Low Power Single Laser Activated Synergistic Cancer Phototherapy

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Low Power Single Laser Activated Synergistic Cancer Phototherapy Using Photosensitizer Functionalized Dual Plasmonic Photothermal Nanoagents Muhammad Rizwan Younis, Chen Wang, Ruibing An, Shouju Wang, Muhammad Adnan Younis, Zhong-Qiu Li, Yang Wang, Ayesha Ihsan, Deju Ye, and Xing-Hua Xia ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09552 • Publication Date (Web): 07 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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Graphic Abstract

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Low Power Single Laser Activated Synergistic Cancer Phototherapy Using Photosensitizer Functionalized Dual Plasmonic Photothermal Nanoagents Muhammad Rizwan Younis1, Chen Wang1,2, Ruibing An1, Shouju Wang3, Muhammad Adnan Younis4, Zhong-Qiu Li1, Yang Wang1, Ayesha Ihsan5, Deju Ye1, and Xing-Hua Xia1* 1

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and

Chemical Engineering, Nanjing University, Nanjing 210023, China 2

Department of Physical Chemistry, School of Science, China Pharmaceutical University,

Nanjing 210009, China 3

Department of Radiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing

210000, China 4

Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of

Chemical and Biological Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310058, China 5

National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box No. 577,

Jhang Road, Faisalabad, Pakistan

* Corresponding authors: [email protected].

1 ACS Paragon Plus Environment

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ABSTRACT: Combination therapy especially photodynamic/photothermal therapy (PDT/PTT) has shown promising applications in cancer therapy. However, sequential irradiation by two different laser sources and even the utilization of single high-power laser to induce either combined PDT/PTT or individual PTT will be subjected to prolonged treatment time, complicated treatment process, and potential skin burns. Thus, low power single laser activatable combined PDT/PTT is still a formidable challenge. Herein, we propose an effective strategy to achieve synergistic cancer phototherapy under low power single laser irradiation for short duration. By taking advantage of dual plasmonic PTT nanoagents (AuNRs/MoS2), a significant increase in temperature upto 60 oC with an overall photothermal conversion efficiency (PCE) of 68.8 % has been achieved within 5 mins under very low power (0.2 W/cm2) NIR laser irradiation. The enhanced PCE and PTT performance is attributed to the synergistic plasmonic PTT effect (PPTT) of dual plasmonic nanoagents, promoting simultaneous release (85 %) of electrostatically bonded indocyanine green (ICG) to induce PDT effects, offering simultaneous PDT/synergistic PPTT. Both in vitro and in vivo investigations reveal complete cell/tumor eradication, implying that simultaneous PDT/synergistic PPTT effects induced by AuNRs/MoS2-ICG are much superior over individual PDT or synergistic PPTT. Notably, synergistic PPTT induced by dual plasmonic nanoagents also demonstrates higher in vivo antitumor efficacy than either individual PDT or PTT agents. Taken together, under single laser activation with low power density, the proposed strategy of simultaneous PDT/synergistic PPTT effectively reduces the treatment time, achieve high therapeutic index, and offers safe treatment option, which may serve as a platform to develop safer and clinically translatable approaches for accelerating cancer therapeutics.

KEYWORDS: single laser activation, synergistic cancer phototherapy, plasmonic photothermal nanoagents, photothermal conversion efficiency, therapeutic index


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Although significant advancements in cancer research have been progressed, cancer therapy remains a great challenge for the scientific community.1-3 Recently, light triggered therapeutic approaches like photothermal therapy (PTT) and photodynamic therapy (PDT) have attained considerable attention to treat malignant tumors and surpass conventional treatment technologies like chemotherapy, radiotherapy, and surgery, because of their minimal invasive nature, high spatio-temporal precision, controllability, and localized treatment.4-9 However, as PDT strictly relies on the availability of tissues oxygen to generate 1O2, continuous utilization of tissues oxygen during PDT operation generates severe tumor hypoxia, resulting in unsatisfactory PDT efficacy.1015

While, PTT is an oxygen independent treatment modality, which kills tumor cells via local

hyperthermia generated by either visible or near infrared (NIR) activated PTT agents.16-20 Thus, integration of PTT with PDT holds promising potential to overcome the inherent limitation of PDT as well as achieve high cancer therapeutic outcomes.21-25 Unfortunately, due to the spectral mismatch between a PDT agent (photosensitizer, PS) and a PTT agent at NIR region, sequential irradiations by two different lasers are required to activate PDT and PTT, which leads to prolonged treatment time and complicated treatment process.26-32 In addition, it is very hard to precisely align the two laser beams at the same position. Alternatively, single laser induced simultaneous PDT/PTT has been reported by either PDT agent coupled with a PTT agent (usually Au) or by dual modal PDT/PTT agent.33-39 Although an improved treatment outcome from synergistic PDT/PTT was achieved, the demand of high power laser (>= 1 W/cm2) irradiation for long duration (5-10 min) to activate synergistic PDT/PTT or even individual PTT raises serious safety concerns as high power laser could cause skin burns. On the other hand, due to the low PTT performance of a single PTT agent, high dose of PTT agent usually undergoes laser irradiation for long duration to induce local hyperthermia, which may cause potential cytotoxicity to the normal


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tissues. These limitations seriously restrict their clinical translation and demand a safer strategy to achieve synergistic therapeutic effects (PDT/PTT) under single low power NIR laser (808 nm) irradiation for short duration. Compared with individual nanostructure, plasmonic nanohybrids consisting of dual plasmonic nanoagents demonstrate far superior physicochemical properties and possess striking functionalities, which corresponds to the synergistic effects of two different nanostructures.40-42 For instance, upon integration of noble metals with a semiconductor, the resultant metal/semiconductor hybrid exhibits enhanced light absorption capacity as well as an effective charge separation characteristics owing to the localized surface plasmon resonance (LSPR) effect of noble metal nanostructures, facilitating efficient conversion of light energy into either thermal, magnetic or electrical energy. As a result, plasmonic nanohybrids hold great potential in a wide range of applications including, photocatalysis,43,44 water splitting,45,46 photovoltaics,47 and biomedicine.48-50 Among noble metal nanostructures, Au nanostructures especially Au nanorods (AuNRs) have been extensively explored for biomedical applications particularly in tumor imaging51-53 and PTT because of their higher photothermal conversion performance, tailorable longitudinal LSPR band especially at NIR region, larger extinction coefficients (108-1010 M-1 cm1

), biocompatibility, excellent stability, and facile synthesis protocol.54-57 Recently, different Au

nanostructures based plasmonic nanohybrids have been developed for enhanced PTT performance, but tedious synthesis process and the requirement of high laser power density (1 W/cm2) to generate local hyperthermia limit their practical applications.58-61 In addition, in vivo applications of these recently developed nanohybrids are remain elusive. Therefore, there is a great demand to develop simple yet highly effective plasmonic nanohybrids to coordinate PTT with PDT in vivo,


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ensuring deep penetration depth and achieve excellent therapeutic outcomes under single low power laser activation. Apart from metallic nanomaterials, two dimensional (2D) MoS2 nanosheets (NSs) have shown promising applications in catalysis and nanoelectronics.62,63 Encouraged by the NIR absorption and large surface area, MoS2 NSs have been developed as a PTT agent and as a carrier of different PSs in PDT.64-68 Previously, we have demonstrated improved hydrogen evolution reaction (HER) activity and enhanced photoelectrochemical biosensing performance of Au/MoS2 hybrid by utilizing LSPR phenomenon.69,70 Thus, our previous findings inspired us to explore the synergistic therapeutic potential of AuNRs/MoS2 hybrid, which is yet unexplored to the best of our knowledge. To achieve simultaneous PDT/synergistic PPTT under single low power NIR laser irradiation and overcome the inherent limitation of PDT, herein, we anchor NIR excitable PDT agent (indocyanine green, ICG) onto dual plasmonic PTT nanoagents (AuNRs/MoS2 hybrid) via electrostatic interaction. Due to the ultrahigh surface area of PEGylated MoS2 NSs, plasmonic AuNRs/MoS2 nanohybrid offers high payload of ICG dye for efficient PDT. To ensure deep penetrated synergistic cancer phototherapy under single continuous wave (CW) NIR laser irradiation, we tune the longitudinal LSPR band of Au NRs in the NIR region to couple with that of MoS2 NSs and ICG, respectively. Under single low power CW NIR laser irradiation (0.2 W/cm2), plasmonic AuNRs/MoS2 hybrid continuously generates extreme heat due to the synergistic plasmonic PTT effect (PPTT), enabling thermal release of the electrostatically bonded ICG dye into the system to generate 1O2 (Scheme 1). Since the photobleaching of ICG leads to decrease in PDT efficiency, the synergistic PPTT effect accumulates over time and gradually increases upon irradiation due to the excellent stability of plasmonic AuNRs/MoS2 hybrid. Moreover, in vitro and in vivo results verify the enhanced therapeutic potential of synergistic


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PDT/PPTT by AuNRs/MoS2-ICG nanoplatform and synergistic PPTT by AuNRs/MoS2 hybrid as compared to either individual PTT (Au NRs or MoS2 NSs) or even PDT agent (ICG). Thus, our study clearly demonstrates an effective approach to coordinate PDT/synergistic PPTT under single low power CW NIR laser, offering safe treatment approach with enhanced therapeutic efficacy within short treatment time. RESULTS AND DISCUSSION The present Au/MoS2-ICG nanoplatform was prepared in three synthetic steps as illustrated in Scheme 1. First, Au NRs were prepared by a well-established seed-mediated growth method. TEM characterization of the CTAB stabilized Au NRs reveals that the Au NRs core has an average length and width of 50 nm and 14 nm, respectively, with an aspect ratio of 3.5 as shown in Supporting Information S1. Meanwhile, zeta potential analysis shows +22 mV charge on Au NRs, which confirms the presence of a CTAB layer on the Au NRs (Supporting Information S2A). In addition, both the energy dispersive X-ray (EDX) spectroscopy and SEM elemental mapping of Au NRs indicate the presence of Au element as shown in Supporting Information S3A and S4A. The PEG functionalized MoS2 NSs were prepared by solvothermal approach, offering excellent biocompatibility and water dispersibility as evident by the digital photograph of aqueous dispersion of PEG-MoS2 NSs (Supporting Information S5). Importantly, the presence of both Mo and S as well as C and O elements verifies the successful surface grafting of MoS2 NSs by PEG during the solvothermal process as demonstrated by the SEM elemental mapping and EDX spectroscopy (Supporting Information S3B and S4B). The TEM characterization shows that the prepared 2D PEGylated MoS2 NSs have a piece diameter of approximately 80 nm (Figure 1A). Importantly, the PEG-MoS2 NSs exhibit negative zeta potential (-20 mV), which is possibly due


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Scheme 1. Schematic illustration of the synthesis approach and working mechanism of the Au/MoS2-ICG nanoplatform to induce simultaneous PDT/synergistic plasmonic PTT under low power single CW NIR laser irradiation (808 nm, 0.2 W/cm2). to the PEG functionalization as well as the presence of “S” atoms within “Mo” layers, facilitating an efficient electrostatic interaction with the positive charged Au NRs. Thus, the Au/MoS2 hybrid is formed through an electrostatic interaction between Au NRs and PEG-MoS2 NSs. The TEM image (Figure 1B) of the Au/MoS2 hybrid clearly demonstrates the random decoration of Au NRs onto the surface of PEG-MoS2 NSs. Zeta potential and DLS studies show that the prepared hybrid has an overall positive charge (15.8) and a mean diameter of 103 nm, respectively (Supporting Information S2A-B). As Au NRs clearly exhibit a characteristic longitudinal and transverse band at 780 nm and 515 nm, respectively, the Au/MoS2 hybrid shows an obvious red-shifted LSPR


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Figure 1. TEM morphological characterization of (A-C) PEG-MoS2 NSs, plasmonic Au/MoS2 hybrid, and Au/MoS2-ICG nanoplatform. White arrows indicate the presence of PEG-MoS2 NSs. (D) SEM elemental mapping of the Au/MoS2 hybrid. The presence of Au within PEGylated (presence of C and O) MoS2 NSs (presence of Mo and S) confirms the formation of plasmonic Au/MoS2 hybrid. band at 788 nm, indicating the electronic interaction between the plasmonic metal and semiconductor (Figure 2A). Whereas, the PEG-MoS2 NSs do not show any obvious peak from UV-vis to NIR region. Notably, the SEM elemental mapping and EDX clearly indicate the presence of Au, Mo, and S as well as C and O elements, which suggests the successful formation of plasmonic Au/MoS2 hybrid (Figure 1D and Supporting Information S4C). The amount of Au present in the Au/MoS2 hybrid is calculated as 26 %. The X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy were employed to ensure the formation of Au/MoS2 hybrid. Compared to MoS2 NSs, the XPS binding energy peaks of Mo 3d are positively shifted by 2 eV in Au/MoS2 hybrid, whereas, a positive shift of 2.4 eV and 1.3 eV is observed for S 2p3/2 and 2p1/2 peaks,


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respectively. The presence of Au 4f peaks in the Au/MoS2 hybrid at 82.8 eV (4f 7/2) and 86.8 eV (4f

5/2

), respectively, clearly indicate that Au NRs are bounded with PEG-MoS2 NSs via

electrostatic interaction (Supporting Information Figure S6A-C). Similarly, the Raman spectrum of the Au/MoS2 hybrid represents a blue shift of ca. 3 and 5 cm-1, respectively, in the characteristic Raman peaks of both Mo-S bond vibration (335 nm and 445 nm) and A2g phonon modes (480 nm), respectively (Supporting Information Figure S6D). Hence, optical characterizations evidence the successful formation of plasmonic Au/MoS2 hybrid and suggest strong electrostatic interaction between Au NRs and PEG-MoS2 NSs, enabling them to effectively promote synergistic PPTT effect. Finally, ICG dye was adsorbed onto PEG-MoS2 NSs via hydrophobic interactions and π-π stacking (Figure 1C) due to the high surface area to volume ratio and sp2 bond. Due to the loading of an anionic dye, the zeta potential value changes from positive value to -10 mV, and the resultant Au/MoS2-ICG nanocomposite possesses a mean diameter of 115 nm (Supporting Information S2A-B). After ICG loading, the UV-vis spectrum of the Au/MoS2 hybrid is further red-shifted to 810 nm, whereas an aqueous solution of unbounded ICG shows one distinct absorption peak at 780 nm and a shoulder peak at 710 nm, respectively (Figure 2A). Notably, the fluorescence signal of ICG is significantly quenched upon loading onto the surface of Au/MoS2 hybrid, confirming the successful loading of ICG onto the Au/MoS2 hybrid. However, under NIR light irradiation, the fluorescence signal of ICG is fully recovered, which suggests that the ICG dye could be effectively released from the Au/MoS2 hybrid under the influence of laser irradiation (Figure 2B). We next investigated the dye loading capacity of the Au/MoS2 hybrid. It has been noted that the loading efficiency increases with an increasing amount of ICG, suggesting a linear relationship between the amount of loaded ICG and the loading efficiency of the Au/MoS2 hybrid. The loading


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efficiency is calculated as 58.03 wt % at ICG : Au/MoS2 ratio of 45 % (Figure 2C). To prove our concept of simultaneous PDT, we further studied the ICG release behavior from the Au/MoS2ICG nanocomposites either under laser irradiation or without laser irradiation (as a control). Impressively, a more than 80 % of ICG is released from the nanocomposite under laser irradiation compared to the control group (15 %) (Figure 2D). Notably, a linear relationship has been noticed between the concentration and the absorbance of the released ICG in a given detection range as shown in Supporting Information S7. The rapid release behavior of ICG could be possibly due to the synergistic photothermal effect induced by the plasmonic Au/MoS2 hybrid, leading to break the π-π interactions between the loaded ICG and Au/MoS2 hybrid. Based on these findings, we conclude that the Au/MoS2-ICG nanocomposite has been successfully synthesized and the spectral overlap between Au/MoS2 hybrid and ICG allows single CW NIR laser trigger simultaneous PDT/synergistic PPTT. To confirm that the ICG release is owing to the synergistic plasmonic photothermal effect induced by the Au/MoS2 hybrid, we evaluated the in vitro and in vivo PTT efficiency under NIR laser irradiation (808 nm, 0.2 W/cm2) for 5 min. The real time increase in temperature was monitored by IR thermal camera. A maximum increase in temperature up to 60 oC and 57 oC is recorded by the Au/MoS2-ICG and Au/MoS2 hybrid, respectively. These results suggest that ICG loading neither increases nor decreases the PTT efficiency of the Au/MoS2 hybrid as indicated by the PTT profile of free ICG. On the other hand, the Au/MoS2 hybrid demonstrates a synergistic PPTT effect compared to the PTT performance of individual Au NRs (45 oC) and MoS2 NSs (42 o

C), respectively (Figure 2E, Supporting Information S8A). Notably, the plasmonic hybrid

exhibits superior photothermal conversion efficiency (68.8 %) over individual Au NRs (36.16 %) and 2D-PEGylated MoS2 NSs (32.96 %), which indicates that the plasmonic hybrid could


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Figure 2. Characterization of the Au/MoS2-ICG nanoplatform and photothermal trigger release of ICG from the Au/MoS2-ICG nanoplatform. (A) UV-vis absorption spectra of different materials as mentioned. (B) Fluorescence quenching and fluorescence recovery of ICG in Au/MoS2-ICG nanoplatform verified by fluorescence spectra of free ICG, Au/MoS2 hybrid, and Au/MoS2-ICG nanoplatform without and with laser irradiation (808 nm, 0.2 W/cm2). (C) Loading content of ICG in Au/MoS2-ICG nanoplatform at increasing concentrations of ICG. (D) Photothermal trigger release profiles of ICG from Au/MoS2-ICG nanoplatform from 0 h to 24 h without and with the influence of laser irradiation (808 nm, 0.2 W/cm2) at specific time points as indicated. (E) In vitro PTT profiling of different materials as mentioned at a concentration of 100 µg/mL, following 808 nm laser irradiation (0.2 W/cm2). (F) Photothermal stability of Au/MoS2 hybrid for successive four cycles of on/off laser irradiation.


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efficiently convert laser energy into heat to induce local hyperthermia under low laser power density (Supporting Information S9A-F). Meanwhile, the high PCE is attributed to the synergistic plasmonic effect as well as low scattering loss of laser energy and enhanced local electric field.59 In addition, concentration dependent temperature increase is also noticed by the Au/MoS2-ICG composite as the temperature increases from 22 oC to 60 oC with an increased concentration from 7 μg/mL to 100 μg/mL, respectively. Importantly, under very low power laser irradiation (0.2 W/cm2), the Au/MoS2 hybrid shows significant temperature increase (57.2 oC) owing to the synergistic photothermal effect of dual plasmonic PTT agents, while a maximum temperature increase up to 106.8 oC is observed at a laser power density of 0.75 W/cm2 (Supporting Information S8B-C). Therefore, to avoid the risk of skin burns associated with the high laser power density and following the guidelines of American National Standard Institute (ANSI), 0.2 W/cm2 laser power was chosen for further in vitro and in vivo therapeutic investigations, which is still lower than the standard set (330 mW/cm2) by the ANSI for NIR laser (808 nm).71 As the stability of the PTT agent is highly desirable to ensure high PTT efficacy, we determined the photothermal stability of the Au/MoS2 hybrid under four on/off laser cycles. There is no obvious change observed in the photothermal efficacy after four consecutive laser on/off cycles, demonstrating the excellent stability of the Au/MoS2 hybrid (Figure 2F). Moreover, in vivo PTT efficiency was also assessed using HeLa tumor model and similar results are obtained as those from in vitro photothermal measurements (Supporting Information S10). Briefly, a significant temperature increase is observed at the tumor region, while no apparent temperature increase is noticed in other body parts of the mice. As a control, PBS does not show any distinct temperature changes both in vitro and in vivo, respectively. Thus, in vitro and in vivo photothermal investigations reveal significant synergistic PPTT effect, which is sufficient to ablate tumor cells under low power laser irradiation


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within short duration. Moreover, these results also demonstrate that NIR laser induced local hyperthermia by the Au/MoS2 hybrid could favor the thermal release of ICG from the nanocomposite, allowing to elicit simultaneous PDT. To verify simultaneous PDT by thermally released ICG, 1O2 generation capability of the Au/MoS2-ICG composite under NIR light irradiation was first estimated by 1,3-diphenyl isobenzofuran (DPBF), a well-known 1O2 indicator. The 1O2 generation was determined by gradual decrease in the DPBF absorption peak at 410 nm. The Au/MoS2-ICG nanocomposite under 808 nm light irradiation (0.2 W/cm2) demonstrates noticeable time dependent decrease in the DPBF absorption peak at 410 nm, suggesting efficient 1O2 generation (Figure 3A). In contrast, the Au/MoS2 hybrid alone does not show any obvious decrease in the DPBF absorption (Figure 3B), which confirms that the plasmonic material does not generate 1O2. As a control, pure DPBF does not display photodegradation of DPBF even under NIR light irradiation, confirming the photostability of DPBF (Figure 3C, Supporting Information S11A). Next, we investigated the 1O2 generation ability of the Au/MoS2-ICG composite using singlet oxygen sensor green (SOSG), a highly sensitive 1O2 indicator. The 1O2 generation was evaluated by an increase in the fluorescence signal intensity of SOSG at 530 nm. Interestingly, the Au/MoS2-ICG composite displays considerably high SOSG fluorescence intensity, which is almost comparable to free ICG (Figure 3D, Supporting Information S11B), confirming the 1O2 generation by thermally released ICG under NIR laser irradiation, leading to simultaneous PDT effects. However, a slight decrease in SOSG fluorescence intensity in the case of Au/MoS2-ICG composite suggests that a partial amount of ICG remains bonded with Au/MoS2 hybrid, while a major portion of the loaded ICG releases into the system. To ensure that the thermally released ICG is responsible for an increase in SOSG fluorescence intensity by the Au/MoS2-ICG composite under laser irradiation, we next


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Figure 3. Proof of concept of simultaneous PDT induced by released ICG from Au/MoS2-ICG nanoplatform. (A-B) Time dependent decrease in the UV-vis absorption spectra of DPBF treated with Au/MoS2-ICG (50 µg/mL) and Au/MoS2 hybrid (50 µg/mL). (C) Normalized absorbance profile of only DPBF as a control, Au/MoS2 hybrid, and Au/MoS2-ICG under 808 nm laser irradiation (0.2 W/cm2). (D-E) Time dependent increase in SOSG fluorescence intensity treated with 50 µg/mL Au/MoS2-ICG nanoplatform and 808 nm laser irradiation (0.2 W/cm2) in the absence or presence of 100 mM NaN3. (F) Comparison of the fluorescence intensity (ᴧF=F5minF0min) of SOSG (20 µM) in the presence of different materials as indicated, following irradiation with 808 nm laser (0.2 W/cm2) for 5 mins added sodium azide (NaN3), a specific 1O2 quencher. Interestingly, the SOSG fluorescence signal from Au/MoS2-ICG nanocomposite has been significantly quenched, which provides a direct


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evidence of 1O2 generation from the released ICG to elicit simultaneous PDT (Figure 3E). In contrast, the Au/MoS2 hybrid alone does not increase the fluorescence intensity of SOSG, which further confirms that the Au/MoS2 hybrid is unable to generate 1O2. Similarly, pure SOSG without any material also does not show any noticeable increase in the fluorescence intensity of SOSG (Figure 3F). The SOSG fluorescence spectra of Au/MoS2 hybrid and a pure SOSG under light irradiation as a control are displayed in Supporting Information S11C-E. These findings prove our concept of photothermal release of ICG to simultaneously induce PDT effects under the influence of synergistic PPTT effect. Encouraged by the excellent demonstration of 1O2 generation in aqueous solution as detected by DPBF and SOSG, we then studied the intracellular 1O2 generation efficiency of the Au/MoS2-ICG composite using a fluorescent probe (DCFH-DA). The in vitro 1O2 generation was determined by monitoring the green fluorescence emitted from the oxidized DCF using an inverted fluorescence microscope. Under laser irradiation, both the free ICG and Au/MoS2-ICG composite exhibit highly intense DCFH signals (Figure 4A), implying that ICG is effectively released from the nanocomposite, resulting in a high level of intracellular 1O2 generation. However, a slight DCFH signal is observed in the case of Au/MoS2 hybrid alone, which might be owing to the generation of electron-hole pairs during laser excitation, resulting in the generation of hydroxyl ions. These results agreed well with the DPBF photodegradation by Au/MoS2 hybrid under laser irradiation. Similarly, HeLa cells neither treated with laser irradiation nor with any material do not show noticeable fluorescence of DCFH. Before investigating the in vitro cell killing ability, we examined the stability of the Au/MoS2-ICG and Au/MoS2 hybrids in different media e.g. DI water, PBS, DMEM with 10 % FBS, and DMF. As shown in Supporting Information S12, both the Au/MoS2 hybrid and Au/MoS2-ICG present good solubility in the given media without any


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Figure 4. Singlet oxygen generation and enhanced cytotoxicity in vitro. (A) Fluorescence images of HeLa cells incubated with DCFH (20 µM) as a control (i) and 50 µg/mL of (ii) Au/MoS2 hybrid, (iii) ICG (10 µg/mL), and (iv) Au/MoS2-ICG [ICG = 10 µg/mL], followed by laser irradiation for


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5 min. Scale bar: 20 µm. (B) Concentration dependent cytotoxicity of the Au/MoS2-ICG nanoplatform incubated with HeLa and HUVECs in dark. (C) Concentration dependent light trigger enhanced cytotoxicity of different materials with or without 100 mM NaN3 as stated, incubated with HeLa cells, following laser irradiation for 5 min. (D) Fluorescence images of Calcein AM/PI co-stained HeLa cells incubated with DMEM media without or with laser treatment for 5 min (i-ii), and 100 µg/mL of MoS2 NSs (iii), Au NRs (iv), 20 µg/mL ICG (v), Au/MoS2 hybrid (vi), and Au/MoS2-ICG [ICG = 20 µg/mL] (vii). Scale bar: 50 µm. Low power (0.2 W/cm2) 808 nm laser was used for the above mentioned experiments.

obvious aggregation. Similarly, we also performed the UV-vis characterization of the Au/MoS2 hybrid and Au/MoS2-ICG before (0 h) and after (1, 4, 12, 24, and 48 h) post-incubation in PBS with 10 % FBS. In the case of Au/MoS2, no obvious change is observed, while a negligible change occurs in the UV-vis absorption spectra of the Au/MoS2-ICG at longer time periods, which is possibly due to the slight release of the electrostatically bonded ICG from Au/MoS2. However, in terms of the stability of Au/MoS2-ICG for short time duration, we conclude that Au/MoS2-ICG is stable in biological media (Supporting Information S13A-B). Afterwards, in vitro phototherapeutic performance of the designed nanoplatform was quantitatively assessed by a standard MTT assay. First, dose dependent cytotoxicity of the Au/MoS2-ICG composite against both HeLa and HUVEC cells was evaluated under dark conditions, and the results indicate negligible dark cytotoxicity (Figure 4B). However, under single 808 nm laser excitation (0.2 W/cm2), more than 95 % HeLa cells are killed at a dose of 100 µg/mL, suggesting the dose dependent phototoxicity behavior of the designed Au/MoS2-ICG nanoplatform. It is noteworthy to mention that the therapeutic efficacy of the Au/MoS2-ICG is much higher than either the free ICG (PDT alone) or the Au/MoS2 hybrid


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(synergistic PPTT). In addition, cell killing ability of the Au/MoS2 hybrid is also far superior than both individual Au NRs and MoS2 NSs, respectively, which verifies the synergistic PPTT (Figure 4C). To ensure that the simultaneous PDT/synergistic PPTT really participate in the Au/MoS2ICG induced therapeutic effects, we incubated the Au/MoS2-ICG composite treated HeLa cells with varying concentrations of sodium azide (NaN3) prior to irradiation. Interestingly, the cell killing efficiency of Au/MoS2-ICG is markedly inhibited at 100 mM NaN3 and reaches to the level of the Au/MoS2 hybrid, suggesting that both simultaneous PDT/synergistic PPTT are responsible for the enhanced phototherapeutic killing efficiency. Given that, PPTT treatment does not rely on 1

O2, the addition of NaN3 does not alter the cell killing efficiency of the Au/MoS2 hybrid at all

(Supporting Information S14). Meanwhile, the individual therapeutic efficacy of PDT/PTT is calculated as 25.13:74.87. Furthermore, in vitro PDT efficiency was further examined by cellular staining using Calcein AM/PI. As expected, the Au/MoS2-ICG induces complete destruction of HeLa cells as shown by the red fluorescence of PI. However, only a small fraction of the cells exhibits green fluorescence, while more than 85 % of the cells are died after synergistic PPTT treatment as revealed by the red fluorescence. By contrast, the free ICG, individual Au NRs, and MoS2 NSs present a partial cells destruction as indicated by 50 % live/dead cells ratio (Figure 4D). These findings suggest a strong agreement with the MTT phototoxicity results, demonstrating that the combined simultaneous PDT/synergistic PPTT are much superior than any individual modality either PDT or PTT and thus, can effectively induce tumor cell destruction. Importantly, the current strategy of simultaneous PDT/synergistic PPTT shows quite appreciable in vitro therapeutic effects even under single NIR laser irradiation with extremely low power density (0.2 W/cm2), which is far superior over previously reported works. For comparison, we have also provided a detailed summary of the most recently reported works in Supporting information (Table S1),


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suggesting the synergistic PDT/PTT by different nanotherapeutic agents under either single laser or double laser irradiation. Owing to the commendable in vitro phototherapeutic efficacy demonstrated by the Au/MoS2ICG composite, we explored its potential in vivo anti-cancer application using HeLa tumor bearing mouse model. We chose the HeLa tumor model due to the following reasons: 1) HeLa cells are the human cervical cancer cells and as the human cervix is the lower part of the uterus and connected with vagina, the cervical cancer can be irradiated by a laser via colposcopy directly. Therefore, we thought that the PPTT treatment could be applied to cervical cancer more easily than the other deep-seated cancers in future clinical practices. 2) The cervical cancer is one of the most common cancer affecting women. In developing countries, cervical cancer was the most frequent malignant tumor among women until the early 1990s. Therefore, considering the huge potential benefit of the designed treatment strategy to patients with cervical cancer, we chose HeLa cells as our study object. In general, when the mean tumor volume exceeded 80 mm3, 35 mice were randomly divided into control groups (Group 1-2) and experimental groups (Group 3-7), respectively. Group 1 neither received any material nor laser irradiation, while Group 2 was just treated with NIR laser irradiation for 5 min. For experimental groups, each mouse in Group 3-7 received an intratumoral injection of 50 µL of 100 µg/mL Au NRs, PEG-MoS2 NSs, Au/MoS2 hybrid, Au/MoS2-ICG (ICG = 20 µg/mL), and free ICG (20 µg/mL), respectively, followed by 808 nm laser excitation (0.2 W/cm2) for only 5 min at 4-hour post-injection. The body weight and the tumor volume of each mouse were monitored every other day after treatment. It is worthwhile to mention that the Au/MoS2-ICG treated group demonstrates complete tumor inhibition within the first 4th days. Therefore, we neither provide any treatment nor intratumoral injection of Au/MoS2-ICG to Group 7 after day 4th except follow up to investigate the local tumor recurrence.


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Figure 5. Single low power NIR laser trigger simultaneous PDT/synergistic plasmonic PTT in vivo. (A-C) Relative tumor volume, body weight, and tumor weight of different groups (n = 5) as mentioned from day 0 to day 14th. All the values indicate the mean and SD (**** P < 0.0001). (D) Digital micrographs of the representative mice from control (i-ii) and the Au/MoS2-ICG treated groups (iii-iv) at day 0 and 14th. All the experimental groups were administered with 100 µg/mL (50 µL) of the respective nanomaterial and PDT/PPTT was conducted by laser irradiation for only 5 min after 4 h post-injection. For laser treatment, single low power CW NIR 808 nm laser (0.2 W/cm2) irradiation was used throughout the study. (E) Photographs of representative tumors resected from different groups at day 14th as indicated.


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Surprisingly, a complete tumor eradication with no obvious recurrence is noticed at day 14th in the Au/MoS2-ICG treated group due to the combined PDT/PPTT effects. Similar approach was also carried out for the Au/MoS2 hybrid treated group, which also demonstrates an effective tumor suppression due to the synergistic PPTT effect. These findings are in good accordance with the in vitro phototoxic results, confirming the excellent in vivo antitumor efficacy of the Au/MoS2-ICG and Au/MoS2 hybrids. Additionally, it is notable to mention that simultaneous PDT/synergistic PPTT triggered by single low power NIR laser offers significant therapeutic outcomes within short treatment time. Except Group 6 and 7, all the experimental groups (3-5) receive both intratumoral injection of the respective agent and laser treatment every other day. Compared to the combined PDT/PPTT, the individual PDT performed by free ICG (Group 5) reveals partial tumor growth inhibition. Whereas, the individual PTT groups (Group 3-4) treated with Au NRs and PEG-MoS2 NSs, respectively, slightly delay the tumor growth rather than growth inhibition. These results suggest that neither individual PDT nor PTT are sufficient to completely eradicate the tumor. On contrary, tumors of Groups 1-2 grow progressively during the entire treatment period (Figure 5A). Importantly, body weights of all the mice either in control or experimental groups do not indicate any noticeable change, which verifies that the designed nanocomposite does not induce acute toxic effects to other body parts (Figure 5B). After 14th day, the tumor sections and major body organs (lungs, kidneys, spleen, heart, and liver) from both control and experimental groups were collected, and the mice were sacrificed. Compared to other groups, the tumor sections obtained from the Au/MoS2-ICG treated group (Group 7) demonstrate a negligible tumor weight as further evidenced by representative digital photographs shown in (Figure 5C-E). For comparison, the representative photographs of the control and Au/MoS2-ICG treated mice at day 0 and day 14th are also shown in Figure 5D, which evidently confirm the highest PDT/synergistic PPTT effects by the developed


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Figure 6. In vivo biocompatibility study. (A-B) Levels of different blood biochemical indicators (ALT, AST, ALP, BUN, and Crea) after exposure to saline and Au/MoS2-ICG for 14 days. (C) Changes in the body weight of healthy nude mice after intravenous injection of saline and Au/MoS2-ICG. (D) H&E stained images of the indicated major organs collected from both the saline and Au/MoS2-ICG treated mice after 14 days. nanocomposites. Moreover, tumor H&E staining analysis reveals an extensive cancer necrosis and apoptosis by either Au/MoS2-ICG or Au/MoS2 hybrid treated groups (Group 6-7), illustrating that the combined PDT/PTT or synergistic PPTT significantly damages the tumor tissues. Meanwhile, a partial necrosis is observed by free ICG. Similarly, a few necrotic cells are noticed by individual PTT groups either Au NRs or MoS2 NSs, respectively, which indicate a slight damage to the tumor cells. In stark contrast, H&E staining of the control groups does not show any noticeable tumor necrosis as shown in Supporting Information S15A. Subsequently, organ H&E staining was also


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performed which neither shows any potential damage or inflammation nor any tumor necrotic cells, thus confirming the non-cytotoxicity of the designed nanoconstruct (Supporting Information S15B). Finally, the in vivo biocompatibility of the Au/MoS2-ICG was evaluated. It has been noted that Au/MoS2-ICG neither reduces the body weight of the mice nor increases the level of different blood biochemical indicators related to the normal functioning of the kidney and liver such as blood urea nitrogen (BUN), creatinine (Crea), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). In addition, histopathological investigation of the major organs collected from the Au/MoS2-ICG treated mice does not reveal any pathological changes (Figure 6A-D). Thus, these findings clearly demonstrate that the Au/MoS2-ICG nanoplatform has enhanced in vivo therapeutic capability without any apparent systemic cytotoxicity and is suitable to trigger simultaneous PDT/synergistic PPTT of tumors in vivo under single low power laser irradiation for short duration. CONCLUSIONS In summary, we have successfully constructed an effective phototherapeutic nanoplatform based on ICG loaded plasmonic Au/MoS2 hybrid. We demonstrate single low power CW NIR laser trigger simultaneous PDT/synergistic PPTT owing to the spectral overlap between ICG (PDT agent) and the plasmonic Au/MoS2 nanohybrid (PTT agent). Impressively, the combined PDT/PPTT effects have been proven to completely eradicate the cell/tumor both in vitro and in vivo within short irradiation time, compared to single treatment modality either PDT or synergistic PPTT, respectively. The designed nanoplatform offers several advantages in the following aspects: 1) high ICG loading owing to the high surface area of MoS2 NSs; 2) plasmonic Au/MoS2 nanohybrid constantly enhances the PPTT effect for antitumor therapy under NIR light irradiation owing to the synergistic PPTT effect; 3) strong synergistic PPTT effect induces the thermal release


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of ICG to simultaneously generate 1O2, achieving the combined PDT/PPTT effects with an effective skin protection. Compared with the previously reported photosensitizer-modified nanostructures, the current strategy of single laser trigger simultaneous PDT/synergistic PPTT shows significantly enhanced in vivo antitumor performance under low power laser irradiation (0.2 W/cm2) for only 5 min, offering safer treatment approach which holds great promise for clinical translation. METHODS Materials. All commercial chemicals and reagents were used as received unless otherwise stated. Gold chloride (HAuCl4.4H2O) hydrated was obtained from the First Reagent Factory (Shanghai, China). Sodium borohydride (NaBH4), sodium oleate (NaOL), and polyethylene glycol (PEG, MW = 400 Da) were obtained from Sinopharm Chemical Reagent Co., Ltd. Ethanol and silver nitrate (AgNO3) were bought from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Ascorbic acid (AA) and hexadecyl trimethylammonium bromide (CTAB) were from Johnson Matthey Corporation. Ammonium tetrathiomolybdate ((NH4)2MoS4), 1,3-diphenylisobenzofuran (DPBF), indocyanine green (ICG), sodium azide (NaN3), and singlet oxygen sensor green (SOSG) were bought from Sigma Aldrich and Thermo Fisher Scientific, Inc., respectively. Dimethyl sulfoxide (DMSO), calcein AM, propidium iodide, 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA), phosphate buffer saline (PBS, pH 7.4), and methyl thiazolyl-tetrazolium (MTT) were obtained from Keygen Biotech. Co., Ltd. Dulbecco Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone. Deionized water (DI, resistivity of 18.2 MΩ·cm) was used throughout the study. Synthesis of Gold Nanorods (NRs) with SPR Band at 775 nm. Au NRs were prepared following the well-developed seed-mediated approach.72 First, Au seeds solution was prepared by


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reducing HAuCl4 with NaBH4. CTAB solution (0.1 M, 0.005 L) was mixed with HAuCl4 (25 mM, 100 μL), following the addition of ice-cold NaBH4 (0.01 M, 0.6 mL) under vigorous stirring. Immediately, the solution color was turned from bright yellow to brown, which suggests the formation of Au seeds. To make sure the hydrolysis of unreacted sodium borohydride, the prepared Au seeds solution was maintained at room temperature (RT) for about 120 min. For the growth of Au NRs, the solutions of different reagents (0.1 M CTAB (0.1 L), 0.1 M AgNO3 (0.1 mL), 25 mM HAuCl4 (0.02 L), and 0.2 mL of 1 M HCl) were sequentially added into the 250 mL flask. Then, 0.0788 M AA (800 μL) was added as a reducing agent to the above mixed solution and stirred gently to make a homogenized solution. At the end, the Au seeds solution (120 μL) was added and left the solution undisturbed at 30 oC for about 12 h. Afterwards, the Au NRs were centrifuged for about 3 times (15 min, 8000 rpm) and stored at RT for further use. Synthesis of PEGylated MoS2 Nanosheets. PEGylated MoS2 NSs were prepared by a slight modification of the previously reported hydrothermal approach by Wang et al.67 Briefly, (NH4)2MoS4 (300 or 150 mg) and PEG-400, respectively, were thoroughly dissolved into 0.03 L of DI water. The resultant mixture was transferred to a Teflon-lined stainless-steel autoclave and kept at 200 oC for 18 h. After cooling down to RT, the final product was washed for several times with DI water and freeze dried for further use. Fabrication of Au NRs Modified PEG-MoS2 NSs. For the preparation of Au/MoS2 hybrid, both Au NRs and PEG-MoS2 NSs solutions of same concentration were mixed at a volume ratio of 5:1. Then, the mixture was sonicated for about 2 h, followed by centrifugation and freeze drying for overnight. Characterization. UV-vis spectroscopic characterization and photodegradation of DPBF were analyzed using UV-1800 spectrophotometer (Shimadzu). Transmission electron microscopy


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(TEM, JEM-2100, Japan) was used to perform the morphological characterization by drying a small volume of each sample solution on Ni-grids with carbon film. Malvern Zetasizer Nano ZS90 analyzer was used to perform dynamic light scattering (DLS) and zeta potential analysis at RT. All the SOSG fluorescence spectra were performed on a HORIBA Jobin Yvon Fluoromax-4 fluorescence spectrophotometer. All the fluorescence images were acquired using an Olympus IX73 fluorescence inverted microscope (Olympus, Japan). ICG Loading Capacities. To estimate the dye loading capacity of Au/MoS2 hybrid, 0.5 mg/mL Au/MoS2 hybrid (0.005 L) were mixed with different volumes (10 - 80 μL) of 1 mg/mL ICG. Next, the resultant mixtures were gently stirred at RT for about 6 h. Thereafter, the Au/MoS2-ICG composite was washed with DI water for three times by centrifugation (8000 rpm, 15 min). The remaining unbound ICG was removed through dialysis (dialysis bag MW = 12 kDa). Then, UVvis spectroscopy was used to quantitate the amount of free ICG by determining the UV-vis absorption peak of ICG at 780 nm. Finally, the ICG loading capacity was calculated by the following equation: drug loading content = (amount of drug in Au/MoS2 hybrid) / (total weight of Au/MoS2 hybrid) × 100%. Measurement of Photothermal Performance. To investigate the in vitro photothermal performance, aqueous solutions (100 μg/mL) of each Au NRs, MoS2 NSs, Au/MoS2 hybrid, ICG (20 μg/mL), and Au/MoS2-ICG composite were taken into the eppendorf tubes, following irradiation with 808 nm laser (0.2 W/cm2) for 300 sec. The real time temperature increase by each material was recorded every 1 min using an IR thermal camera (Fortic 225-1, Fortic), and the AnalyzIR software was used to quantify the temperature from IR thermal images. Meanwhile, the concentration dependent photothermal performance of the Au/MoS2-ICG (7, 15, 30, 60, and 100 μg/mL) and the PTT stability of the Au/MoS2 hybrid (100 μg/mL) under four consecutive laser


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on/off cycles were also evaluated under similar conditions. To investigate the NIR laser power dependent PTT efficiency, 100 μg/mL aqueous solution of Au/MoS2-ICG was irradiated under NIR laser of different powers (0.1, 0.2, 0.4, and 0.75 W/cm2) and record the temperature every 1 min. In vivo PTT efficiency was estimated using HeLa tumor model. When the tumor size reaches 100 mm3, all the mice were randomly divided into six groups (1. PBS; 2. Au NRs; 3. MoS2 NSs; 4. Au/MoS2 hybrid; 5. ICG (20 μg/mL); and 6. Au/MoS2-ICG composite) (n=3 per group). Then, 100 μg/mL (50 μL) of the above-mentioned sample solutions were intratumorally injected into the relevant mice group. Afterwards, all the groups were irradiated with 808 nm laser (0.2 W/cm2) for 300 sec and the real time changes in in vivo temperature were monitored every 1 min by a thermal IR camera. Only PBS was used as a control in both in vitro and in vivo experiments, respectively. Measurement of Thermal Release of ICG. Thermal release of ICG from the Au/MoS2-ICG composite was evaluated at room temperature. In detail, the Au/MoS2-ICG (1 mL, ICG = 10 μg/mL) was placed in a dialysis membrane bag (MW = 12 kDa) which contains PBS (50 mL, pH 7.4). The solution was kept under gentle stirring at RT for about 24 h. After specific time intervals, the solution was irradiated by an NIR laser (808 nm, 0.2 W/cm2) for about 120 sec. Afterwards, 2 mL solution was taken from outside the dialysis bag and the fresh medium of the same volume was added, followed by using UV-vis spectroscopy to quantitate the amount of thermally released ICG. For the control, 1 mL of Au/MoS2-ICG composite without NIR laser irradiation was used. Meanwhile, fluorescence quenching of ICG after loading onto Au/MoS2 hybrid and the recovery of ICG fluorescence upon thermal release were also studied by a fluorescence spectrophotometer. Singlet Oxygen Detection Methods. First, photodegradation of DPBF was used to detect 1O2. All the experiments involving DPBF were carried out in the dark. Keeping in mind the high


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penetration depth (10 mm) of 808 nm NIR laser,73 continuous wave (CW) laser with a wavelength of 808 nm and a power of 0.2 W/cm2 was used for irradiation experiments throughout the study unless otherwise stated. A DPBF solution (1 mg/mL) in ethanol was freshly prepared and mixed with 50 μg/mL each of Au/MoS2 hybrid and Au/MoS2-ICG composite ([ICG] = 10 μg/mL) with stirring for 5 min in the dark. The resulting mixture was subjected to CW laser irradiation for specific time intervals and the decrease in DPBF absorbance at 410 nm was recorded by UV-vis spectroscopy. The DPBF solution under near infrared (NIR) light irradiation without any material was used as a control. Second, SOSG was used as an indicator of 1O2. Typically, all the sample solutions such as free ICG (10 μg/mL), and 50 μg/mL each of Au/MoS2 hybrid and Au/MoS2-ICG composite ([ICG] = 10 μg/mL) were mixed with SOSG (20 μM, 8 μL) and irradiated for different times. Only SOSG in PBS under NIR light irradiation was used as a control. For 1O2 quenching experiments, 100 mM NaN3 was used. Except this, all the conditions were the same as mentioned above. The fluorescence spectra of SOSG were collected with an excitation at 488 nm and an emission at 525 nm, respectively. Cell Culture Experiments. For in vitro experiments, HeLa and human umbilical vein endothelial cells (HUVECs) were bought from Procell Life Science and Technology Co., Ltd (Wuhan, China), and were kept at 37 oC in a 5 % CO2 humidifier incubator. The DMEM media supplemented with 10 % FBS were used as the culture media. In Vitro ROS Detection Assay. In vitro ROS generation under NIR light irradiation was investigated by DCFH-DA assay. HeLa cells were seeded in a 35 mm culture dish at a density of 1 × 105 and incubated with 1 mL DMEM medium containing 10 % FBS for 24 h. Then, the fresh DMEM medium containing different samples including ICG (10 μg/mL), Au/MoS2 hybrid (50 μg/mL), and Au/MoS2-ICG (50 μg/mL, ICG = 10 μg/mL) was added and incubated for 6 h. After


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incubation, DCFH-DA (20 μM) was added and incubated for further 20 min. Then, the cells were irradiated with NIR laser for 5 min, washed with PBS, and the fluorescence images were captured on an inverted fluorescence microscope. Moreover, the cells without any treatment (no light, no material) were used as a control. Except this, all the conditions were the same for both the control and experimental groups, respectively. In Vitro Stability. To determine the in vitro stability, Au/MoS2 and the Au/MoS2-ICG were incubated in PBS with 10 % fetal bovine serum (FBS) and their UV-vis absorption were measured after specific time periods (0, 1, 4, 12, 24, and 48 h) using UV-1800 spectrophotometer (Shimadzu). In Vitro Synergistic PTT/PDT. The in vitro cytotoxicity induced by synergistic PTT/PDT was evaluated by MTT assay. Typically, HeLa cells were seeded in 96-well plates at a density of 5 × 103 cells/well and incubated at 37 °C for 24 h. Then, the cells were incubated for 4 h with different concentrations of Au/MoS2 hybrid (7, 15, 30, 60, 100 μg/mL), ICG (2-20 μg/mL), and Au/MoS2ICG composite (Au/MoS2 = 7-100 μg/mL, 2-20 μg/mL ICG). To induce synergistic PTT/PDT, each well was irradiated with a CW 808 nm laser for 300 sec and incubated for 24 h. Next, MTT agent was added and incubated for another 4 h. After incubation, the cell culture medium was replaced with PBS containing 100 μL DMSO to dissolve the purple formazan crystals. The absorbance at 550 nm was recorded for each well by microplate reader to quantitatively determine the cell viability. Dark cellular toxicity of different concentrations of Au/MoS2-ICG composite (5, 12, 25, 50, and 100 μg/mL) against HeLa cells and human umbilical vein endothelial cells (HUVECs) was also examined. In addition, cell viabilities were also assessed in the presence of different concentrations of NaN3 (0, 20, 50, 100 mM) to verify the participation of simultaneous PDT as well as determine the individual therapeutic efficacy (TE) of PDT/PTT. In detail, the therapeutic efficacy without NaN3 was investigated, which corresponds to the combined


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therapeutic efficacy of PDT and PTT (TE), while at 100 mM NaN3 (TE NaN3), the obtained TE corresponds to only plasmonic PTT. Thus, the ratio of PDT to PTT is calculated by using the following formula: (TE - TE NaN3)/TE NaN3. Similarly, in vitro synergistic PTT/PDT performance was further examined by Live and Dead assay using Calcein AM and PI co-staining approach. Briefly, HeLa cells were seeded in a 35 mm culture dish and incubated with above mentioned different materials at their maximum concentration for 6 h. After incubation, each well was subjected to NIR laser irradiation for 5 min. Then, the cells were co-stained with Calcein AM and PI for 20 min. After that, the cells were washed with PBS and imaged on an inverted fluorescence microscope. Importantly, the HeLa Cells either under dark conditions or under illumination but in the absence of any material were used as a control. All the conditions were kept identical for both the control and experimental groups, respectively, except as mentioned. In Vivo Animal Studies. For in vivo investigations, 4-5 weeks old mice with a HeLa tumor in their armpit were obtained from the Model Animal Research Center (MARC) of Nanjing University (Nanjing, China) and maintained under standard housing conditions. All the experimental procedures were in compliant with the regulations of the Institutional Animal Care and Use Committee (IACUC) of Nanjing University. When the tumor volume reached approximately 100 mm3, all the mice were randomly divided into 7 groups (n=5 per group). The control groups were Group 1 and Group 2. Group 1 neither received any treatment nor any material (-L, -M), while Group 2 was only treated with light irradiation (+L, -M), respectively. However, twenty-five mice of 5 different treatment groups (Group 3-7) were intratumorally injected with 50 μL of ICG (20 μg/mL), and 100 μg/mL of each Au NRs, MoS2 NSs, Au/MoS2 hybrid, and Au/MoS2-ICG composite (ICG = 20 μg/mL), respectively. After 4 h post-injection, the mice in


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Group 2-7 were anesthetized using isoflurane and subjected to CW 808 nm laser (0.2 W/cm2 or 0.2 J/sec) for 5 min (Supporting Information S16). The laser spot was adjusted to target the center of the tumor and the margin of the irradiation field was set to exceed the tumor margin for at least 2 mm to make sure that the whole tumor is irradiated by the laser. The body weight and the tumor volume of each mouse were determined every other day after treatment and measured up to 14 d. At 14th day, all the mice were sacrificed and the tumors as well as the major organs (liver, spleen, kidneys, heart, and lungs) were collected, weighed, photographed, and stored for further histological examination. The tumor volume was calculated by the following formula: Volume = (Length × Width2)/2. All the data related to the tumor volume and body weight of each group was normalized to that of initial treatment. Hematoxylin and Eosin (H&E) Staining. For histopathological examination (H&E), tumors and major organs obtained from both the control and experimental groups, respectively, were fixed in formalin solution, stored in paraffin, cryosectioned into 10 μm slices, and mounted onto glass slides. Then, H&E staining was performed according to the manufacturer instructions and examined by an IX73 optical microscope (Olympus, Japan). In Vivo Biocompatibility Studies. For the in vivo biocompatibility investigation, healthy nude mice (n=12) were randomly divided into 2 groups, followed by an intravenous injection of saline (100 µL) in group 1 (Control) and Au/MoS2-ICG (100 µg/mL, 100 µL) in group 2 (Experimental). The body weight of all the mice was recorded every other day and at day 14th, all the mice were sacrificed and their major organs (lungs, spleen, intestine, kidneys, stomach, liver, and heart) and blood were collected for H&E staining and blood biochemical analysis, respectively. Statistical Analysis. The results of atleast three independent experiments were expressed as a mean and the standard deviation (S.D.) unless otherwise mentioned. Student’s t-test was used for


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statistical comparisons between different groups by using Prism 6 (Prism GraphPad Software, Inc., San Diego). A P value of less than 0.0001 (****< 0.0001) was considered as statistically significant.

ASSOCIATED CONTENT Supporting Information. Detailed characterizations of Au/MoS2 and Au/MoS2-ICG, e.g., TEM, DLS, zeta potential, SEM elemental mapping, EDX, XPS and Raman characterization, in vitro and in vivo PTT profiling, calculation of photothermal conversion efficiency, colloidal stability of the Au/MoS2 and Au/MoS2-ICG, H&E staining, irradiation setup (S1-S16), and summary of the previously reported work (Table S1) are included. “This material is available free of charge via the Internet at http://pubs.acs.org.” The authors declare no competing financial interests.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ACKNOWLEDGMENTS This work was supported by the grants from the National Key R&D Program of China (2017YFA0700500) and the National Natural Science Foundation of China (21635004).


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