%, v/v) irradiated by red light (λ > 610 nm 0.2 mW/cm2). (c) Relative absorbance of DPBF (A0 and A are the absorbance of DPBF at 415 nm before and after irradiation). (d) Photo images of GO and SiPc-GO dispersion in H2O without sonification. (e) DLS analysis of SiPc-GO in physiological solutions and (f) the ξ potentials of SiPc-GO.

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Figure 5. (a) Thermal images of SiPc-GO (20 μg mL−1), GO (6.8 μg mL−1), SiPc-NH2 (13.2 μg mL−1), and deionized water under 808 nm laser (12.5 W/cm2) respectively. (b) The corresponding time-dependent photothermal curves of the samples.

Figure 6. (a) Confocal fluorescence images of HeLa cells after being incubated by SiPc-GO (10 μg mL−1) and PBS for 24 h. (b) Confocal fluorescence images of HeLa cells stained by CM-H2DCFDA (10 μM) after being incubated by SiPc-GO (10 μg mL−1) for 24 h and irradiated by LED light (λ > 660 nm, 0.2 mW/cm2) for 5 min.

recorded and compared by a thermal imager. We first used an 808 nm NIR laser (12.5 W/cm2) for irradiation to verify their photothermal efficacy in NIR region. The temperature of SiPcGO readily increased over 50 °C within 5 min of irradiation

(ΔT ∼ 25 °C), which is considered as the photoablation limit of living cells (Figure 5). In contrast, the thermal signals of GO and SiPc-NH2 showed much smaller changes (ΔT < 10 °C) under the same conditions. As a control, no obvious change 2791

DOI: 10.1021/acsanm.8b00449 ACS Appl. Nano Mater. 2018, 1, 2785−2795

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Figure 7. (a) HeLa cells treated with SiPc-GO at different concentrations and irradiated by different lasers (808 nm, 12.5 W/cm2; 671 nm, 2.5 W/ cm2) for 5 min. (b) Comparison of SiPc-GO (10 μg mL−1), GO (3.4 μg mL−1), and PBS in HeLa cells by different lasers for 5 min. Dark cytotoxicity of SiPc-GO, SiPc-NH2 and GO against (c) HeLa and (d) MCF-7 cells.

insufficient energy to initiate a photodynamic reaction.6,43 Cells were first incubated with different concentrations of SiPcGO for 24 h. Then cell viabilities were monitored 24 h after the photothermal treatment by an 808 nm laser (12.5 W/cm2, 5 min) using MTT assay. Cells without samples were used as a control. The cell viabilities dropped to ∼70% and ∼45% for the cells incubated with SiPc-GO at concentrations of 10 and 20 μg mL−1, respectively. For comparison, the viabilities for cells irradiated by a 671 nm laser (2.5 W/cm2, 5 min), of which the laser power was adjusted to cause comparable photothermal heating as that of the 808 nm laser, decreased significantly to 50% and 35%, respectively (Figure 7a). As aforementioned, both singlet oxygen and photothermal heating could be initiated for SiPc-GO by a red light. Hence, this significant phototoxicity increase revealed that a combined therapeutic effect of PDT/PTT was triggered by the 671 nm laser. Furthermore, the successively use of both 671 and 808 nm lasers gave even lower cell viabilities of ∼30% and ∼20% at SiPc-GO concentrations of 10 and 20 μg mL−1, respectively, which further confirmed the good therapeutic efficacy of SiPcGO. At the equivalent concentration of GO, remarkable enhanced cell killing effect could be observed for SiPc-GO comparing to that of GO when irradiated either by 671 or 808 nm lasers (Figure 7b), which further evidenced the synergistic effect of PDT/PTT in red region and efficient photothermal effect in NIR region. To give insight into the biocompatibility of SiPc-GO, the dark toxicity of SiPc-GO, SiPc-NH2, and GO were evaluated by the standard MTT assay. As shown in Figure 7c and d, the cell viabilities were >95% for SiPc-GO at the concentrations up to 20 μg mL−1 after incubation for 24 h, indicating the satisfactory biocompatibility of SiPc-GO. The cell viability of GO was also >95% at the concentration of 6.8 μg mL−1, and >90% at the concentration of 15 μg mL−1. On the contrary, only 25% cell viability was left for free SiPc-NH2 at the equivalent concentration of 13.2 μg mL−1. These results obviously

could be observed for pure water. The photothermal heating by a 671 nm laser followed the same order of SiPc-GO ≫ SiPcNH2 > GO > H2O as that of 808 nm laser. As a result, it could be concluded that both SiPc and GO contribute to the photothermal effect of SiPc-GO. The photothermal conversion efficiency of SiPc-GO was measured and calculated to be 20.20% according to the published procedure (Figure S4).35 3.4. Intracellular Fluorescence. The intracellular fluorescence of SiPc-GO in HeLa cancer cells was conducted by a confocal microscope to evaluate its cellular uptake and subcellular localization. As shown in Figure 6a, bright intracellular fluorescence images could be observed for SiPcGO after incubation for 24 h at a concentration of 10 μg mL−1, showing SiPc-GO could enter the cells and cause cellular fluorescence in cytoplasm. In contrast, no fluorescence could be detected for the control cells. 3.5. Intercellular ROS Generation. The intracellular generation of reactive oxygen species (ROS) was investigated in HeLa cells using CM-H2DCFDA as the probe. CMH2DCFDA is a cell-permeant indicator for ROS that turns into fluorescent by oxidation in the cells. After incubation with SiPcGO for 24 h, the cells were then incubated with CMH2DCFDA for another 15 min before illumination. Upon illumination by a 660 nm red light, it was found that the cells incubated with SiPc-GO could produce significant amount of ROS. However, the cells incubated without SiPc-GO could not generate ROS, indicating SiPc-GO is an efficient PDT photosensitizer inside cancer cells (Figure. 6b). 3.6. In Vitro Anticancer Efficacy. For evaluation of in vitro therapeutic efficacy, we studied the anticancer effects of SiPc-GO in both HeLa and MCF-7 cancer cells. Two sets of laser system with wavelengths of 808 or 671 nm were utilized to proof the synergistic effect of PDT and PTT. First, an 808 nm NIR laser was used for irradiation, of which the cell killing effect was assumed solely by the NIR photothermal heating since lights with wavelengths longer than 800 nm have 2792

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Figure 8. (a) IR thermal images of MCF-7 tumor-bearing mice exposed to 671 nm laser (2.5 W/cm2). (b) Temperature change curves of mice along with irradiation time. (c) Representative photos of mice from each experimental group during the treatments. (d) Relative tumor volume curves of mice.

manifest that the grafting of SiPc-NH2 onto GO could significantly reduce the cytotoxicity of SiPc-NH2. On the other hand, these cytotoxicity changes could further certify the successful conjugation between SiPc-NH2 and GO via covalent bond. 3.7. In Vivo Anticancer Efficacy. In vivo anticancer efficacy was investigated via intravenous injection of SiPc-GO (3.8 mg/kg) into NSG female mice bearing MCF-7 tumors. At 24 h post injection, the mice were irradiated with a 671 nm laser (2.5 W/cm2, 5 min) and thermal imaging was recorded every 1 min with an infrared thermal imager along with irradiation time. The infrared thermal mapping images are show in Figure 8a. After irradiation for 5 min, the temperature of the tumor rapidly increased to 66.5 °C (ΔT = 32.5 °C), which caused irreversible destruction toward cancer cells. In contrast, the control group that was injected with PBS instead exhibited much lower temperature increase with a ΔT = 15.2 °C, indicating that SiPc-GO accumulated in tumor region and played a crucial role in photothermal heating. Subsequently, tumor growth rates were monitored to verify the therapeutic efficacy of combined PTT and PDT. The tumor size was measured every 3 days with a caliper and tumor volume was calculated. As shown in Figure 8c and d, the tumor growth was violently inhibited in the SiPc-GO group. On the contrary, the tumor size of control group continuously increased without shrinkage of tumor volume. The comparison of tumor size between the control and SiPc-GO groups clearly demonstrated the excellent phototherapeutic effect of SiPc-GO. In addition, the mouse weights were also monitored to evaluate the toxicity of SiPc-GO in vivo. No obvious weight changes were observed for mice in either control or SiPc-GO groups,

indicating that SiPc-GO had negligible toxicity in vivo. Overall, these results show that SiPc-GO is an effective anticancer agent with synergistic effect of PDT and PTT, along with satisfactory biocompatibility.

4. CONCLUSIONS In summary, we developed a biocompatible nanosized photomedicine SiPc-GO via covalent amide bond between GO and SiPc. SiPc-GO could exhibit intrinsically fluorescence and synergistic PDT/PTT properties in red region, along with efficient PTT efficacy in NIR region. In vitro cell studies showed that SiPc-GO could cause cellular fluorescence, intracellular temperature increase and reactive oxygen species (ROS) synchronously by lasers. Significant phototoxicity could be observed for SiPc-GO in HeLa cancer cells because of the synergistic effect of PDT/PTT driven by a 671 nm laser. In addition, effective PTT result could also be triggered by a NIR 808 nm laser. In vivo anticancer efficacy in NSG female mice bearing MCF-7 tumors showed the tumor temperature rapidly increased to 66.5 °C (ΔT = 32.5 °C) within 5 min laser irradiation and the tumor growth was violently inhibited in the mice group injected with SiPc-GO. For comparison, the control group exhibited much lower temperature increase and negligible tumor growth inhibition. Our both in vitro and in vivo results show that SiPc-GO is an efficient and biocompatible multimodality anticancer agent for fluorescence imaging, synergistic PDT/PTT therapy and NIR PTT therapy. 2793

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b00449. 1 H and 13C NMR spectra of SiPc-NH2, additional details on the photothermal conversion efficiency of SiPc-GO, and comparison of UV−vis absorption of DPBF in water with and without Cremophor EL (0.1%, v/v) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] Tel: (+86) 0531-88365606. *E-mail: [email protected]. ORCID

Wei Liu: 0000-0002-8653-8228 Author Contributions ‡

J.P. and Y.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from Natural Science Foundation of Shandong Province (ZR2016BM16), Science and Technology Development Plan Project of Shandong Province (2014GGX102004), and The Fundamental Research Funds of Shandong University (2015JC036). We thank Dr. Gang Lian for his beneficial discussion with GO.

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