Indocyanine Green-Loaded Silver Nanoparticle@Polyaniline Core

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Indocyanine Green-Loaded Silver Nanoparticle@Polyaniline Core/Shell Theranostic Nanocomposites for Photoacoustic/ Near-Infrared Fluorescence Imaging-Guided and SingleLight Triggered Photothermal and Photodynamic Therapy Xiaoxiao Tan, Jinping Wang, Xiaojuan Pang, Li Liu, Qi Sun, Qing You, Fengping Tan, and Nan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11262 • Publication Date (Web): 30 Nov 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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ACS Applied Materials & Interfaces

Indocyanine Green-Loaded Silver Nanoparticle@Polyaniline Core/Shell Theranostic Nanocomposites for Photoacoustic/Near-Infrared Fluorescence Imaging-Guided and Single-Light Triggered Photothermal and Photodynamic Therapy Xiaoxiao Tan, Jinping Wang, Xiaojuan Pang, Li Liu, Qi Sun, Qing You, Fengping Tan*, and Nan Li*

Tianjin Key Laboratory of Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, P.R. China *E-mail: [email protected]; Tel.: +86-022-27405160. *E-mail: [email protected]; Tel.: +86-022-27404986.

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ABSTRACT: Photoacoustic

(PA)/near-infrared

fluorescence

(NIRF)

dual-modal

imaging-guided phototherapy has been wide explored very recently. However, the development of high-efficiency and simplified-performed theranostic system for amplifying imaging guided photothermal therapy/photodynamic therapy (PTT/PDT) is still a great challenge. Herein, a single-light triggered indocyanine green (ICG)-loaded PEGylation silver nanoparticle core/polyaniline shell (Ag@PANI) nanocomposites (ICG-Ag@PANI) for PA/NIRF imaging guided enhanced PTT/PDT synergistic effect has been successfully constructed. In this study, the synthesized Ag@PANI nanocomposites are not only utilized as the promising photothermal agent, but also as the potential nanovehicles for loading photosensitizer ICG via π-π stacking and hydrophobic interaction. The as-prepared ICG-Ag@PANI possesses many superior properties such as strong optical absorption in near-infrared (NIR) region, enhanced photo-stability of ICG as well as outstanding NIR laser-induced local hyperthermia and reactive oxygen species (ROS) generation. In the in vivo study, PA/NIRF dual-modal imaging confirms the accumulation and distribution of ICG-Ag@PANI in tumor region via enhanced permeability and retention (EPR) effect. Moreover, the PTT effect of ICG-Ag@PANI rapidly raised the tumor temperature to 56.8 °C within 5 min. It is also demonstrated that the cytotoxic ROS generation ability of ICG is well maintained after being loaded onto Ag@PANI nanocomposites. Remarkably, in comparison with PTT or PDT alone, the single 808 nm NIR laser-triggered combined PTT/PDT therapy exhibits the enhanced HeLa cells lethality 2 ACS Paragon Plus Environment

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in vitro and tumor growth inhibition in vivo.

KEYWORDS: ICG, Ag@PANI nanocomposites, dual-imaging guided, single-light triggered, PTT/PDT, theranostic system



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1. Introduction Traditional cancer therapy generally depending on a single therapeutic treatment remains unsatisfying. In comparison with chemotherapy, radiotherapy and surgery, phototherapy is a promising alternative administration with minimal invasion for the tumor treatment.1-5 Miscellaneous near-infrared (NIR) laser-induced interventional treatments, for example, photothermal therapy (PTT), photodynamic therapy (PDT), have been widely explored very recently.5-10 In the PTT protocol, several types of photothermal agents (PTAs), such as gold nanostructures,11 carbon nanotubes12, graphene13, copper sulfide nanostructures,14 organic NIR dyes,15 and porphysomes,16 have been widely explored by numerous groups for effective cancer cells ablation both in vitro and in vivo.17Alternatively, polyaniline nanoparticles (PANI NPs), a kind of conductive polymers with conjugated molecular structures, have received significant attention as novel organic PTAs for photothermal cancer ablation since 2011.18 According to the reports, PANI NPs exhibit diverse advantages such as good biocompatibility, excellent conductivity, mechanical flexibility, low cost and mild synthesis conditions.19-21 Besides, PANI NPs can be transformed from the emeraldine base (EB) to emeraldine salt (ES) by doping protonic acids, alkali ions and transition metals.22, 23 After appropriate doping, the electrons within the conduction bands of PANI become mobile with the significant decrease of the energy gap. Therefore, the main absorbance peak of PANI red-shift towards the NIR region, leading to strong NIR absorption and high photo-thermal conversion efficiency.24 4 ACS Paragon Plus Environment

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However, the transition pH value of PANI from EB to ES is too low (pH < 3) to be realized in tumor microenvironment.25 Therefore, it is a good choice to incorporate transition metals, such as gold nanoparticles, into PANI matrix to increase the electron-delivery efficiency and transform EB to ES, performing the potential PTT effect with 808 nm NIR laser irradiation. For example, Qu’s group25 has successfully constructed PEGylation gold nanoparticle core/PANI shell nanostructures (Au@PANI) as an efficient photothermal agent in tumor acidic microenvironment (pH 6.5). Besides, there are several reports about Ag@PANI composite being used as conductive material26, 27, surface-enhanced Raman scattering (SERS) substrate28 and antibacterial agent29. To the best of our knowledge, however, Ag@PANI has not been applied in antitumor research. In this work, we introduce silver nanoparticles (AgNPs) into PANI matrix to obtain the metal-organic nanocomposites as high-efficiency photothermal agents for tumor treatment for the first time. Because of the charge transfer (from AgNPs to PANI) and incorporating procedure induced enhanced electron-delivery efficiency, Ag@PANI nanocomposites are able to act as efficient photothermal agents avoiding extremely acidic condition. In comparison with PTT or PDT alone, combination therapy is demonstrated to improve therapeutic efficacy and minimize side effects.30-32 A current hot spot of combination therapy is to develop a system with synchronous PTT and PDT effect triggered by a single NIR laser for the simplified performance.4, 33 However, a great challenge remains in the particular requirement of the absorption match between PTAs and photosensitizers (PSs) in the NIR window (700-1100 nm).4, 5 ACS Paragon Plus Environment

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masses of PSs respond to visible or ultraviolet light, such as Photofrin (630 nm)34 and Chlorin e6 (660 nm)1, have been widely explored. In such region, efficient light penetration through tissue is hampered because of endogenous chromophores (mainly haemoglobin) and light scattering34, which remarkably limits the applications of PSs in vivo. In particular, indocyanine green (ICG), an NIR dye approved by Federal Drug Administration (FDA)35, can be activated by 808 nm NIR light to produce local heat and reactive oxygen species (ROS), thus realizing effective tissue penetration and NIR-activated PTT and PDT.36 Nevertheless, because of the poor water stability, concentration dependent aggregation and rapid elimination of free ICG37-39, it is critical to load it to efficient nanovehicles to maximize the combined PTT and PDT effect. Previous studies reported that hydrosoluble conjugated polymers with delocalized π-electrons, for example,

polypyrrole

(PPy),

poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS), could be utilized as nanovehicles to load aromatic molecules such as Chlorin e6 and Doxorubicin via π-π stacking and hydrophobic interaction.40, 41 Hence, we wonder whether the PANI, a conjugated polymer also with delocalized π-electrons, could be utilized for drug delivery and tumor ablation at the same time. Interestingly, it seems expected but justified that the ICG molecules with aromatic structures could be effectively loaded onto Ag@PANI nanocomposites with great stability and excellent loading capacity. In this study, a single-light triggered ICG-loaded PEGylation silver nanoparticle 6 ACS Paragon Plus Environment

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core/PANI shell nanocomposites (ICG-Ag@PANI) has been constructed, aiming to obtain an enhanced and combined PTT/PDT effect for tumor ablation with PA/NIRF dual-modal imaging guidance. PA imaging offers both increased imaging depth and high resolution, while NIRF imaging provides high sensitivity, excellent superficial resolution and real-time imaging feasibility.42 Hence, the combination of both imaging modalities can complement each other for a more precise diagnosis and offer comprehensive physiological and pathological information for cancer therapy. Nanoparticles can enter the tumor interstitial space easily because of the high permeability of tumor vessel, while they can retain here owing to the poor lymphatic drainage. Consequently, in this work, ICG-Ag@PANI nanocomposites could passively target and accumulate in tumor site due to the enhanced permeability and retention (EPR) effect. After the nanocomposites being internalized by tumor cells, a single 808 nm NIR laser was then acted as a critical factor to induce on-demand mechanisms at 3 steps (Scheme 1): (1) Ag@PANI displayed efficient photothermal effect under irradiation; (2) ICG released and dequenched from the surface of Ag@PANI owing to the hyperthermia, thus performing NIRF imaging; (3) Cytotoxic ROS and efficient hyperthermia were then simultaneously generated from ICG upon the photoirradiation. Therefore, such single light activated ICG-Ag@PANI nanocomposites could efficiently produce enhanced local hyperthermia and cytotoxic ROS by the guidance of dual-imaging. Overall, ICG-Ag@PANI-induced tumor PA/NIRF imaging as well as tumor ablation and obliteration under single 808 nm NIR laser irradiation shows great promise of ICG-Ag@PANI for the further exploitation as 7 ACS Paragon Plus Environment


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a theranostic system. 2. Experimental section 2.1 Materials Silver nitrate (AgNO3, 99.8%) was obtained from Tianjin Hongqiao Chemical Trading Co., Ltd. (China). Aniline (99+ %) was provided by Alfa Aesar (China) Chemicals Co., Ltd. Indocyanine green (ICG, 98+ %) was purchased from Beijing Ouhe Technology Co., Ltd (China). NH2-PEG2000 was obtained from Xi’an ruixi Biological

Technology

Co.,

Ltd

(China).

Trisodium

citrate

dihydrate

(Na3C6H5O7 · H2O), L-ascorbic acid (AA), potassium iodide (KI), ammonium persulfate ((NH4)2S2O8), sodium dodecyl sulfate (SDS) and hydrochloric acid (HCl, 36-38%) were provided by Tianjin Jiangtian Chemical Technology Co., Ltd (China). 1, 3-Diphenylisobenzofuran

(DPBF),

4’,6-diamidino-2-phenylindole

(DAPI),

2’,7’-dichlorodihydro-fluorescein diacetate (DCFH-DA), calcein-AM/PI double stain kit, and Annexin V-FITC/PI apoptosis detection kit were bought from Sigma-Aldrich (USA). High glucose Dulbecco’s modified Dagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were bought from Procell (China). 2.2 Preparation of ICG-Ag@PANI nanocomposites 2.2.1 Synthesis of Ag nanoparticles (AgNPs) AgNPs were prepared according to the KI-assisted AA/citrate reduction protocol reported by Li et al.43 Briefly, the respective solutions of sodium citrate (1 wt %, 1 mL), AgNO3 (1 wt %, 0.25 mL) and KI (0.06 µM, 50 µL) were sequentially introduced into 1.25 mL of deionized water with vigorous stirring for 5 min. Just after 8 ACS Paragon Plus Environment

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4 min incubation of citrate/AgNO3/KI mixture solution, the solution of AA (0.1 mM, 50 µL) was injected into boiling water (47.5 mL) and then boiled for an additional 1 min. After 1 min boiling, the mixture solution, just incubated for 5 min completely at that time, was introduced into the boiling solution of AA. Then, it was boiled for 1 h with vigorous stirring in order to form monodisperse quasi-spherical AgNPs. Finally, the transparent and yellow solution with opalescence was obtained. 2.2.2 Synthesis of PEGylation AgNPs core/PANI shell (Ag@PANI) nanocomposites The preparation of Ag@PANI is modified according to the synthesis of Au@PANI by Ju et al.25 The prepared AgNPs solution was centrifuged at 10 000 rpm for 10 min, then the supernatant was removed carefully. The separated AgNPs were added into the mixture solution of aniline (2 mM, 30 mL) and SDS (40 mM, 6 mL), followed by sonicating for 1 min. Immediately, the (NH4)2S2O8 solution (2 mM in 10 mM 30 mL HCl) was injected into the AgNPs/aniline/SDS mixture solution, followed by vortexing for 10 s. The final solution was stirred for additional 24 h for complete doping and polymerization. Thus the nanocomposites with Ag core and PANI shell (Ag-PANI) were obtained. As for PEGylation, 4 mL pristine Ag-PANI were mixed with NH2-PEG2000 (1 mmol, 0.36 mL) and stirred for 1 h. Eventually, the emeraldine solution was centrifuged at 10 000 rpm for 10 min to remove excess PANI and other reactants and redispersed in PBS (pH 7.4). The synthesis of PANI was conducted following the same protocol used for Ag@PANI without adding AgNPs into aniline/SDS mixture solution. 2.2.3 ICG loading onto Ag@PANI 9 ACS Paragon Plus Environment


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In this experiment, the Ag@PANI solution was mixed with ICG at various concentrations under overnight stirring. For purification, excess ICG was removed by dialysis against redistilled water in a 12-14 KD cut-off membrane (Cellu-sep, USA) for 1 h in the dark. The collected ICG-Ag@PANI was stored at 4 °C in the dark. 2.3 Characterization Transmission electron microscopy (TEM) images were taken with a JEM-100CXII electron microscope (JEOL Ltd., Japan). The particle size, polydispersity index (PDI) as well as ζ potential of Ag@PANI were detected by dynamic light scattering (DLS) with Zetasizer Nano ZS (Malvern, UK) at room temperature.

UV-vis-NIR

spectra

were

recorded

using

Cary

60

UV-vis

spectrophotometer (Agilent, USA). The FT-IR spectra were measured on KBr (Aladdin, China) pressed pellet samples with a TESOR 27 spectrophotometer (Bruker, Germany). The Raman spectra were recorded by a DXR Raman microscope (Thermo Fisher Scientific, USA) with a He-Ne laser emitting at 633 nm. Laser irradiation was conducted using an LSR808NL-2W semiconductor power-tunable laser (Laserver, China). The solution temperature was gauged with TASI-8620 digital thermometer (TASI, China). Infrared thermographic images were obtained using a TI400 infrared thermal camera (Fluke TiR, USA). 2.4 Photothermal effect and singlet oxygen detection PBS, AgNPs, PANI, Ag@PANI, free ICG and ICG-Ag@PANI were placed in the centrifuge tubes and irradiated with continuous NIR laser (808 nm, 1.0 W cm-2) for 5 min individually. The solution temperature was recorded using a digital 10 ACS Paragon Plus Environment

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thermometer every 30s. The generation of 1O2 was measured with the method reported by Wöhrle et al.44 with little modifications using DPBF under continuous irradiation (808 nm, 1.0 W cm2). Briefly, 1 mL of blank solvent, free ICG (10 µg/mL), Ag@PANI (200 µg/mL) and ICG-Ag@PANI (10 µg/mL for free ICG and 200 µg/mL for Ag@PANI) in PBS were respectively mixed with 100µL of DPBF (0.5 mg/mL, freshly prepared in acetonitrile), and then diluted by acetonitrile to a final volume of 3mL. After vortexing for 10 s, the mixture solutions were stirred and irradiated for 15 min. The generation of 1O2 accompanying absorbance decrease of DPBF was detected at 410 nm. 2.5 Cell culture HeLa cells were cultured in high glucose DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin in a humidified standard incubator (37 °C, 5% CO2). 2.6 Cellular uptake study HeLa cells (1 × 105) were seeded in CLSM dishes and cultured overnight. Cells were then washed with PBS and treated with designed formulations (10 µg/mL of ICG) for 4 or 12 h with or without 1 min irradiation. Cells were washed thoroughly, fixed with 4% paraformaldehyde (1 mL) for 20 min, and successively stained DAPI (5 µg/mL, 1 mL) for 15 min and Lysotracker Green DND-26 (500 nM, 1 mL) for 30 min in darkness. Eventually, cells were washed 3 times at least and observed immediately using confocal laser scanning microscopy (CLSM, Leica) (DAPI: 11 ACS Paragon Plus Environment


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360/460 nm; Lysotracker: 504/511 nm; ICG: 633/700 nm). 2.7 Detection of intracellular ROS HeLa cells (1 × 105) were seeded in CLSM dishes and cultured overnight. Cells were washed and treated with designed formulations (10 µg/mL of ICG) for 12 h. After washing twice, cells were successively incubated with DAPI (5 µg/mL, 1 mL) for 15 min and DCFH-DA (10 µM, 1 mL) for 50 min. Subsequently, cells were washed 3 times and irradiated (808 nm, 1.0 W cm-2, 5 min). Intracellular ROS images were acquired by CLSM at once (DAPI: 360/460 nm; DCF: 488/525 nm; ICG: 633/700 nm). Averaged DCF fluorescence intensities of individual samples were analyzed with ImageJ software. 2.8 In vitro cytotoxicity evaluation MTT assay was utilized in appraising the cellular toxicity of designed formulations. HeLa cells (5 000 per well) were pre-seeded into 96-well plate and cultured overnight. Then cells were treated with designed formulations for 24 h in darkness. For phototoxicity, cells were exposed to laser (808 nm, 1.0 W cm-2, 5 min) after 6 h incubation and further incubated for 18 h. Afterwards, MTT solution (20 µL) was introduced to per well of the plate and incubated for additional 4 h. The supernatant was removed and dimethyl sulfoxide (DMSO, 150 µL) was added to dissolve the intracellular formazan crystals. Optical density (OD) was examined at 490 nm with a microplate reader. HeLa cells (1 × 106) were pre-seeded into CLSM dishes and cultured overnight. Cells were incubated with various formulations (ICG equivalent concentration of 10 12 ACS Paragon Plus Environment

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µg/mL, Ag@PANI equivalent concentration of 200 µg/mL) for 12 h with irradiation . After washing three times, calcein-AM (4 µM) stained live cells green and PI (4 µM) stained dead/late apoptotic cells red, respectively, according to the manufacturer’s suggested protocol (Sigma-Aldrich). Eventually, cells were washed 3 times at least and CLSM images were acquired immediately (calcein-AM: 488/515 nm; PI: 535/617 nm). For flow cytometry, HeLa cells (1 × 106) were seeded into six-well plate and cultured for 24 h. After washing, cells were treated with fresh culture medium containing various formulations (ICG equivalent concentration of 10 µg/mL, Ag@PANI equivalent concentration of 200 µg/mL) for 12 h with 808 nm irradiation (1.0 W cm-2, 5 min). Afterwards, the cells were harvested, detected and quantified apoptosis by Annexin V-FITC/PI apoptosis detection kit using BD Accuri C6 flow cytometer. 2.9 Animals and tumor models Female Balb/c nude mice (6 weeks) were obtained from Beijing Huafukang Biological Technology Co., Ltd. (China) and received care according to the protocols for Care and Use of Laboratory Animals approved by Tianjin University. To construct the tumor model, 100 µL of HeLa cells (1 × 106) suspension was injected hypodermically into the right axilla of mice. Tumor volume (V) was computed as V = width2 × length/2. Animal studies were performed when tumor volume reached to 100 mm3. 2.10

NIRF, PA and infrared thermal imaging and ex vivo detection of ROS 13 ACS Paragon Plus Environment


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200 µL of free ICG or ICG-Ag@PANI (both containing 50 µg/mL ICG) was intravenously injected into the tumor-bearing nude mice. In vivo NIRF images were acquired at pre-determined time point (2, 8, 24 or 48 h) by ex/in vivo imaging system (Cri, Woburn, MA) with a 704 nm excitation wavelength and 735 nm filter. The mice after injection at 24 h were sacrificed. Organs including heart, liver, spleen, lung, kidney and tumors were harvested for the ex vivo imaging and semiquantitative biodistribution analysis. 200 µL of PBS, free ICG, PANI, Ag@PANI or ICG-Ag@PANI (ICG equivalent concentration of 50 µg/mL, Ag@PANI equivalent concentration of 1 000 µg/mL) was intravenously injected into nude mice. Photoacoustic imaging was conducted with Vevo LAZR PAI System (808 nm) at 24 h and the signal intensities were analyzed. The nude mice injucted with 200 µL of designed formulations were irradiated with NIR laser for predetermined time intervals after injection at 24 h and simultaneously photographed with an infrared thermal camera. Two-dimensional (2D) and three-dimensional (3D) infrared thermographic images were processed with SmartView 3.7 software (Fluke, USA). For ex vivo ROS detection, the tumor-bearing nude mice for thermal imaging had been pre-injected intratumorally of DCFH-DA. After laser irradiation for thermal imaging, tumors were isolated and frozen at - 80 °C immediately. Frozen sections were prepared and observed using CLSM. Cell nuclei were stained blue with DAPI. Intratumoral ROS were stained green with DCFH-DA. ICG distribution within the tumor was indicated with red. 14 ACS Paragon Plus Environment

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2.11

In vivo antitumor efficiency 100 µL of H22 tumor cells (2 × 106) suspension was inoculated hypodermically

into the right axilla of female Kunming mice. Mice were divided into six groups (seven per group) randomly and injected with 200 µL of PBS, free ICG, PANI, Ag@PANI or ICG-Ag@PANI (ICG equivalent concentration of 50 µg/mL, Ag@PANI equivalent concentration of 1 000 µg/mL) with or without 5 min irradiation as indicated 24 h post-injection, respectively. Tumor volumes and body weights were recorded every 2 days. Relative tumor volume was computed as V/V0 (V0 means initial tumor volume). At the end of antitumor therapy, organs (heart, liver, spleen, lung and kidney) and tumors were excised, sliced and stained with hematoxylin and eosin (H&E). Ultimately, each slice was observed and photographed by a digital microscope. 2.12

Statistics Data are expressed as mean ± standard deviation (SD). Two-tailed Student’s

t-tests or one-way ANOVA was applied for statistical evaluations. 3.

Results and discussion

3.1 Synthesis and characterization of Ag@PANI core/shell nanocomposites In our study, monodisperse quasi-spherical AgNPs (Figure 1a) were prepared firstly according to the KI-assisted AA/citrate reduction protocol reported by Li et al.43 The nanocomposites with Ag core and PANI shell (Ag-PANI) were synthesized by SDS-aided oxidative polymerization of aniline in the presence of AgNPs.25, 45 In this oxidative polymerization process, neutral aniline molecules were strongly 15 ACS Paragon Plus Environment


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assembled onto AgNPs by ligand-exchange to replace the negatively charged citrate ions.25, 46 The hydrophobic dodecyl tail of SDS was inset into the aniline layer to expose the negative sulfate radical outside, thus stabilizing the assemblages against aggregation. Once adding (NH4)2S2O8 as the oxygenant, the PANI shell was coated onto the surface of AgNPS due to the polyreaction. PEGylation Ag-PANI core/shell (Ag@PANI) nanocomposites were obtained via electrostatic interaction of electropositive NH2-PEG2000 and electronegative Ag-PANI NPs (Figure 1b). The Ag@PANI dispersion exhibited emeraldine color of the conductive ES form. In order to obtain the accurate particle sizes of AgNPs and Ag@PANI nanocomposites, DLS was conducted. The average particle size of AgNPs, Ag-PANI and Ag@PANI nanocomposites was 21.8 nm (PDI: 0.188), 109.2 nm (PDI: 0.194) and 115.7 nm (PDI: 0.236), with the ζ potential of - 22.0 mV, -20.5 mV and - 13.5 mV, respectively. The absolute ζ potential value of Ag@PANI was relatively decreased compared with AgNPs and Ag-PANI because of the polymerization of PANI shell onto the surface of AgNPs and the absorption of positively charged NH2-PEG2000. Size distributions of AgNPs, Ag-PANI as well as Ag@PANI NPs were shown in Figure 1c and Figure S1. PEGylation process improved the solubility and stability of Ag@PANI. As shown in Figure S2, Ag-PANI aggregated and precipitated out of PBS solution after 5 h, however, Ag@PANI remained stable. The UV-vis-NIR spectra of AgNPs, PANI and Ag@PANI in aqueous solution were displayed in Figure 1d. The maximum absorption peak of the KI-assisted AA/citrate reduced AgNPs was at ~ 400 nm, which was consistent with previous 16 ACS Paragon Plus Environment

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report43. In our study, PANI exhibited a strong absorption peak around 810 nm (π-polaron transitions) with two shoulders around 350 (π-π* transitions) and 410 nm (polaron-π* transitions). With the incorporation of AgNPs into PANI matrix, the strong absorption peak at NIR region red-shifted to ~ 820 nm with the increased maximum absorbance compared to pure PANI. FT-IR spectra of PANI and Ag-PANI were displayed in Figure 1e. The spectrum of PANI displayed bands attributed to the EB form at 3445 cm-1 (N-H stretching), 2926 cm-1 (C-H stretching), 1580 cm-1 (C=C stretching of quinoid ring), 1493 cm-1 (C=C stretching of benzenoid ring), 1302 cm-1 (C-N stretching), 1254 cm-1 (C=N stretching), 1126 cm-1 (C-H in-plane bending) and 802 cm-1 (C-H out-of-plane bending), while that of Ag-PANI displayed at 3435, 2924, 1578, 1491, 1298, 1246, 1124 and 797 cm-1, respectively. Doping AgNPs into PANI matrix led to slight red shifts and decreased intensity in the region of 2000 - 500 cm-1, which was in good agreement with previous report26. In order to get more information of molecular structure and charge transfer behavior, Raman spectroscopy was employed to characterize pure PANI and Ag-PANI (Figure 1f). The functional groups of PANI were observed at ~ 1598 cm-1 (C=C stretching of quinoid ring), ~ 1475 cm-1 (C=N stretching of quinoid ring), ~ 1386 cm-1 (C-N+ polaron), ~ 1350 cm-1 (C-N+• vibration of delocalized polaronic structure), ~ 1224 cm-1 (C-N stretching of benzenoid ring), and ~ 1174 cm-1 (C-H bending of benzenoid ring). The assignments of the characteristic peaks were consistent with previous reports.27, 47 Incorporation of AgNPs into PANI matrix led to 17 ACS Paragon Plus Environment


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slight blue shifts, which corroborated the above IR observations and previous report25 that doping of transition metal nanoparticles would increase the electron-delivery efficiency. It was noteworthy that the intensity of Ag-PANI was much higher than that of PANI because of the SERS effect after doping. In a word, the above UV-vis-NIR spectra, IR spectra and Raman spectra all confirmed the interactions and charge transfer behavior between AgNPs and PANI. The enhanced NIR absorption of Ag@PANI induced more effective PTT effect. As shown in Figure 1g, under the same concentration and irradiation conditions, temperature of PBS, AgNPs, PANI and Ag@PANI reached to 27.7 °C, 29.4 °C, 40.7 °C and 50.3 °C, respectively. The AgNPs exhibited similar PTT performance to PBS, which could be ignored. While the Ag@PANI displayed more effective PTT effect than pure PANI, proving the enhanced PTT effect of doping AgNPs into PANI matrix. Infrared thermal images of centrifuge tubes with PBS, AgNPs, PANI and Ag@PANI were captured at 5 min and shown in Figure 1h. 3.2 ICG loading onto Ag@PANI and stability It was previously illustrated that water soluble conjugated polymers with delocalized π-electrons, for example, PPy, PEDOT:PSS, could act as drug nanocarriers to load aromatic drugs such as doxorubicin and Chlorin e6 via π-π stacking and hydrophobic interaction.40, 41 Hence, we detected the possibility of PANI NPs, a conjugated polymer also with delocalized π-electrons, to be used as drug delivery vehicles. TEM image and size distribution of ICG-Ag@PANI were shown in Figure S3 and S4. As indicated in Figure 2a, the absorbance of ICG-Ag@PANI was 18 ACS Paragon Plus Environment

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much higher than that of Ag@PANI within 600-900 nm. In addition, the absorption region of ICG-Ag@PANI was much broader than free ICG (at the same concentration), suggesting the enhanced PTT effect after loading ICG. Besides, the maximum absorption peak of ICG-Ag@PANI exhibited an obvious red-shift (~ 30 nm) compared with free ICG, demonstrating the different local environment within the nanovehicles.15, 48 Meanwhile, it was found that the fluorescence signal of ICG was obviously quenched after loading onto Ag@PANI (Figure S5). Given the strong optical

absorbance

of

ICG

and

Ag@PANI,

photoacoustic

signals

from

ICG-Ag@PANI were evaluated (Figure S6). The ICG loading capacity was improved with augment of feeding ICG amount, reaching a maximum above 50% at the weight ratio of 2.0 (Figure 2b). It was probative that the loading level of ICG was feasible to be controlled. The calibration curve between the absorbance and the concentration of ICG at 780 nm was shown in Figure S7. Moreover, there were no obvious difference of ICG release profile among pH 5.0, 6.5 and 7.4, illustrating that pH value barely affected the release behavior (Figure S8). Considering the poor aqueous stability and concentration-dependent aggregation properties, we investigated the stability of free ICG and loaded ICG. It was observed that the absorbance of free ICG at 780 nm rapidly declined to 7.5% of its original value after 10 min irradiation, while sustained 73.7% for the loaded ICG (Figure 2c). After illumination, color of free ICG solution changed from cyan to tawney, while for ICG-Ag@PANI solution was green throughout (Inset of Figure 2c). Furthermore, the absorbance of free ICG significantly dropped to 23.7% after 10 days storage under the 19 ACS Paragon Plus Environment


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normal indoor environment (room light at 25°C), while that of the loaded ICG maintains 80.5% (Figure S9). These results proved that loading ICG onto Ag@PANI nanovehicles endowed ICG with outstanding photo-stability and storage stability. 3.3 Photothermal effect and singlet oxygen detection In order to explore the photothermal effect of this therapeutic system, the temperature increase was examined with 808 nm laser irradiation (1.0 W cm-2) for 5 min. As indicated in Figure 2d, the temperature of PBS, Ag@PANI, free ICG and ICG-Ag@PANI reached to 27.7 °C, 50.3 °C, 53.1 °C and 62.9 °C, respectively. It was obvious that ICG-Ag@PANI exhibited the best effective PTT effect as compared to Ag@PANI and free ICG, confirming the further enhanced PTT effect of conjugating ICG onto Ag@PANI. Infrared thermal images of centrifuge tubes with PBS, Ag@PANI, free ICG and ICG-Ag@PANI were photographed at 5 min and shown in Figure 2e. The capability of ICG-Ag@PANI as potential PDT agents was investigated by measuring the production of 1O2 under laser irradiation with DPBF. As shown in Figure 2f, the absorbance of DPBF sharply dropped to 1.9% of its original value of free ICG treated group with 15 min irradiation. Besides, ICG-Ag@PANI showed similar result to free ICG with the normalized absorbance declining to 19.6%. However, the decrease of DPBF absorbance was negligible in either pure DPBF or Ag@PANI/DPBF treated group. As illustrated, the 1O2 production of ICG-Ag@PANI was lower than free ICG under the same condition, owing to the part consumption of ICG-Ag@PANI on heat generation. 20 ACS Paragon Plus Environment

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3.4

Cellular uptake study CLSM was used to investigate the subcellular localization of free ICG and

ICG-Ag@PANI in HeLa cells (Figure 3a). The fluorescence (FL) signals of ICG were barely observed in free ICG treated group after 4 or 12 h incubation. While relatively stronger FL signals were detected in endolysosomes of ICG-Ag@PANI treated group, demonstrating the heightened internalization level of ICG by nanocarrier formulation. Notably, ICG-Ag@PANI with irradiation not only markedly enhanced the FL intensity in endolysosomes, but also facilitated ICG into the nuclei and cytosol (white arrows indicated), revealing the endolysosomal escape behavior for NIR laser-triggered cytosolic delivery49. These phenomena could be explained that the laser-induced hyperthermia improved cellular permeability and fluidity, thus increasing the intracellular ICG accumulation.50 What’s more, the hyperthermia also seriously caused cellular damage including nucleus loss, cell shrinkages or coagulation.51 We also conjectured that the heat could break the π-π stacking and hydrophobic interaction between ICG and Ag@PANI to accelerate the release and diffusion of ICG all over the cell. On the whole, the ICG signals with 12 h incubation was stronger than 4 h, suggesting that the extension of incubation time could increase the internalization at a certain degree. 3.5 In vitro detection of ROS The ROS generation induced by ICG-Ag@PANI with laser irradiation was also assessed. Non-fluorescent 2’,7’-dichlorodihydro-fluorescein diacetate (DCFH-DA) would be hydrolysed the diacetate group by intracellular esterases after entering the 21 ACS Paragon Plus Environment


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cells. In the presence of ROS, the remaining part would be further oxidized to DCF which could emit bright green fluorescence.52 As indicated in Figure 3b, it was difficult to observe the green fluorescence of DCF in PBS and ICG-Ag@PANI treated cells. In the free ICG + 808 nm laser irradiation treated cells, however, relatively weak fluorescence signals could be detected because of the poor uptake ability of free ICG. While in the ICG-Ag@PANI plus NIR laser irradiation group, HeLa cells exhibited strong DCF fluorescence intensity, suggesting the excellent ROS generation capability of ICG-Ag@PANI nanocomposites. Besides, the averaged intensity of ICG-Ag@PANI plus NIR laser treated group was 150.3-, 3.6- and 7.7-fold higher than PBS control, free ICG + NIR laser and ICG-Ag@PANI treated groups, respectively (Figure 3c). 3.6 In vitro cytotoxicity evaluation To intuitively evaluating the therapeutic efficiency of ICG-Ag@PANI, calcein-AM and PI were used to stain HeLa cells treated with different formulations to distinguish the live and dead/later apoptosis cells. As indicated in Figure 4a, it was obvious that laser irradiation alone barely affected cell viability, suggesting the safety of irradiation for HeLa cells at power density of 1.0 W cm-2. However, the cell viability and density decreased to different extent in other formulations treated groups. In other words, compared with PANI, Ag@PANI and free ICG, the most amounts of cell apoptosis and necrosis were induced by laser-activated PTT/PDT synergistic effect from ICG-Ag@PANI. Additionally, flow cytometry was utilized to further quantify the extent of cell 22 ACS Paragon Plus Environment

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apoptosis by Annexin V-FITC/PI staining (Figure 4b). Late apoptotic/necrotic cells were generally considered to localize in upper right (PI+ Annexin V+) quadrant. The results illustrated that ICG-Ag@PANI with irradiation caused the most late apoptosis/necrosis (74.1%) as compared to PBS (1.2%), free ICG (9.4%), PANI (5.9%) and Ag@PANI (45.9%). MTT assay was further conducted to quantitatively evaluate the dark or photo toxicity for HeLa cells as a function of ICG (or Ag@PANI) concentration. After cells were cultured with PBS (namely the concentration of ICG or Ag@PANI was 0 µg/mL) with or without irradiation, cell viability reached up to 95.8% or 99.1%, respectively. Although at the maximum concentration (20 µg/mL of ICG and 400 µg/mL of Ag@PANI), cell viabilities of ICG-Ag@PANI treated group were kept near 70% after 24 h treatment in darkness (Figure 4c), suggesting the biocompatibility of as-prepared ICG-Ag@PANI. However, it led to a drastic decreased percent of cell viabilities in all of the treated groups upon NIR laser for 5 min (Figure 4d). Interestingly, ICG-Ag@PANI exhibited significantly enhanced cell lethality in a dose-dependent manner. For instance, cell viabilities of ICG-Ag@PANI + 808 nm laser irradiation treated group tempestuously dropped from 99.3% to 11.7% as the ICG concentration increased from 2.5 µg/mL (50 µg/mL of Ag@PANI) to 20 µg/mL (400 µg/mL of Ag@PANI). Moreover, the results of phototoxicity of free ICG, PANI, Ag@PANI as well as ICG-Ag@PANI at the concentration of ICG 10 µg/mL (Ag@PANI 400 µg/mL) were in a good agreement with the calcein-AM/PI co-staining and flow cytometry study. All the results evidently demonstrated the superior PTT/PDT combination 23 ACS Paragon Plus Environment


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therapeutic efficiency of ICG-Ag@PANI nanocomposites in vitro. 3.7 In vivo NIRF and PA imaging In vivo tumor accumulation and distribution behavior of ICG-Ag@PANI were investigated using HeLa tumor model in female Balb/c nude mice. As presented in Figure 5a, the fluorescence signal in free ICG treated mice after 2 h injection was whole body distributed. At 8 h post-injection, the fluorescence signal was mainly located in the liver and intestine. As expected, no distinct fluorescence signals were observed after 24 or 48 h, which showed the rapid clearance process of free ICG. However, the ICG-Ag@PANI exhibited a different biodistribution behavior. Fluorescence signals of ICG-Ag@PANI progressively accumulated and strengthened toward tumor site from 2 to 24 h. While the fluorescent intensity in tumor region decreased after 48 h injection. These results suggested that ICG-Ag@PANI could significantly enhance the EPR effect, slow down the body clearance and reduce the degradation compared with free ICG. For further validation, organs and tumors were harvested after 24 h injection. As illustrated in Figure 5b, the majority of ICG concentrated in liver after 24 h injection. However, ICG-Ag@PANI significantly boosted the tumor accumulation, followed by liver, kidneys and lung. The semiquantitative fluorescence intensity analysis suggested that the intratumoral average intensity of ICG-Ag@PANI was 4.1-fold stronger than free ICG (Figure 5c). In order to quantitatively understand the biodistribution of ICG-Ag@PANI, Ag contents in major organs at different time points were determined using ICP-MS. As displayed in Figure S10, ICG-Ag@PANI progressively accumulated in tumor site 24 ACS Paragon Plus Environment

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within the first 24 h and then decreased. The remaining ICG-Ag@PANI was ingested predominantly by liver, spleen and kidney in the beginning 12 h. Afterwards, the level of Ag in organs declined over time. Compared with NIR fluorescence imaging, photoacoustics (PA) imaging is a newly developed and hopeful approach for prospective clinical utilization in carcinomatosis patients due to the enhanced imaging depth and elevated resolution for detecting the deep-seated tumors.53 In this research, the tumor accumulation behavior of ICG-Ag@PANI could be monitored by PA imaging because of its strong absorbance. In order to achieve the optimal imaging results, PA imaging was conducted after 24 h injection. It was seen that the PBS treated tumor exhibited low PA signals due to the presence of endogenous PA contrast agent haemoglobin (Figure 5d). While the ICG-Ag@PANI treated tumor showed the strongest PA signals as compared to free ICG, PANI or Ag@PANI treated tumors. Besides, the quantitative analysis results accurately manifested that the PA signals of ICG-Ag@PANI in tumor site was approximately 7.6-fold and 2.5-fold stronger than free ICG and Ag@PANI, respectively, indicating the highly sensitive PA imaging property of the optimal formulation (Figure 5e). 3.8 In vivo synergistic antitumor efficacy Encouraged by the above mentioned in vivo imaging results, PTT/PDT combination treatment efficacy of ICG-Ag@PANI was then carried out. The tumor region was exposed to irradiation for 5 min after 24 h injection of different formulations. The temperature changes during irradiation were dynamically 25 ACS Paragon Plus Environment


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monitored using an infrared thermal camera. The central temperature of ICG-Ag@PANI treated tumor reached 56.8 °C within 5 min, exceeding the destructive threshold for irreversible tumor ablation (Figure 6a). An inefficient temperature increase from 33.4 °C to 38.7 °C was measured in free ICG treated group because of the inadequate tumor accumulation. The three-dimensional (3D) infrared thermal images showed the temperature distribution of HeLa tumor-bearing nude mice. Having evaluated in vitro ROS generation ability of ICG-Ag@PANI, we then explored the potential of ICG-Ag@PANI to produce ROS within tumor for PDT effect (Figure 6b). After intratumorally pre-injecting DCFH-DA and receiving 5 min laser irradiation, mice were sacrificed, and tumors were sliced for histopathologic analysis. It was obvious that no fluorescence of ROS was found in PBS or free ICG plus laser treated group. The histopathologic image of ICG-Ag@PANI without laser treated tumor exhibited the intratumoral distribution. Moreover, it was worthy to note that ICG-Ag@PANI with laser irradiation strongly triggered ROS generation and accumulation within tumor. After phototherapy, the tumor volume and body weight of mice were monitored every other day for two weeks. As indicated in Figure 6c, ICG-Ag@PANI without laser or free ICG plus laser barely affected tumor growth. Besides, PANI or Ag@PANI plus laser group had a slight inhibitory effect on tumor growth in the early several days, but grew normally later on. Owing to the effective accumulation of ICG-Ag@PANI in tumor site, ICG-Ag@PANI with laser irradiation induced an 26 ACS Paragon Plus Environment

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apparent tumor ablation, leaving a black hard scab on the original tumor surface after two weeks’ treatment (Figure 7). Tumors that harvested from ICG-Ag@PANI plus laser treated mice were the smallest among all the excised tumors, which was in agreement with the results of tumor growth inhibition (Figure 6d). Body weight of each treatment group did not significantly change (Figure S11). In order to confirm the ICG-Ag@PANI induced PTT/PDT synergistic effect as well as the potential biological toxicity to normal tissues and organs, we harvested the organs and tumors from the sacrificed mice after two weeks’ treatment for histological analysis (Figure 7). Apparent extensive tumor necrosis, such as coagulative necrosis, pyknosis and sporadic karyolysis were observed in H&E staining slice of ICG-Ag@PANI plus laser treated tumor. However, only slight necrosis could be found in free ICG, PANI or Ag@PANI plus laser treated tumors. No apparent destruction was observed in PBS and ICG-Ag@PANI without irradiation treated tumors. Neither evident damage nor obvious inflammation was observed in H&E staining images of major organs from ICG-Ag@PANI plus laser treated mice compared to PBS treated group. These results demonstrated that ICG-Ag@PANI was an efficient PTT/PDT synergistic therapeutic agent without evident systemic toxicity to normal organs. 4.

Conclusions In summary, a single-light triggered ICG-loaded PEGylation silver nanoparticle

core/PANI shell nanocomposites have been constructed for enhanced and combined PTT/PDT effect for tumor ablation under NIRF and PA dual-modal imaging guidance. 27 ACS Paragon Plus Environment


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This single-light triggered ICG-Ag@PANI nanocomposites had the following advantages: (1) the as-prepared Ag@PANI metal-organic nanocomposites acted as not only high-efficiency photothermal agents for PTT effect, but also promising nanocarriers for photosensitizer ICG loading via π-π stacking and hydrophobic interaction for PDT effect; (2) this ideal theranostic system generated strong photoacoustic/fluorescence

signals

for

sensitive

dual-modal

PA/NIRF

imaging-guidance, facilitating the accurate phototherapy of ICG-Ag@PANI; (3) PTT/PDT was synchronously triggered by a single 808 nm laser, thus simplifying the therapeutic process. The ICG-Ag@PANI mediated PTT/PDT synergistic treatment efficiently improved the anticancer effects without evident side effects, leading to superior tumor ablation and obliteration. All the results clearly demonstrated that the ICG-Ag@PANI nanocomposites synthesized in this work were promising theranostic platforms for dual-modal imaging-guided tumor phototherapy and had great potential for further clinical translation. Acknowledgment We are grateful for financial support from National Natural Science Foundation of China (Grant No.81503016), Tianjin Research Program of Application Foundation and Advanced Technology (Grant No.15JCQNJC13800) and National Basic Research Program of China (973 Program, Grant No.2014CB932200). Supporting information Size distribution and zeta potential, stability, TEM image, fluorescence spectra, in vitro PA images, calibration curve, time-dependent release profile, time-dependent 28 ACS Paragon Plus Environment

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variation of normalized absorbance values, and in vivo biodistribution References (1) Tan, X. X.; Pang, X. J.; Lei, M. Z.; Ma, M.; Guo, F.; Wang, J. P.; Yu, M.; Tan, F. P.; Li, N. An Efficient Dual-Loaded Multifunctional Nanocarrier for Combined Photothermal and Photodynamic Therapy Based on Copper Sulfide and Chlorin e6. Int. J. Pharm. 2016, 503, 220-228. (2) Huang, P.; Lin, J.; Li, W. W.; Rong, P. F.; Wang, Z.; Wang, S. J.; Wang, X. P.; Sun, X. L.; Aronova, M.; Niu, G.; Leapman, R. D.; Nie, Z. H.; Chen, X. Y. Biodegradable Gold Nanovesicles with an Ultrastrong Plasmonic Coupling Effect for Photoacoustic Imaging and Photothermal Therapy. Angew. Chem. 2013, 125, 14208-14214. (3) Menon, J. U.; Jadeja,P.; Tambe, P.; Vu, K.; Yuan, B.; Nguyen, K. T. Nanomaterials

for

Photo-Based

Diagnostic

and

Therapeutic

Applications.

Theranostics 2013, 3, 152-166. (4) Wang, G. H.; Zhang, F.; Tian, R.; Zhang, L. W.; Fu, G. F.; Yang, L.; Zhu, L. Nanotubes-Embedded Indocyanine Green-Hyaluronic Acid Nanoparticles for Photoacoustic-Imaging-Guided Phototherapy. ACS Appl. Mater. Interfaces 2016, 8, 5608-5617. (5) Rai, P.; Mallidi, S.; Zheng, X.; Rahmanzadeh, R.; Mir, Y.; Elrington, S.; Khurshid, A.; Hasan, T. Development and Applications of Photo-Triggered Theranostic Agents. Adv. Drug Delivery Rev. 2010, 62, 1094-1124. (6) Master, A.; Livingston, M.; Gupta, A. S. Photodynamic Nanomedicine in the 29 ACS Paragon Plus Environment


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Page 30 of 48

Treatment of Solid Tumors: Perspectives and Challenges. J. Controlled Release 2013, 168, 88-102. (7) Chu, K. F.; Dupuy, D. E. Thermal Ablation of Tumours: Biological Mechanisms and Advances in Therapy. Nat. Rev. Cancer 2014, 14, 199-208. (8) Deng, K. R.; Hou, Z. Y.; Deng, X. R.; Yang, P. P.; Li, C. X.; Lin, J. Enhanced Antitumor Efficacy by 808 nm Laser-Induced Synergistic Photothermal and Photodynamic

Therapy

Based

on

a

Indocyanine-Green-Attached

W18O49

Nanostructure. Adv. Funct. Mater. 2015, 25, 7280-7290. (9) Lyu, Y.; Fang, Y.; Miao Q. Q.; Zhen X.; Ding D.; Pu K. Y. Intraparticle Molecular Orbital Engineering of Semiconducting Polymer Nanoparticles as Amplified Theranostics for in Vivo Photoacoustic Imaging and Photothermal Therapy. ACS Nano 2016, 10, 4472-4481. (10) Wang, J. P.; Tan, X. X.; Pang, X. J.; Liu, L.; Tan, F. P.; Li, N. MoS2 Quantum Dot@Polyaniline Inorganic-Organic Nanohybrids for in Vivo Dual-Modal Imaging Guided Synergistic Photothermal/Radiation Therapy. ACS Appl. Mater. Interfaces 2016, 8, 24331-24338. (11) Choi, W. I.; Kim, J.-Y.; Kang, C.; Byeon, C.C.; Kim, Y. H.; Tae, G. Tumor Regression In Vivo by Photothermal Therapy Based on Gold-Nanorod-Loaded, Functional Nanocarriers. ACS Nano 2011, 5, 1995-2003. (12) Liu, Z.; Cai, W. B.; He, L. N.; Nakayama, N.; Chen, K.; Sun, X. M.; Chen, X. Y.; Dai, H. J. In Vivo Biodistribution and Highly Efficient Tumour Targeting of Carbon Nanotubes in Mice. Nat. Nanotechnol. 2007, 2, 47-52. 30 ACS Paragon Plus Environment

Page 31 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13) Zhang, W.; Guo, Z. Y.; Huang, D. Q.; Liu, Z. M.; Guo, X.; Zhong, H. Q. Synergistic Effect of Chemo-Photothermal Therapy Using PEGylated graphene Oxide. Biomaterials 2011, 32, 8555-8561. (14) Zhou, M.; Zhang, R.; Huang, M.; Lu, W.; Song, S. L.; Melancon, M. P.; Tian, M.; Liang, D.; Li, C. A Chelator-Free Multifunctional [64Cu] CuS Nanoparticle Platform for Simultaneous Micro-PET/CT Imaging and Photothermal Ablation Therapy. J. Am. Chem. Soc. 2010, 132, 15351-15358. (15) Yu, J.; Javier, D.; Yaseen, M. A.; Nitin, N.; Richards-Kortum, R.; Anvari, B.; Wong, M. S. Self-Assembly Synthesis, Tumor Cell Targeting, and Photothermal Capabilities of Antibody-Coated Indocyanine Green Nanocapsules. J. Am. Chem. Soc. 2010, 132, 1929-1938. (16) Lovell, J.F.; Jin, C. S.; Huynh, E.; Jin, H. L.; Kim, C.; Rubinstein, J. L.; Chan, W. C. W.; Cao, W. G.; Wang L. V.; Zheng, G. Porphysome Nanovesicles Generated by Porphyrin Bilayers for Use as Multimodal Biophotonic Contrast Agents. Nat. Mater. 2011, 10, 324-332. (17) Cheng, L.; Wang, C.; Feng, L. Z.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869-10939. (18) Yang, J.; Choi, J.; Bang, D.; Kim, E.; Lim, E.-K.; Park, H.; Suh, J.-S.; Lee, K.; Yoo, K.-H.; Kim, E.-K.; Huh, Y.-M.; Haam, S. Convertible Organic Nanoparticles for Near-Infrared Photothermal Ablation of Cancer Cells. Angew. Chem., Int. Ed. 2011, 50, 441-444. (19) Bidez, P. R.; Li, S. X.; MacDiarmid, A. G.; Venancio, E. C.; Wei, Y.; Lelkes, P.I. 31 ACS Paragon Plus Environment


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Polyaniline, an Electroactive Polymer, Supports Adhesion and Proliferation of Cardiac Myoblasts. J. Biomater. Sci., Polym. Ed. 2006, 17, 199-212. (20) Kang, E. T.; Neoh, K. G.; Tan, K. L. Polyaniline: A Polymer with Many Interesting Intrinsic Redox States. Prog. Polym. Sci. 1998, 23, 277-324. (21) Hong, Y.; Hwang, S.; Heo, D.; Kim, B.; Ku, M.; Lee, E.; Haam, S.; Yoon, D. S. Yang, J.; Suh, J.-S. A Magnetic Polyaniline Nanohybrid for MR Imaging and Redox Sensing of Cancer Cells. Nanoscale 2015, 7, 161-1666. (22) Wang, J. P.; Guo, F.; Yu, M.; Liu, L.; Tan, F. P.; Yan, R.; Li, N. Rapamycin/DiR Loaded Lipid-Polyaniline Nanoparticles for Dual-Modal Imaging Guided Enhanced Photothermal and Antiangiogenic Combination Therapy. J. Cnotrol. Release 2016, 237, 23-34. (23) Dimitriev, O. P. Doping of Polyaniline by Transition-Metal Salts. Macromolecules 2004, 37, 3388-3395. (24) Xu, L. G.; Cheng, L.; Wang, C.; Peng R.; Liu, Z. Conjugated Polymers for Photothermal Therapy of Cancer. Polym. Chem. 2014, 5, 1573-1580. (25) Ju, E. G.; Dong, K.; Liu, Z.; Pu, F.; Ren J. S.; Qu, X. G. Tumor Microenvironment Activated Photothermal Strategy for Precisely Controlled Ablation of Solid Tumors upon NIR Irradiation. Adv. Funct. Mater. 2015, 25, 1574-1580. (26) Gupta, K.; Jana P.C.; Meikap, A. K. Optical and Electrical Transport Properties of Polyaniline-Silver Nanocomposite. Synthetic Met. 2010, 160, 1566-1573. (27) Roussel, F.; King, R. C. Y.; Kuriakose, M.; Depriester, M.; Hadj-Sahraoui, A.; Gors, C.; Addad, A.; Brun, J.-F. Electrical and thermal Transport Properties of 32 ACS Paragon Plus Environment

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Polyaniline/Silver Composites and Their Use as Thermoelectric Materials. Synthetic Met. 2015, 199, 196-204. (28) Mondal, S.; Rana, U.; Malik, S. Facile Decoration of Polyaniline Fiber with Ag Nanoparticles for Recyclable SERS Substrate. ACS Appl. Mater. Interfaces 2015, 7, 10457-10465. (29) Poyraz, S.; Cerkez, I.; Huang, T. S.; Liu, Z.; Kang, L. T.; Luo J. J.; Zhang, X. Y. One-Step

Synthesis

and

Characterization

of

Polyaniline

Nanofiber/Silver

Nanoparticle Composite Networks as Antibacterial Agents. ACS Appl. Mater. Interfaces 2014, 6, 20025-20034. (30) Yong, Y.; Zhou, L. J.; Gu, Z. J.; Yan, L.; Tian, G.; Zheng, X. P.; Liu, X. D.; Zhang, X.; Shi, J. X.; Cong, W. S.; Yin, W. Y.; Zhao, Y. L. WS2 Nanosheet as a New Photosensitizer Carrier for Combined Photodynamic and Photothermal Therapy of Cancer Cells. Nanoscale 2014, 6, 10394-10403. (31) Liu, T.; Wang, C.; Gu, X.; Gong, H.; Cheng, L.; Shi, X. Z.; Feng, L. Z.; Sun, B. Q.; Liu, Z. Drug Delivery with PEGylated MoS2 Nano-sheets for Combined Photothermal and Chemotherapy of Cancer. Adv. Mater. 2014, 26, 3433-3440. (32) Zheng, M. B.; Yue, C. X.; Ma, Y. F.; Gong, P.; Zhao, P. F.; Zheng, C. F.; Sheng, Z. H.; Zhang, P. F.; Wang, Z. H.; Cai, L. T. Single-Step Assembly of DOX/ICG Loaded Lipid-Polymer Nanoparticles for Highly Effective Chemo-Photothermal Combination Therapy. ACS Nano 2013, 7, 2056-2067. (33) Yu, M.; Guo, F.; Wang, J. P.; Tan, F. P.; Li, N. A pH-Driven and Photoresponsive Nanocarrier: Remotely-Controlled by Near-Infrared Light for 33 ACS Paragon Plus Environment


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Stepwise Antitumor Treatment. Biomaterials 2016, 79, 25-35. (34) Sternberg, E. D.; Dolphin, D. Porphyrin-Based Photosensitizers for Use in Photodynamic Therapy. Tetrahedron 1998, 54, 4151-4202. (35) Zha, Z. B.; Deng, Z. J.; Li, Y. Y.; Li, C. H.; Wang, J. R.; Wang, S. M.; Qu, E. Z.; Dai, Z. F. Biocompatible Polypyrrole Nanoparticles as a Novel Organic Photoacoustic Contrast Agent for Deep Tissue Imaging. Nanoscale 2013, 5, 4462-4467. (36) Sheng, Z. H.; Hu, D. H.; Xue, M. M.; He, M.; Gong, P.; Cai, L. T. Indocyanine Green Nanoparticles for Theranostic Applications. Nano-Micro Lett. 2013, 5, 145-150. (37) Zheng, X. H.; Zhou, F. F.; Wu, B. Y.; Chen, W. R.; Xing, D. Enhanced Tumor Treatment using Biofunctional Indocyanine Green-Containing Nanostructure by Intratumoral or Intravenous Injection. Mol. Pharm. 2012, 9, 514-522. (38) Yaseen, M. A.; Yu, J.; Wong, M. S.; Anvari, B. Laser-Induced Heating of Dextran-Coated Mesocapsules Containing Indocyanine Green. Biotechnol. Prog. 2007, 23, 1431-1440. (39) Kirchherr, A.-K.; Briel, A.; Mäder, K. Stabilization of Indocyanine Green by Encapsulation within Micellar Systems. Mol. Pharm. 2009, 6, 480-491. (40) Wang, C.; Xu, H.; Liang, C.; Liu, Y. M.; Li, Z. W.; Yang, G. B.; Cheng, L.; Li, Y. G.; Liu, Z. Iron Oxide@Polypyrrole Nanoparticles as a Multifunctional Drug Carrier for Remotely Controlled Cancer Therapy with Synergistic Antitumor Effect. ACS Nano 2013, 7, 6782-6795. (41) Gong, H.; Cheng, L.; Xiang, J.; Xu, H.; Feng, L. Z.; Shi, X. Z.; Liu, Z. 34 ACS Paragon Plus Environment

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Near-Infrared Absorbing Polymeric Nanoparticles as a Versatile Drug Carrier for Cancer Combination Therapy. Adv. Funct. Mater. 2013, 23, 6059-6067. (42) Peng, D.; Du, Y.; Shi, Y. W.; Mao, D.; Jia, X. H.; Li, H.; Zhu, Y. K.; Wang, K.; Tian, J. Precise Diagnosis in Different Scenarios Using Photoacoustic and Fluorescence Imaging with Dual-Modality Nanoparticles. Nanoscale 2016, 8, 14480-14488. (43) Li, H. S.; Xia, H. B.; Wang, D. Y.; Tao, X. T. Simple Synthesis of Monodisperse, Quasi-spherical, Citrate-Stabilized Silver Nanocrystals in Water. Langmuir 2013, 29, 5074-5079. (44) Wöhrle, D.; Shopova, M.; Müller, S.; Milev, A. D.; Maantareva, V. N.; Krastev, K. K. Liposome-Delivered Zn (II)-2, 3-naphthalocyanines as Potential Sensitizers for PDT: Synthesis, Photochemical, Pharmacokinetic and Phototherapeutic Studies. J. Photoch. Photobio. B 1993, 21, 155-165. (45) Jiang, N. N.; Shao, L.; Wang, J. F. (Gold Nanorod Core)/(Polyaniline Shell) Plasmonic Switches with Large Plasmon Shifts and Modulation Depths. Adv. Mater. 2014, 26, 3282-3289. (46) Kundu, J.; Neumann, O.; Janesko, B. G.; Zhang, D.; Lal, S.; Barhoumi, A.; Scuseria, G. E.; Halas, N. J. Adenine-and Adenosine Monophosphate (AMP)-Gold Binding

Interactions

Studied

by

Surface-Enhanced

Raman

and

Infrared

Spectroscopies. J. Phys. Chem. C 2009, 113, 14390-14397. (47) Bogdanović, U.; Pašti, I.; Ćirić-Marjanović, G.; Mitrić, M.; Ahrenkiel, S. P.; Vodnik, V. Interfacial Synthesis of Gold-Polyaniline Nanocomposite and Its 35 ACS Paragon Plus Environment


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Electrocatalytic Application. ACS Appl. Mater. Interfaces 2015, 7, 28393-28403. (48) Han, L.; Zhang, Y.; Chen, X.-W.; Shu, Y.; Wang, J. H. Protein-Modified Hollow Copper Sulfide Nanoparticles Carrying Indocyanine Green for Photothermal and Photodynamic Therapy. J. Mater. Chem. B 2016, 4, 105-112. (49) Lee, C.-S.; Na, K. Photochemically Triggered Cytosolic Drug Delivery Using pH-Responsive Hyaluronic Acid Nanoparticles for Light-Induced Cancer Therapy. Biomacromolecules 2014, 15, 4228-4238. (50) Tang, Y.; Lei, T. J.; Manchanda, R.; Nagesetti, A.; Fernandez-Fernandez, A.; Srinivasan, S.; McGoron, A. J. Simultaneous Delivery of Chemotherapeutic and Thermal-Optical Agents to Cancer Cells by a Polymeric (PLGA) Nanocarrier: An In Vitro Study. Pharm. Res. 2010, 27, 2242-2253. (51) Moon, H. K.; Lee, S. H.; Choi, H. C. In Vivo Near-Infrared Mediated Tumor Destruction by Photothermal Effect of Carbon Nanotubes. ACS Nano 2009, 3, 3703-3713. (52) Jiang, C. X.; Cheng, H.; Yuan, A.; Tang, X. L.; Wu, J. H.; Hu, Y. Q. Hydrophobic IR780 Encapsulated in Biodegradable Human Serum Albumin Nanoparticles for Photothermal and Photodynamic Therapy. Acta Biomater. 2015, 14, 61-69. (53) Zheng, M. B.; Zhao, P. F.; Luo, Z. Y.; Gong, P.; Zheng, C. F.; Zhang, P. F.; Yue, C. X.; Gao, D. Y.; Ma, Y. F.; Cai, L. T. Robust ICG Theranostic Nanoparticles for Folate Targeted Cancer Imaging and Highly Effective Photothermal Therapy. ACS Appl. Mater. Interfaces 2014, 6, 6709-6716. 36 ACS Paragon Plus Environment

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Scheme 1. Schematic illustration of the preparation procedure and function mechanism

of

ICG-Ag@PANI

theranostic

nanocomposites

for

photoacoustic/fluorescence imaging-guided photothermal and photodynamic therapy.



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Figure 1. Characterization of Ag@PANI. TEM images of AgNPs (a) and Ag@PANI (b). (c) DLS data of AgNPs and Ag@PANI. (d) UV-vis-NIR absorption spectra of AgNPs, PANI and Ag@PANI. (e) FT-IR spectra of PANI (blue curve) and Ag-PANI (red curve). (f) DXR Raman spectra of PANI (blue curve) and Ag-PANI (red curve). 38 ACS Paragon Plus Environment

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(g) Temperature profiles of PBS, AgNPs, PANI and Ag@PANI at the same concentration with laser irradiation (808 nm, 1.0 W cm-2). (h) Infrared thermographic images of centrifuge tubes with PBS, AgNPs, PANI and Ag@PANI were captured at 5 min with an infrared thermal imaging camera after continuous laser irradiation.



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Figure 2. Characterization of ICG-Ag@PANI. (a) UV-vis-NIR absorption spectra of free ICG, Ag@PANI and ICG-Ag@PANI. (b) The drug loading capacity in ICG loaded Ag@PANI system as the function of feeding drug concentrations. Data are presented as means ± SD (n = 3). (c) Normalized absorbance values of free ICG and ICG-Ag@PANI under laser irradiation (808 nm, 1.0 W cm-2) with different duration. Inset: Photos of free ICG (1, 2) and ICG-Ag@PANI (3, 4) solutions before (1, 3) and after (2, 4) laser irradiation. (d) Temperature profiles of PBS, Ag@PANI, free ICG and ICG-Ag@PANI with laser irradiation (808 nm, 1.0 W cm-2). (e) Infrared 40 ACS Paragon Plus Environment

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thermographic images of centrifuge tubes with PBS, Ag@PANI, free ICG and ICG-Ag@PANI were measured at 5 min. (f) Normalized absorbance value of DPBF in different solutions as a function of irradiation time of 808 nm laser (1.0 W cm-2). Data are presented as means ± SD (n = 3).



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Figure 3. (a) CLSM images of HeLa cells incubated with free ICG, ICG-Ag@PANI, and ICG-Ag@PANI + NIR for 4 or 12 h. NIR means the cells were exposed to 1.0 W cm-2 808 nm laser irradiation for 1 min after incubation with ICG-Ag@PANI for 2 h. The white arrows indicate subcellular localization of ICG. Scale bar: 25 µm. (b) CLSM images of HeLa cells treated with PBS, free ICG + NIR, ICG-Ag@PANI, and ICG-Ag@PANI + NIR for ROS detection. NIR means the cells were exposed to 1.0 W cm-2 808 nm laser irradiation for 5 min after incubation for 12 h. Scale bar: 25 µm. (c) Averaged DCF fluorescence intensity of individual samples measured from intracellular ROS detection imaging. Data are presented as means ± SD (n = 3); (*) P < 0.05, (**) P < 0.01.


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Figure 4. (a) CLSM images of HeLa cells treated with PBS, free ICG, PANI, Ag@PANI and ICG-Ag@PANI with 1.0 W cm-2 808 nm NIR laser irradiation for 5 min. Viable cells were stained green with calcein-AM, and dead/later apoptosis cells were floating and eluted, or stained red with PI. Scale bar: 250 µm. (b) Flow cytometry analysis of HeLa cells apoptosis induced by different formulations for 12 h with NIR laser irradiation (1.0 W cm-2, 808 nm, 5 min) by using Annexin V-FITC/PI staining. Cell viability of HeLa cells incubated with various concentrations of free ICG, PANI, Ag@PANI and ICG-Ag@PANI without (c) or with (d) NIR laser irradiation (808 nm, 1.0 W cm-2, 5 min). Data are presented as means ± SD (n = 3); (*) P < 0.05, (**) P < 0.01.



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Figure 5. (a) In vivo NIR fluorescence images of HeLa tumor-bearing nude mice receiving intravenous injection of free ICG or ICG-Ag@PANI. Red dash line circles indicated tumor regions. (b) NIR fluorescence images of the isolated major organs and tumors 24 h post-injection with free ICG or ICG-Ag@PANI. (c) Averaged ICG fluorescence intensity of individual organs and tumor 24 h post-injection with free ICG or ICG-Ag@PANI. (d) In vivo PA imaging of tumor treated with PBS, free ICG, PANI, Ag@PANI or ICG-Ag@PANI. Red dash line circles indicated tumor regions. (e) Photoacoustic intensity of tumor tissues measured from in vivo PA imaging. Data are presented as means ± SD (n = 3); (*) P < 0.05, (**) P < 0.01.


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Figure 6. (a) Infrared thermographic images of HeLa tumor-bearing nude mice exposed to NIR laser irradiation (808 nm, 1.0 W cm-2) for determined time intervals after 24 h post-injection with PBS, free ICG, PANI, Ag@PANI and ICG-Ag@PANI. The 3D temperature distribution of HeLa tumor-bearing nude mice were detected after 5 min laser irradiation. (b) ROS production test depended on ICG (red) and DCFH-DA that fluorescence when oxidized in the presence of ROS (green). Nuclei stained with DAPI (blue). Scale bar: 100 µm. (c) Tumor growth inhibition profiles of the mice bearing HeLa tumor injected with various formulations indicated, followed by NIR laser irradiation (808 nm, 1.0 W cm-2, 5 min) 24 h post-injection or without any laser treatment. Data are presented as means ± SD (n = 7); (*) P < 0.05, (**) P < 0.01. (d) Representative morphology and size of the tumors of each group isolated 45 ACS Paragon Plus Environment


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from the sacrificed mice at day 14 (the end point) after the treatment.


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Figure 7. Histology staining. Hematoxylin and eosin (H&E) staining of major organs and tumors of HeLa tumor-bearing mice with various formulations indicated with or without NIR laser irradiation (808 nm, 1.0 W cm-2, 5 min). Scale bar: 200 µm.



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Table of Contents


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Indocyanine Green-Loaded Silver Nanoparticle@Polyaniline Core

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