Porphyrin Derivative

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Aptamer-Conjugated Graphene Quantum Dots/Porphyrin Derivative Theranostic Agent for Intracellular Cancer-Related MicroRNA Detection and Fluorescence -Guided Photothermal/Photodynamic Synergetic Therapy Yu Cao, HaiFeng Dong, Zhou Yang, Xiangmin Zhong, Yi Chen, Wenhao Dai, and Xueji Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13150 • Publication Date (Web): 13 Dec 2016 Downloaded from http://pubs.acs.org on December 14, 2016

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ACS Applied Materials & Interfaces

Aptamer-Conjugated Graphene Quantum Dots/Porphyrin Derivative Theranostic Agent for Intracellular Cancer-Related MicroRNA Detection and Fluorescence -Guided Photothermal/Photodynamic Synergetic Therapy Yu Cao,[a] Haifeng Dong,* [a] Zhou Yang,[b] Xiangmin Zhong,[b] Yi Chen,[c] Wenhao Dai,[a] Xueji Zhang*[a] a

Beijing Key Laboratory for Bioengineering and Sensing Technology, Research Center for

Bioengineering and Sensing Technology, School of Chemistry & Biological Engineering, University of Science & Technology Beijing, Beijing 100083, P.R. China b

Beijing Key Laboratory of Function Materials for Molecule & Structure Construction, School of

Materials Science and Engineering, University of Science & Technology Beijing, Beijing 100083, P.R. China c

Department of Nanoengineering, University of California, San Diego, CA La Jolla 92093,

United States

ACS Paragon Plus Environment

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ABSTRACT: Multifunctional theranostic platform coupling diagnostic and therapeutic functions holds great promise for personalized nanomedicine. Nevertheless, integrating consistently high performance in one single agent is still challenging. This work synthesized a sort of porphyrin derivatives (P) with high singlet oxygen generation ability and graphene quantum dots (GQDs) possessing good fluorescence properties. The P was conjugated to polyethylene glycol (PEG)ylated and aptamer-functionalized GQDs to gain a multifunctional theranostic agent (GQD-PEG-P). The resulting GQD-PEG-P displayed good physiological stability, excellent biocompatibility and low cytotoxicity. The intrinsic fluorescence of the GQDs could be used to discriminate cancer cells from somatic cells, while the large surface facilitated gene delivery for intracellular cancer-related microRNA (miRNA) detection. Importantly, it displayed a photothermal conversion efficiency of 28.58% and a high quantum yield of singlet oxygen generation up to 1.08, which enabled it to accomplish advanced photothermal therapy (PTT) and efficient photodynamic therapy (PDT) for cancer treatment. The combined PTT/PDT synergic therapy led to an outstanding therapeutic efficiency for cancer cell treatment.

KEYWORDS: theranostic nanostructure, graphene quntaum dots, porphyrin derivative, intracellular microRNA detection, photothermal/photodynamic therapy


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1. INTRODUCTION Nowadays, integrating diagnostic and therapeutic functions in one single nanostructure to develop theranostic platform has attracted intense attention in the personalized nanomedicine and clinical application.1-3 However, it is still changing to fabricate consistently high performance theranostic platform. Various novel therapeutic strategies including chemotherapy, radiotherapy and phototherapy are combined with diagnostic tools such as fluorescence imaging, photoacoustic imaging (PAI), X-ray computed tomography (CT) imaging and magnetic resonance imaging (MRI) to develop multifunctional theranostic platforms.4-6 As a noninvasive technology, photodynamic therapy (PDT) attracted remarkable attention due to the declined side effects and improved selectivity compared to traditional therapy.7-9 In a PDT process, the photosensitizer (PS) molecule transfers the photon energy to surrounding oxygen molecules to generate reactive oxygen species (ROS) such as singlet oxygen (1O2) to kill cancer cells under the irradiation of light with appropriate wavelengths.10-12 Three pivotal factors are accounted for PDT therapeutic efficiency: (1) PS photo-transferring efficiency, (2) light of the wavelength that activates the PS, and (3) molecular oxygen. Porphyrins are sort of tetrapyrrolic conjugated macrocycles. It is consisted of four pyrrole subunits interconnected via methane bridges (-CH-), which has large absorption and strong emission characteristic in the visible region.13 Porphyrin and its derivatives have received great attention as the second generation of PS since their effective singlet oxygen (1O2) generation ability and low toxicity.14,15 However, the poor psychochemical stability, hydrophobic property and low cell-uptake efficiency limit its widespread application in biomedicine. Various nanocarriers have been actively developed to satisfy its biomedical application.16-18


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As a particularly versatile type of graphene, nano-sized graphene quantum dots (GQDs) and its derivatives possess enormous unique physicochemical properties due to the quantum confinement and edge effects.19-21 It has emerged as effective cargo nano-vector because of the ultrahigh surface area available for highly efficient loading of molecules via π-π stacking.22-24 Meanwhile, the photoluminescent (PL) GQDs is a type of remarkable bioimaging and fluorescent label in term of their low toxicity and eco-friendly nature.25-28 Moreover, the high NIR absorption and flexibility of functionalization make it attractive in photothermal therapy (PTT).29 For example, Kai and co-workers reported that the polyethylene glycol (PEG) functionalized nanosize graphene oxide could effectively ablate tumor under 808 nm laser for 5 min.30 Additionally, a sort of GQDs with size of 2~6 nm could produce 1O2 for PDT through a multistate sensitization process has been reported.31 In term of these advantages, intense efforts are continuously devoted to the biomedical application of GQDs. Herein, a sort of porphyrin derivatives (P) possessing high singlet oxygen generation ability and GQDs with good optical properties of strong was designed. The synthesized P was conjugated on the polyethylene glycol (PEG)ylated and aptamer-functionalized GQDs to produce a multifunctional theranostic platform (Scheme 1). The resulting GQD-PEG-P exhibited good feasibility of delivery for intracellular cancer-related microRNA (miRNA) detection and fluorescent imaging of cancer cells. Importantly, it simultaneously accomplished good photothermal conversion efficiency for PTT and high singlet oxygen generation ability for advanced PDT. The PTT/PDT synergic treatment led to a highly efficient elimination of cancer cells, which provided great promise for clinical diagnosis and therapeutic.


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Scheme 1. Synthesis of GQD-PEG-P and schematic presentation of GO-PEG-P theranostic platform for intracellular miRNA detection and the combined photothermal/photodynamic therapy.

2. EXPERIMENTAL SECTION Synthesis of GODs. The GO was prepared from microcrystalline graphite with some modification.32 In brief, graphite oxide (XFnano, 40 mg) was sonicated in 40 mL of a 3:1 mixture of H2SO4 (18 M) and HNO3 (17 M) for 10 h in a sonicator at a power level of 200 W maintaining the temperature at 70 oC. After that, the mixture was diluted with an excess amount of water. The sediment was collected by centrifugation at 10,000 rpm for 30 min to remove the supernatant, then the sediment was washed for 4-5 times until pH turned to 3. The solution was further dialyzed for 2-3 days to pH 7 to remove the acids. To obtain graphene quantum dots


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(GQDs), the resulting suspension was then transferred to a poly (tetrafluoroethylene) (Teflon)lined autoclave (50 mL) and heated at 200 oC for 10 h. The resulting solution was then dialyzed for 3 days to obtain purified GQDs. Synthesis of GOD-PEG-P. The P was synthesized according to the previous synthesis methods with some modification as shown in Scheme S1. In order to conjugate the PEG to GQDs, 40 mg 1-ethyl-3- (3-(dimethylamino)propyl) carbodiimide (EDC) and 8.6 mg Nhydroxy-succinimide (NHS) were added to GQDs solution (2 mg/mL) and stirred at room temperature for 30 min to activate the carboxylic groups presenting on the surface of GQDs. Then 100 mg NH2-PEG-NH2 (Mw=2kDa) was added to the solution and stirred for another 24 h. The resulting solution was dialyzed in a 3500 Da dialysis membrane for 3 days to remove the unreacted PEG. The purified GQD-PEG (1 mg) and the as-prepared porphyrin (1 mg) were mixed in 1 mL aqueous solution to sonicate for 30 min and incubate overnight. Unabsorbed porphyrin was removed by filtration through a 100 kDa filter and washed with water for 3 times to get the purified GQD-PEG-P. Material characterizations. The morphologies of GO sheets were examined with atomic force microscopy (AFM) (Nanoscope IIIa, USA) and a FEIF20 transmission electron microscope (TEM) (FEI, USA). AFM measurements were carried out under tapping mode. The X-rayphotoelectron spectroscopy (XPS) analysis was recorded with an ESCALAB 250 spectrometer (Thermo-VG Scientific, USA). Fourier transform infrared spectra (FTIR) was recorded on Nicolet 400 Fourier transform infrared spectrometer (Madison, WI). Raman spectroscopy was done at room temperature using a HR-800 Jobin-Yvon with 532 nm Nd-YAG excitation source to study the carbon structure of the graphene sheets and nano-platelets. The UV-Visible (UV-vis) absorption analysis was recorded with an UV-1800 spectrophotometer (Shimadzu, Japan). Zeta


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potential analysis was performed on Nano ZS (Malvern, UK). All fluorescence measurements were performed on a confocal laser scanning fluorescence microscope (CLS, FV1200, Olympus, Japan). Cell viability. A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% penicillin and streptomycin at 37 oC in a humidified atmosphere containing 5% CO2. For cell viability analysis, A549 cells and MCF7 cells (1.0×105 cells per well) were firstly cultured for 24 h in a 96-well plate containing DMEM with 10% (v/v) FBS (100 µL) in each well, and then the medium was replaced with fresh Opti-MEM alone or medium containing GQD-PEG-P at different concentrations (10, 25, 50, 100 and 200 µg/mL) and incubated for another 4 h. Afterwards, the cells were cultured for another 12 h. Then, fresh DMEM (100 µL) containing 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl -2-Htetrazolium bromide (MTT) (10 µL, 5 mg/mL) were then added to each well. The media was removed 4 h later, and dimethylsulfoxide (DMSO) (100 µL) was added to solubilize the crystal violet. After shocking for 15 min, the absorbance of each well was measured using an Anthos 2010 microplate reader (biochrom, USA) at 492 nm. Intracellular miRNAs Detection. Molecular beacon (MB) detection probe for miRNAs155: 5’ FAM-TCTAGC CCC CTA TCA CGA TTA GCA TTAA GCTAGA -Dabcyl 3’. A549 cells (1.0 × 104) were cultivated in confocal dish containing DMEM (1mL) for 12 h. The medium was then replaced with fresh Opti-MEM (1mL) containing GQD-PEG-P (100 µg/mL, 50 nM loaded MB) and cultivated for 4 h. After washing each dish twice by PBS (10 mM, pH 7.4), the fresh DMEM medium (1 mL) was added and cultured for another 12 h, and the cells were imaged using a confocal laser scanning fluorescence microscope.


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Photothermal performance of GQD-PEG-P. In vitro PTT performances of different GQD-PEG-P were analyzed by irradiating a quartz cuvette containing aqueous dispersion of GQD-PEG-P (1 mL, 100 µg/mL) using a 980 nm multimode pump laser (Hi-Tech Optoelectronics Company) at a power of 0.72 W/cm2. The temperature of the aqueous dispersion was measured and recorded by an OMEGA 4-channel datalogger thermometer. The thermal images were taken with a TiS65 infrared camera (Fluke, USA). Detection of singlet oxygen. The chemical probe 1,3-diphenylisobenzofuran (DPBF) was selected to detect 1O2. In a typical experiment, 20 uL DPBF solution (10 mM) was added to 2 mL GQDs or GQD-PEG-P solutions (100 µg/mL). The mixture was irradiated with a 635 nm laser for 10 min, and the absorption intensity of DPBF at 420 nm was recorded every minute. For comparison, the absorbance of the water was also measured as the same condition. Furthermore, a singlet oxygen sensor green (SOSG) regent was employed to study the production of 1O2 by confocal analysis. A549 cells were treated as same as the previous steps. After 635 nm laser irradiation for 10 min, the cells were then washed three times with PBS (10 mM, pH 7.4) and stained with 2 mM of SOSG in PBS for 15 min. The cells were imaged using a CLSM. The dye was excited at 488 nm and observed through a 525 nm emission band-pass. In vitro photothermal and photodynamic therapy. The centrifuge tubes (1.5 mL) containing A549 cells or MCF-7 cells (5 × 104) in 1 mL in DMEM were incubated at 4 oC with GQD-PEG-P (100 µg/mL) or PBS for 1 h and then irradiated for 10 min with a 980 nm laser at a power of 0.72 W/cm2 (LOS-BLD-0980-2W-C lasers) or 635 nm at a power of 0.16 W/cm2 (LOS-BLD-0980-2W-C lasers).33 The cells were then immediately plated in a 96-well plate


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containing 100 µL of media. The cell viability was determined by MTT assay after incubation at 37 oC for another 24 h. PDT and PTT on multicellular tumor spheroids (MCTS). The MCTS was generated according previous reports.34 MCF-7 cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin at 37 oC in a humidified atmosphere containing 5% CO2. Then, 20 µL single cell suspension of MCF-7 cells with density of 25000/mL was loaded into the inner surface of the lid (10 cm dish) for hanging drop culture, and 2mL PBS was added to the bottom of the 10 cm dish. Then the handing drop cultures were incubated at 37 oC for 72 h to generate spheroids. The resulting MCTS were incubated with GQD-PEG-P (100 µg/mL) for 4 h. After incubation, MCTS were washed with PBS and cultured for another 24h. Then, MCTS were stained with Calcein-Am and Propidium Iodide (PI) for confocal imaging. 3. Results and Discussion Characteristics of GO, GQDs and GQD-PEG-P. GQDs which synthesized from graphite oxide presented good PL properties (Fig. S1). TEM and AFM were employed to analyze surface morphologies, lateral dimensions and height distributions of as-prepared GO and GQDs. The TEM images showed that the main lateral size of GO was about 1-2 µm (Fig. 1A). The size of GODs was about 8 ± 3 nm (Fig. 1C) after cleavage from the GO. According to AFM analysis, the thickness of the as-prepared GQDs was about 1 nm (Fig. 1E), which was similar to the single or two-layered GO (Fig. 1B).35 Meanwhile, the high-resolution TEM (HRTEM) image (Fig. 1D) revealed the lattice constant of GQDs was 0.241 nm, in agreement with previous report.36 The Xray-photoelectron spectroscopy (XPS) measurements were performed to investigate the composition. Both GO and GQDs presented obvious peaks located at 284.45, 286.4, 287.06, and


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288.4 eV, which was ascribed to the C-C/C=C, C-OH, C=O, and O=C-OH, respectively, indicating the successful synthesis of GO and GQDs (Fig. S2).37 In comparison with the GO (Fig. S2A), GQDs displayed higher oxygen content (Table, S1) and stronger intensity of O=COH characteristic peak (Fig. S2B), illustrating that carboxyl groups and edge structure were formed during the cleavage. Similar result was obtained from the Raman spectra analysis. The GQDs showed higher intensity of the D peak and D/G ratio than GO (Fig. S3).38 The functionalization of GQDs with PEG and P led to the height of GQD increase from 1.5 nm to 2.5 nm (Fig. 1F), meaning that successful loading of P and PEG on the surface of GQDs. The HNMR analysis (supporting information) confirmed the successful synthesis of P. Specially, it showed pronounced singlet oxygen superoxide generation ability (Fig. S4).

Figure 1. TEM, AFM and XPS characterization images of as-prepared GO and GQDs. (A) TEM image of GO; (B) AFM image of GO; (C) TEM image of GQDs; (D) HRTEM image of GQDs; (E) AFM image of GQDs; (F) AFM image of GQD-PEG-P. Inset B, E, F: the height of GO, GQD, and GQD-PEG-P. Inset C: the size distribution of GQDs.


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Intracellular miRNA Imaging. The conjugation of the GQD-PEG-P was characterized by UV-vis absorption, FT-IR and Zeta potential analysis (Fig. S5). It presented good PL property, excellent biocompatibility and superb physiological stability for nanomedical application (Fig. S6). As shown in Fig. 2, the 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI) dyed the A549 cell membrane to red color (red field). The GQD-PEG-P enabled targetcell-specific transfection and discrimination of A549 cancer cells and human fibroblast (HDF) cells from the intrinsic blue fluorescence of GQDs, no obvious blue fluorescence which induced by GQD-PEG-P was observed in the HDF cells due to the target ability of the GQD-PEG-P. (Fig. 2, blue field). Moreover, it effectively loaded and delivered molecular beacon (MB) gene probe into cancer cell for cancer-related microRNA (miRNA) detection. After the GQD-PEG-P loaded with MB were taken up by A549 cells through clathrin-mediated endocytosis (CME),39 the MB hybridized with intracellular miRNA-155 and dissociated from the surface of GQDPEG-P. The fluorescence dye of MB separated from the quencher, producing green fluorescence signal for detection (green field). In contrary, no obvious green fluorescence was observed in the HDF cells due to the low expression of miRNA. These results confirmed the suggested that the GQD-PEG-P could effectively discriminate cancer cells from somatic cells by cell imaging label and intracellular cancer-related molecules detection, providing an efficient cancer cell diagnostic tool.


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Figure 2. Confocal microscopic images of A549 cells transfected by GQD-PEG-P (100 µg/mL, 50 nM linked MB). Scale bars: 20 µm.

Photothermal Properties of GQD-PEG-P. The photothermal conversion ability of GQDPEG-P was then systematically studied. As shown in Fig. 3A, after irradiation by a low power 980 nm laser at 0.72 W/cm2 for 10 min, the temperature of the GQD-PEG-P solution (100 µg/mL) reached 53.6 oC, while the temperature of control water remained 33.2 oC. The temperature increased monotonically with the increase of GQD-PEG-P concentration in the range of 10 to 200 µg/mL (Fig. 3B). To study the photothermal stability, the GQD-PEG-P solution was irradiated with NIR laser for 10 min (laser on), followed by naturally cooling of temperature to room temperature upon the switch off the NIR laser (laser off) and then repeated the cycle of laser on/off. It illustrated good stability and excellent reproducibility of GQD-PEG-P in these cycles (Fig. 3C). The photothermal conversion efficiency was calculated to 28.58% (Fig. S7) following Zhu’s report.40 It is higher than those widely studied PTT agents such as Cu2-


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xSe

(22%) and Cu9S5 (25.7%).41,42 Furthermore, the temperature of GQD-PEG-P-exposed A549

cells was investigated. As shown in Fig. 3D, the temperature was readily reached the photoablation limit of 46 oC within 5 min of irradiation, and the temperature achieved at 56.3 oC after 10 min irradiation. The high temperature could produce enormous localized heat for effective cancer cells ablation (Fig. S8). Early stage of apoptosis and terrible cell membrane destroy was observed in GQD-PEG-P-exposed A549 cells even if the laser power decrease to 0.36 W/cm2 (Fig. S9). These results revealed that GQD-PEG-P was a promising agent for PTT of cancer treatment.

Figure 3. Photothermal profile of GQD-PEG-P. (A) Thermal images of vials containing water and GQD-PEG-P solution (100 µg/mL) (B) Photothermal heating curves of water (a) and solutions containing GQD-PEG-P at different concentration (b-f: 10, 25, 50, 100 and 200 µg/mL); (C) Photothermal effect of the NIR irradiation to GQD-PEG-P solutions (100 µg/mL) with the NIR laser was shut off after irradiation for 10 min. (D) Thermal images taken with 2 min interval of vials containing pellets of GQD-PEG-P-exposed A549 cells at the concentration of 100 µg/mL.


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Photodynamic Properties of GQD-PEG-P. The singlet oxygen production capability of GQD-PEG-P was evaluated by the 1, 3-diphenylisobenzofuran (DPBF) chemical probe, whose absorbance intensity at 420 nm would be reduced in the presence of 1O2.43 Fig. 4A showed the absorbance of DPBF solutions containing GQD-PEG-P (100 µg/mL) as function of exposure time under 635 nm laser irradiation. The absorbance intensity of DPBF displayed a continuous decrease within 10 min, indicating good 1O2 production ability. As depicted in Fig. 4B, the PEGlyated GODs (GQDs-PEG) also displayed a 1O2 production ability, in agreement with previous report.31 Superior 1O2 generation capability of GQD-PEG-P to GQD-PEG was observed from the decline trend of DPBF absorption, which was resulted from high 1O2 generation capability of P. The 1O2 generation capability was further assessed by the DPBF consumption ratio (1-A/A0), where A0 is the original absorption of DPBF, and A is the absorption after 10 min irradiation under a 635 nm laser. The GQD-PEG-P exhibited good 1O2 production ability with the DPBF consumption ratio of 76.5%. Using Rose Bengal (RB) as the standard photosensitizer (1O2 quantum yield of 0.75 in water), the 1O2 quantum yield was measured to be 1.08,31,44 further confirming the high 1O2-generating efficiency (Fig. S10). The 1O2 production ability of GQDPEG-P was also investigated by using the fluorescent SOSG chemical probe, which can produce strong green fluorescence upon reaction with 1O2. Furthermore, the 1O2 production ability of P and GQD-PEG-P were compared to investigate the influence of the GQD. The GQD-PEG-P displayed a slight slower 1O2 production rate than that of P, and the quantum yield of 1O2 of P decreases from 1.27 to 1.08 of GQD-PEG-P (Fig. S11). The result suggests that the influence of the GQD to the photosensitizers because of the energy resonance transfer is slight, which will not significantly affect the PDT performance. As showed in Fig. 4C, the DiI dyed the A549 cell membrane to red color. As expected, under irradiation with 635 nm laser, the GQD-PEG-P-


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treated A549 cells showed strong SOSG fluorescence(Fig. S12A). 1O2-induced dramatic cell membrane damages of the cells were observed in 12 h-later (Fig. 4D, S12B).

Figure 4. (A, B) Time-course generation of 1O2 by GO nanosheets detecting by the bleaching of DPBF absorption at 420 nm under 635 nm laser irradiation. (C) Confocal fluorescence cell images of SOSG after irradiating at 635 nm laser. (D) Confocal fluorescence cell images of SOSG 12 h after irradiating at 635 nm laser. Scale bars: 40 µm.

PDT/PTT Efficiency of GQD-PEG-P on A549 cells. The efficient photothermal conversion efficiency and pronounced 1O2 generation ability make the GQD-PEG-P promising agent for combined PTT/PDT of cancer treatment. The Calcein-AM/propidium iodide (PI) staining and 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl -2-H-tetrazolium bromide (MTT) were used to investigate the therapeutic efficiency. As shown in Fig. 5, the GQD-PEG-P (100 µg/mL) treated-A549 cells exhibited significantly higher cell death ratio for both 635 nm and 980 nm laser irradiation (Fig. 5A) than the control cells transfected by PBS. Importantly, when the GQD-


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PEG-P treated-A549 cells were irradiated together with 635 nm and 980 nm laser. All the treated cells were almost dead; the death ratio was higher than the group irradiated with 635 or 980 nm single laser. The result was further confirmed by MTT experiment (Fig. 5B). All the control groups exhibited high cell viability above 90%, indicated the NIR laser (635 nm or 980 nm) itself had negligible influence of the to the cell viability. As for the GQD-PEG-P-treated cells, the 635 nm and 980 nm combined irradiation group displayed 14.0% cell viability. It was much lower than the 61.3% of 635 nm single laser irradiated cells and 30.5% of 980 nm single laser irradiated cells. These results demonstrated the excellent therapeutic efficiency of the GQDPEG-P-based photothermal/photodynamic therapy due to the synergetic effect. We added MCF-7 cells as another cancer cell line to evaluate the therapeutic effects. Similar result was observed that the GQD-PEG-P treated-MCF-7 cells were almost dead after sequent irradiation with 635 nm and 980 nm laser (Fig. S13), which further confirmed the advanced anti-cancer efficacy in vitro of the GQD-PEG-P. Furthermore, the treatment sequence to therapeutic effects was investigated, the cell viability for the PTT/PDT and PDT/PTT was 14.8% and 14.1%, respectively (Fig. S14). The treatment sequence had little influence on the final therapeutic effects. The synergistic therapeutic effect was mainly resulted from the two complementary treatments of PTT-caused serious membrane damage and PDT-related nuclear damage.


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Figure 5. (A) Confocal fluorescence images of A549 cells which were treated with GQD-PEG-P (100 µg/mL) or control with different laser irradiation. The cells were all co-stained by Calcein-AM (live: green) and PI (dead: red). Scale bars: 40 µm. (B)Cell viability studies with or without GQD-PEG-P (100 µg/mL) with different laser irradiation.

Additionally, the capability of selective therapeutic of the GQD-PEG-P was also investigated. Compared to the weak apoptotic fluorescence from HDF cells, much stronger apoptotic fluorescence was observed in the A549 cells, which indicated the good target-cellspecific therapeutic of the GQD-PEG-P theranostic agent (Fig. S15). HDF cells also showed high cell viability after synergistic PTT/PDT treatment, which further verified the target ability for synergistic therapy (Fig. S16). PDT/PTT Efficiency of GQD-PEG-P on MCTS. Multicellular tumor spheroids (MCTS) generated by a liquid overlay cultivation technique using MCF-7 cells were employed to evaluate the feasibility of the GQD-PEG-P for in vivo therapeutics. The control groups including MCF-7 MCTS without (Fig. 6A) or with (Fig. 6B) NIR laser irradiation and GQD-PEG-P-


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treated MCF-7 MCTS without NIR laser irradiation (Fig. 6C), strong green fluorescence of the living cells were observed, which meant inappreciable cell death. Contrary, strong red fluorescence from PI was observed for the GQD-PEG-P-treated MCF-7 MCTS sequentially irradiated by 635 nm and 980 nm laser for 10 min, (Fig. 6D). It suggested almost all the MCF-7 cells were killed. These results demonstrated the great potential promise of the GQD-PEG-P for in vivo therapeutics by synergistic photothermal and photodynamic effects.

Figure 6. 3-dimensional confocal fluorescence reconstruction images and corresponding fluorescence images of (A) MCTS with no laser; (B) MCTS with 635/980 nm laser irradiation for 10 min; (C) GQD-PEG-P (100 µg/mL) treated MCTS with no laser; (D) GQD-PEG-P (100 µg/mL) treated MCTS with 635/980 nm laser irradiation for 10 min. The MCTS were all co-stained by Calcein-AM (live: green) and PI (dead: red). Scale bars: 200 µm.

4. Conclusion In summary, a novel theranostic platform of aptamer-conjugated PEGylated GQD loaded with porphyrin derivatives photosensitizer for delivery, intracellular miRNA biomarker detection and fluorescence-guided PTT/PDT synergetic therapy was developed. Firstly, the GQD-PEG-P displayed good capability of discriminating cancer cells fromsomatic cells and cancer-related miRNA detection. Moreover, the as-prepared P showed amazing singlet oxygen generation capability for PDT of cancer cells, while the unique optical properties of the GQD enable


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fluorescence imaging and PTT of cancer cells. Importantly, the theranostic agent presented excellent therapeutic efficiency for both in vitro cancer cells and in vivo MCTS due to the PTT/PDT synergetic effect. The proposed GQD-PEG-P theranostic platform combined efficient cancer cells diagnostics and therapeutics, which holds great potential in biomedical application. ASSOCIATED CONTENT Supporting Information. Additional information as noted in the text, including PL spectra of as-prepared GQDs, XPS characterization of GO and GQDs, raman spectroscopic characterization of GO and GQDs, photostability and singlet oxygen superoxide generation ability of porphyrin, UV-vis absorption, FT-IR spectra and Zeta potential of as-prepared GQDs, GQD-PEG and GQD-PEG-P, PL, cytotoxicity and stability properties of GQD-PEG-P, time constant for heat transfer from the system, confocal fluorescence images of A549 cells which were treated with GQD-PEG-P (100 µg/mL) or control under 980 nm laser irradiation, early stage cell apoptosis of GQD-PEG-Plabelled cells, chemical trapping measurements of 1O2 quantum yield of GQD-PEG-P, PL intensity of blue, green and red fluorescence of A549 cells and specific-target-cell capability of proposed GQD-PEG-P theranostic agent. AUTHOR INFORMATION Corresponding Author *Haifeng Dong. Tel.: +86 10 82375840. E-mail: [email protected]. *Xueji Zhang. Tel./fax: +86 10 82376993. E-mail: [email protected].


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ACKNOWLEDGMENT The work was supported by National Natural Science Foundation of China (Grant No. 21305008, 21475008, 21275017, 51373024), the Fundamental Research Funds for the Central Universities (NO. FRF-BR-15-020A); State Key Laboratory of Analytical Chemistry for Life Science SKLACLS1401; Special Foundation for State Major Research Programe of China (NO. 2016YFC0106602). The authors would like to express their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project No. Prolific Research Group No. 1436-011. REFERENCES [1] Chen, G.; Roy, I.; Yang, C.; Prasad, P. N. Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy. Chem. Rev. 2016, 116, 2826-85. [2] Ma Y.; Huang J.; Song S.; Chen H.; Zhang Z. Cancer-Targeted Nanotheranostics: Recent Advances and Perspectives. Small. 2016. 12, 4936-4954 [3] Li, L.; Chen, C.; Liu, H.; Fu, C.; Tan, L.; Wang, S.; Fu, S.; Liu, X.; Meng, X.; Liu, H. Multifunctional Carbon-Silica Nanocapsules with Gold Core for Synergistic Photothermal and Chemo-Cancer Therapy under the Guidance of Bimodal Imaging. Adv. Funct. Mater. 2016, 26, 4252-4261. [4] Ai, X.; Ho, C. J.; Aw, J.; Attia, A. B.; Mu, J.; Wang, Y.; Wang, X.; Wang, Y.; Liu, X.; Chen, H.; Gao, M.; Chen, X.; Yeow, E. K.; Liu, G.; Olivo, M.; Xing, B. In Vivo Covalent Crosslinking of Photon-converted Rare-earth Nanostructures for Tumour Localization and Theranostics. Nat Commun. 2016, 7, 10432


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Upconversion

Nanoparticles

for

Combined

NIR

Mediated

Imaging

and

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Porphyrin Derivative

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