Multifunctional Photosensitizer Grafted on Polyethylene Glycol and

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A Multifunctional Photosensitizer Grafted on Polyethylene Glycol and Polyethylenimine Dual-Functionalized Nanographene Oxide for Cancer-Targeted Near-Infrared Imaging and Synergistic Phototherapy Shenglin Luo, Zhangyou Yang, Xu Tan, Yang Wang, Yiping Zeng, Yu Wang, Chang Ming Li, Rong Li, and Chunmeng Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05383 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 26, 2016

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ACS Applied Materials & Interfaces

A Multifunctional Photosensitizer Grafted on Polyethylene Glycol and Polyethylenimine Dual-Functionalized Nanographene Oxide for Cancer-Targeted

Near-Infrared

Imaging

and

Synergistic

Phototherapy Shenglin Luo Li‡, Rong Li †

†,§

†,

†,§

, Zhangyou Yang

†

†

†

†

, Xu Tan , Yang Wang , Yiping Zeng , Yu Wang , Changming

*and Chunmeng Shi†,*

Institute of Combined Injury, State Key Laboratory of Trauma, Burns and Combined Injury,

Chongqing Engineering Research Center for Nanomedicine, Department of Preventive Medicine, Third Military Medical University, Chongqing, 400038, China. ‡

Institute for Clean Energy and Advanced Materials, Southwest University, Chongqing 400715,

China.

ABSTRACT The integration of photodynamic therapy (PDT) with photothermal therapy (PTT) offers improved efficacy in cancer phototherapy. Herein, a PDT photosensitizer (IR-808) with cancer-targeting ability and near-infrared (NIR) sensitivity was chemically conjugated to both polyethylene glycol (PEG)- and branched polyethylenimine (BPEI)-functionalized nanographene oxide (NGO). Because the optimal laser wavelength (808 nm) of NGO for PTT is consistent with that of IR-808 for PDT, the IR-808-conjugated NGO sheets (NGO-808, 20~50 nm) generated both large amounts of reactive oxygen species (ROS) and local hyperthermia as a result of 808 nm laser irradiation. With PEG- and BPEI-modified NGO as the carrier, the tumor cellular uptake of NGO-808 exhibited higher efficacy than that of strongly hydrophobic free IR-808. Through evaluation with both human and mouse cancer cells, NGO-808 was demonstrated to provide significantly enhanced PDT and PTT effects compared to individual PDT using IR-808 or PTT using NGO. Furthermore, NGO-808 preferentially accumulated in cancer cells as mediated by organic-anion 1

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transporting polypeptides (OATPs) overexpressed in many cancer cells, providing the potential for highly specific cancer phototherapy. Using the targeting ability of NGO-808, in vivo NIR fluorescence imaging enabled tumors and their margins to be clearly visualized at 48 h after intravenous injection, providing a theranostic platform for imaging-guided cancer phototherapy. Remarkably, after a single injection of NGO-808 and 808 nm laser irradiation for 5 min, the tumors in two tumor xenograft models were ablated completely, and no tumor recurrence was observed. After treatment with NGO-808, no obvious toxicity was detected in comparison to control groups. Thus, high-performance cancer phototherapy with minimal side effects was afforded from synergistic PDT/PTT treatment and cancer-targeted accumulation of NGO-808.

KEYWORDS: photodynamic therapy, photothermal therapy, graphene oxide, cyanine dye, theranostics

1. Introduction Cancer phototherapy involving PDT and PTT is characterized as local treatment because only the lesion exposed to light is treated, whereas non-irradiated tissues are not affected.1,2Regardless of whether PDT or PTT is used for cancer treatment, a photosensitizer (PS) is needed to convert light energy into excessive ROS or heat to irreversibly destroy tumor cells.3 However, the real application of many PSs has been hindered by their limited therapeutic index.4Once PDT is executed, severe local hypoxia is rapidly induced, and eventually ROS cease to be produced for cancer PDT treatment. However, PTT suffers from the drawback that the PS is generally destroyed under extended light irradiation, resulting in little heat being generated over time. For therapeutic efficacy to be improved, it is of great interest to combine PDT and PTT in cancer treatment.5-9However, such multifunctional PSs fabricated for synergistic PDT and PTT are quite often limited by poor tumor-specific accumulation, leading to an unsatisfactory therapeutic effect for tumor eradication and a high risk of side effects 2


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on adjacent normal cells. In our previous studies, two heptamethine cyanine dyes characterized by preferential tumor accumulation were identified.10-14Further studies15-17have indicated that the tumor accumulation of these dyes could be transported by OATPs, which are over-expressed on the surface of many tumor cells.18,19 Among them, a novel NIR cyanine dye, IR-808, was developed not only with tumor targeting and NIR imaging properties but also with PDT activity under 808 nm laser irradiation.20 In subsequent studies, we and other groups demonstrated that IR-808 can function as a tumor-targeted ligand to carry radionuclides21.22 or anticancer drugs15,23 for tumor specific multimodal imaging or treatment. This new agent may hold promise as an NIR PS for tumor-targeted photodynamic therapy. However, IR-808—as well as many other PSs limited by their inherent hydrophobic nature and singular PDT use—exhibits limited therapeutic efficacy. Therefore, a versatile nanocarrier to deliver the hydrophobic IR-808 and simultaneously introduce PTT capability is highly desired. Graphene oxide nanosheets (NGOs) and graphene quantum dots (GQDs) as novel materials have emerged in industrial24 and biological applications.25In recent years in particular, PEGylated NGO (NGO-PEG) and BPEI-modified NGO(NGO-BPEI) have been demonstrated with good water dispersibility, effective drug delivery and photothermal therapy, drawing intensive attention in biomedicine applications.26-31 In the area of π–π hydrophobic interactions, NGO also has exhibited efficient loading capacity for many water-insoluble drugs.32,33 Notably, it exhibits superior PTT properties under 808 nm laser irradiation,34,35 the wavelength of which is coincidentally consistent with that of IR-808 for PDT.20 This synergistic PDT/PTT treatment triggered by a single light could facilitate continuous and simultaneous cancer phototherapy and has attracted considerable research interest.36-40Inspired by the multifunctional properties of IR-808 and NGO, in this work, we demonstrate novel covalent conjugation of IR-808 to NGO with the aim to achieve highly efficient cancer phototherapy with minimal side effects by combining PDT and PTT treatment 3



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and improving cancer-targeted accumulation (Scheme 1).

Scheme 1. Schematic illustration of the preparation and cancer synergistic phototherapy implementation of NGO-808. The tumor-targeted NIR photodynamic PS (IR-808) was grafted onto the PEG- and BPEI dual-functionalized photothermal PS (NGO) to obtain NGO-808. Under an 808 nm laser irradiation, high-performance PDT/PTT combination therapy was achieved by the enhanced tumor accumulation of NGO via IR-808-mediated OATP transport.

2. MATERIALS AND METHODS 2.1. Materials. N-(3-dimethylamino propyl-N’-ethylcarbodiimide) hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), Hoechst 33258 and Rho123 were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was obtained from GIBCO, Invitrogen Corp. (Carlsbad, USA). Modified RPMI-1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from 4


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HyClone. (Thermo Scientific, USA). The Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences (New Jersey, USA). The Cell Counting Kit-8 (CCK-8) and Calcein AM were purchased from Dojindo (Kumamoto, Japan). Penicillin/streptomycin, propidium iodide (PI) and LysoTracker Green were purchased from Molecular Probes, Invitrogen Corp. (Carlsbad, USA). Dialysis ultrafiltration tubes and bags with 10 kDa molecular weight cutoff were purchased from Millipore (Massachusetts, USA).

2.2. Synthesis of NGO-PEG and NGO-PEG-BPEI. The preparation of NGO began with the oxidization of graphite sheets followed by ultrasonication according to the modified Hummer’s method.41 NGO-PEG and NGO-PEG-BPEI were synthesized according to our newly developed method.42 NGO-PEG-BPEI was washed with 50% isopropanol several times to remove the BPEI physically adsorbed on NGO. To verify complete washing of the free BPEI, the filtrate was analyzed using UV/Vis spectrophotometry based on 630 nm, at which wavelength a cuprammonium complex forms between BPEI and copper ion(II).43

2.3. Synthesis of NGO-808 and NGO@808. Heptamethine indocyanine dye IR-808 was synthesized by our laboratory according to a reported method.20Afterthe synthesis of NGO-PEG-BPEI, IR-808 was loaded on NGO-PEG-BPEI via covalent conjugation to obtain NGO-808or via non-covalent van der Waals interactions to obtain NGO@808. To synthesize NGO-808, IR-808 (3.0 mg in 2 mL of DMSO) was activated by EDC·HCl (40 mg) and NHS (20mg) for 15 min. Then, NGO-PEG-BPEI (2.1 mg/mL, 4.5 mL) was added to the reaction mixture. After 24 h of reaction, the mixture was dialyzed against distilled water for 24 h. The raw product was purified by centrifuge filtration (8,000 rpm for 5 min) through centrifugal filters (10 kDa), washed five times with 50% isopropanol to remove IR-808 non-covalently loaded on NGO, and washed three times with double-distilled water to remove isopropanol. The final product was freeze-dried and stored below 4°C for further use. To synthesize 5



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NGO@808, IR-808 (3.0 mg) was dissolved in 400 µL of DMSO and added to the suspension of NGO-PEG-BPEI (2.1 mg/mL, 4.5 mL). The mixture was stirred for 24 h at room temperature and dialyzed in distilled water for 24 h (molecular weight cutoff: 10 kDa). The final product (NGO@808) was freeze-dried and stored below 4°C for further use. The amount of IR-808 loaded onto the NGO-808 or NGO@808 was measured by the absorbance peak at 787 nm according to the standard curve established in 50% isopropanol aqueous solution.

2.4. Characterization. 1H NMR (Bruker 600 MHz spectrometer) and FT-IR spectra (NICOLET6700) of NGO-808 were obtained to confirm the covalent conjugation of IR-808 on NGO. Atomic force microscopy (AFM, Bruker Dimension Icon) was used to characterize the size and thickness of the graphene oxide sheet (NGO, NGO-PEG-BPEI, NGO-808 andNGO@808).The size of the NGO-808 was further confirmed by transmission electron microscopy(TEM, Tecnai G2 F20S-TWIN). The optical properties of NGO-PEG-BPEI, IR-808 and NGO-808 were characterized by UV-VIS-NIR spectrometry (UV-3600 Scanning Spectrophotometer, Shimadzu, Japan) and NIR fluorescence spectrometry (Lumina Fluorescence Spectrometer, Thermo Fisher USA).

2.5. Single Oxygen Detection. The generation of singlet oxygen was evaluated with singlet oxygen sensor green (SOSG). Typically, 10 µM NGO-808 in PBS and SOSG (1 µM) were mixed. Then, the mixture was immediately irradiated with an 808 nm laser for 5 min (energy density: 2 W/cm2). The singlet oxygen generation was quantified with our previously reported method.44

2.6. Cell Lines and Culture. A549 and Lewis lung cancer cells were purchased from ATCC and cultured in ATCC recommended media (DMEM for A549 cells, RPMI-1640 for Lewis cells) with 10% FBS and 1% penicillin/streptomycin incubated at 37°C with 5% CO2. Human skin dermal mesenchymal cells (hDMSCs) were 6


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harvested from foreskin tissue after prepucectomy, as established previously in our lab.45These were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The hDMSCs used as normal cells for experiments were between passages 2 and 5.

2.7. Determination of the Expression of OATP mRNA in A549 and hDMSCCells. Total RNA from A549 and hDMSC cells was isolated using TRizol reagent to study the expression of OATP1B3 in cancer and normal cells. After reverse-transcription of the RNA to cDNA using a Prime Script RT reagent kit with gDNA Eraser as described in protocols, quantitative polymerase chain reaction (qPCR) was conducted using a SYBR Premix Ex Taq kit and run with CFX Connect Real-Time PCR Systems (BIO-RAD). The PCR data were analyzed by the 2-△△CT method. Primer sequences for OATP1B3 were forward 5′-ATGTTCTTGGCAGCCCTGTC-3′ and reverse 5′-CAATTTCAAAGCTTCCATCAATTA-3′,

and

for

GAPDH

were

forward

5′-GGCATCCTGGGCTACACT -3′ and reverse 5′-CCACCACCCTGTTGCTGT-3′.

2.8. In Vitro PDT/PTT Combination Treatment. Human cancer cells (A549) and mouse cancer cells (Lewis) were seeded in 96-well plates (1 × 103 cells per well) and incubated at 37°C in a humidified 5% CO2 atmosphere for 24 h. The cells were incubated with NGO-PEG-BPEI (0-30 µg/mL), IR-808 (0-10 µM) and NGO-808 (0-10 µM) in medium supplemented with 10% FBS for 24 h at 37°C. After being rinsed with PBS, cells in the irradiation groups were exposed to 808 nm laser light with an energy density of 2 W/cm2 for 5 min and incubated for another 24 h. The cells in the dark groups were rinsed with PBS and incubated for another 24 h without laser irradiation. All the cells were rinsed again with PBS, and the standard CCK-8 assay was performed to evaluate cell viability. The morphological changes of cells induced by irradiation were observed and recorded with an inverted microscope (Olympus CKX41; Olympus Corp.). The synergistic PDT/PTT effects of NGO-808 were further verified on A549 cells using Calcein AM and PI co-staining. After 24 h of laser 7



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irradiation, the cells were incubated with Calcein AM (2µM) and PI (1µM) in 1 mL of PBS for 30 min and observed by fluorescence microscopy.

2.9. Subcellular Localization and Uptake.A549 cells (1 × 105 cells) were cultured with 10 µM NGO-808 or 10 µM IR-808 for 24 h in DMEM. The cells were treated with LysoTracker for 10 min to specifically stain the lysosomes (1:7,500 dilution in PBS). After washing three times with PBS, A549 cells were stained with Hoechst33258 (10 ng mL–1, 200 µL) for another 15 min to highlight the nucleus. Then, the cells were washed three times with PBS, and 500 µL of fixing solution (1% glutaraldehyde and 10% formaldehyde) was added to each well for 30 min, followedby confocal microscopy imaging. To determine whether the uptake of NGO-808 was energy dependent, A549 cells were incubated with 10 µM NGO-808 at 4°C for 4h or 37°C for 24h. In addition, cells were pretreated with 250 µM BSP for 30 min, and NGO-808 was added and incubated for 24 h at 37°C before the cells were analyzed by flow cytometry (BD FACS Verse) and imaged using a Leica NIR fluorescent microscope (excitation: 770 nm; emission;830 nm).

2.10. Detection of Apoptosis and Intracellular ROS. At 6 h post laser irradiation, A549 cells were collected by careful trypsinization and low-speed centrifugation and washed twice with PBS. The cells were resuspended in 100 µL of binding buffer and stained with 3 µL of Annexin V-FITC and 5 µL of PI for 15 min at room temperature in the dark. After being stained, the cells were collected by low-speed centrifugation, washed twice with PBS, and diluted with 400 µL of binding buffer for flow cytometry analysis. After irradiation treatment, cells were promptly washed with PBS and incubated with 10 µM 2’,7’-dichlorofluoreseindiacetate (DCFH-DA) for 30 min, and intracellular ROS generation was evaluated by flow cytometry and confocal microscopy.

2.11.

Detection

of

Mitochondrial

Membrane

Potential.

8


Alterations

in

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mitochondrial membrane potential were probed with Rho123 and detected by flow cytometry. Briefly, A549 cancer cells were cultured with 0, 5, or 10 µM NGO-808 for 24 h; the cells in the irradiation groups were then illuminated using an 808 nm laser with an energy density of 2 W/cm2 for 5 min and incubated for another 24 h. The cells in the dark groups not exposed to laser irradiation were continuously incubated for 48 h under the same conditions. All the cells were washed with PBS three times and further incubated at 37°C with Rho123 (10 mM) for 30 min. Cells were rinsed again with PBS, and the fluorescence intensity of Rho123 was measured by flow cytometry.

2.12. Cellular Uptake and Phototoxicityof NGO-808 on hDMSCs. For studying cellular uptake, hDMSCs (1 × 105 cells) were seeded in 6-well plates and cultured with 10 µM NGO-808 for 24 h in DMEM. After washing three times with PBS, cells were stained with Hoechst33258 for 15 min. Then, the cells were washed three times with PBS and imaged by the NIR fluorescent microscope. For studying photo-induced cytotoxicity, hDMSCs were seeded in 96-well plates (1 × 103 cells per well) and incubated for 24 h. Then, the cells were incubated with NGO-808 (0, 1.25, 2.5, 5, and 10 µM) in medium supplemented with 10% FBS for 24 h at 37°C. After being rinsed with PBS, the cells were irradiated (808 nm, 2 W/cm2, 5 min) and incubated for another 24 h. Finally, the standard CCK-8 assay was carried out to evaluate the cell viability. The morphological changes of cells before and after irradiation were observed and recorded by an inverted microscope.

2.13 Animals and Tumor Xenografts. Male C57 BL/6 mice (8 weeks old, weighing 18-20 g) and athymic nude mice (6 weeks old, weighing 18-20 g) were purchased from the laboratory animal center of Third Military Medical University. All animals received care and treatment in compliance with the Animal Care and Use Committee of Third Military Medical University (Chongqing, China). Lewis tumor xenografts with C57 BL/6 mice and A549 tumor xenografts with athymic nude mice were established according to our previously reported procedure.20 9



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2.14. NIR fluorescence and Thermal Imaging. The athymic nude mice bearing A549 tumor xenografts and C57BL/6 mice bearing LLC tumor xenografts were used to assess the tumor targeting of NGO-808 and were injected with 0.5 mg/kg of NGO-808 via the tail vein. In vivo NIR imaging was taken from 0 to 48 h after injection with an in vivo NIR Imaging System (Kodak). All the sets and imaging conditions were the same as those of the reported method.11Prior to NIR imaging, mice were anesthetized by intra-abdominal injection of 1% pentobarbital sodium. After sacrifice of the mice, dissected organs and tumors were obtained for ex vivo NIR fluorescent imaging. For in vitro assessment of PTT properties, thermal imaging of blank PBS and NGO-808 under irradiation (808 nm, 2 W/cm2, 5 min) was recorded by an infrared thermal imaging camera (Ti32, Fluke, USA). For in vivo assessment of PTT properties, the real-time temperature change of mice was imaged by the infrared thermal camera when the whole tumor tissue was exposed to the NIR laser beam.

2.15. In Vivo PDT/PTT Combinational Treatment. After A549 or Lewis tumor xenografts were established (with tumor sizes reaching ~60 mm3 for theA549 model and ~100 mm3 for the Lewis model), they were randomly divided into six groups: control dark group, control irradiation group, NGO-808-treated dark group, NGO-808-treated

irradiation

group,

IR-808-treated

irradiation

group

and

NGO-PEG-BPEI treated irradiation group. Drugs were injected intravenously with 10 mg/kg of NGO-808, 2 mg/kg of IR-808, and 8 mg/kg of NGO-PEG-BPEI suspended in PBS. The control group was given the same volume of PBS. After 48 h of drug administration, all animals except those in the dark groups were anaesthetized using 1% pentobarbital, and the tumor xenografts were exposed to an 808 nm continuous-wave NIR laser with power density of 1 W/cm2 for 5 min. Tumor size was measured in two dimensions by a caliper every 3-4 days. Tumor volumes were calculated as length×(width)2/2. To investigate whether tumor recurrence occurred in the NGO-808-treated irradiation group, tumor growth in this group was observed for 60 days after laser irradiation. 10


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3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of NGO-808. The synthetic route for NGO-808 is illustrated in Figure 1. First, the preparation of NGO was initiated by the oxidization of graphite sheets and followed by ultrasonication according to a modified Hummer’s method.41 Second, NGO-PEG was prepared by a ring-opening nucleophilic addition reaction between the epoxy groups of NGO and the amine groups of amino-terminated PEG (average molecular weight: 5,000 Da) according to our newly developed method.42 This new PEGylated method creates individual small nanosheets of NGO with carboxyl functional groups at their edges. Third, BPEI (1,800 Da) was covalently conjugated with the

carboxyl-containing NGO-PEG.

With

this

modification, well-dispersed NGO-PEG-BPEI was prepared and functionalized with abundant amine groups. Finally, IR-808 with carboxyl functional groups was covalently conjugated with NGO-PEG-BPEI to obtain NGO-808. To remove non-covalently loaded IR-808, NGO-808 was purified with ultrafiltration and washed repeatedly with 50% isopropanol until the fluorescence of the filtrate could not be detected by NIR imaging (Supporting Information, Figure S1).

Figure 1. Synthetic route of NGO-PEG, NGO-PEG-BPEI and NGO-808. 11



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The size and thickness of NGO-808, including its morphology, were characterized by AFM. As shown in Figure 2a, unmodified NGO measured approximately 100~500 nmon a side (~1 nm thickness). NGO-PEG-BPEI existed as small sheets with a size range of 20-40 nm (~2 nm thick, Figure 2b). After conjugation of IR-808 with NGO-PEG-BPEI, NGO-808 remained almost the same size (20-40 nm) with a slight increase in thickness (~3 nm thick, Figure 2c). TEM of NGO-808 also revealed synthesized nanosheets with sizes ranging from 20 to 40 nm (Figure S2a). 1H NMR and FT-IR spectra of NGO-808 were tested to confirm the covalent conjugation of IR-808 on NGO. 1H NMR revealed characteristic shift values δ at 8.35 for N-H in the amide group (-CONH-), 3.64 for PEG, and low signals of shift values between 1.25 and 7.50 for IR-808 (Figure S2b). The FT-IR spectrum exhibited characteristic peaks of the amide group (-CONH-) at 1715 cm-1, NGO (PEG) at 3,400 cm-1 and IR-808 (indole) at 924-1,553 cm-1 (Figure S2c). Successful conjugation was further evidenced by the ultraviolet-visible-NIR (UV-VIS-NIR) absorbance spectra (Figure 2d) and fluorescence emission spectra (Figure 2e). This demonstrates that NGO-808 exhibits characteristic absorbance peaks of NGO-PEG-BPEI (254 nm) and IR-808 (787 nm) and the characteristic emission peak of IR-808 (808 nm). For comparison, IR-808 was straightforwardly loaded on NGO-PEG-BPEI via π–π stacking and hydrophobic interactions, obtaining NGO@808. AFM of NGO@808 revealed almost the same size (20-40 nm) but with an increase in thickness (~5 nm thick, Figure S2d). The amount of IR-808 conjugated onto NGO-PEG-BPEI was measured by the absorbance peak at 787 nm according to the standard curve established in 50% aqueous isopropanol (Figure S3). The curves indicated that the IR-808 loading rates on NGO-808 and NGO@808 were approximately 20 and 62%, respectively. The stabilities of NGO-808 and NGO@808 in PBS (pH 7.4) or 10% fetal bovine serum (FBS) were compared (Figure 2f). The result indicated that NGO-808 released

Multifunctional Photosensitizer Grafted on Polyethylene Glycol and

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