Gold Nanoclusters-Indocyanine Green Nanoprobes for Synchronous

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Gold Nanoclusters-Indocyanine Green Nanoprobes for Synchronous Cancer Imaging, Treatment and Real-time Monitoring Based on Fluorescence Resonance Energy Transfer Haodong Cui, Dehong Hu, Jingnan Zhang, Guanhui Gao, Ze Chen, Wenjun Li, Ping Gong, Zonghai Sheng, and Lintao Cai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06192 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 4, 2017

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ACS Applied Materials & Interfaces

Gold Nanoclusters-Indocyanine Green Nanoprobes for Synchronous Cancer Imaging, Treatment and Real-time Monitoring Based on Fluorescence Resonance Energy Transfer Haodong Cui,a† Dehong Hu,a,b† Jingnan Zhang,a Guanhui Gao,c Ze Chen,a Wenjun Li,a Ping Gong,a Zonghai Sheng, a,b* and Lintao Cai a*

a

Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and

Biotechnology, Shenzhen Institute of Advanced Technology, Chinese of Academy of Sciences, Shenzhen, 518055, China. b

Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and

Health Engineering, Shenzhen Institute of Advanced Technology, Chinese of Academy of Sciences, Shenzhen, 518055, China. c

Paul-Drude-Institut für Festkörperelektronik, Berlin, 10117, Germany.

†

These authors contributed equally to this work.

* Corresponding Authors: Z. Sheng ([email protected]), L. Cai ([email protected])

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ABSTRACT A well-designed gold nanoclusters-indocyanine green nanoprobes (Au NCs-INPs) have been developed by the conjugation of Au NCs assemblies with indocyanine green (ICG) for the therapeutic real-time monitoring based on fluorescence resonance energy transfer (FRET). The synthesized Au NCs-INPs demonstrated the improved cellular uptake and effective tumor targeting due to the enhanced permeability and retention (EPR) effect and the gp60-mediated SPARC combined transport pathway, suggesting excellent dual-modal near-infrared fluorescent (NIRF) and photoacoustic (PA) imaging. Moreover, the simultaneous photodynamic therapy (PDT) and photothermal therapy (PTT) of Au NCs-INPs exhibited higher cancer cells killing and tumor removal efficiency than these of PDT or PTT alone. More importantly, a promising therapeutic monitoring strategy was performed based on FRET between Au NCs and ICG, suggesting Au NCs-INPs could be utilized to evaluate the therapeutic response by real-time monitoring the change of Au NCs in fluorescence intensity together with ICG supersession. Therefore, Au NCs-INPs as a novel photosensitizer had great potentials for combinational tumor imaging, therapy and therapeutic monitoring in real time. KEYWORDS: Indocyanine green, Gold nanoclusters, Near-infrared fluorescent imaging, Photoacoustic imaging, Synchronous therapy, Fluorescence resonance energy transfer, Real-time monitoring.


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Introduction Imaging-guided nanomedicine is driven by precision medicine and makes it possible to probe optical process from microscopic to nanoscopic regions, for applications in both diagnosis and treatment.1 At present, the medical imaging with various modalities has been developed to provide more comprehensive theranostic information. Compared to conventional optical tomography, in vivo near-infrared fluorescent (NIRF) imaging has high sensitivity owing to eliminating the self-luminescence of tissues in the region of NIR wavelength,2,3 and photoacoustic (PA) imaging has the desired imaging depth of tissues and excellent spatial resolutions.4,5 In addition, as a non-invasive optical process, phototherapy has gained attracted for avoiding damages of normal tissues from chemotherapy or radiotherapy. In the presence of photosensitizer, laser irradiation triggers the photochemical process, which further generates reactive oxygen species (ROS) for photodynamic therapy (PDT)6–8 and heat radiation for photothermal therapy (PTT).9–11 More importantly, the transient vasoconstriction triggered from PDT has increased the time of local hyperthermia, and the penetration efficiency of drugs has been further improved by the heat irradiation. In comparison with PDT or PTT alone, the synergistic phototherapeutic modality achieved the enhancement of anticancer efficacy.12,13 The good contrast agents are necessary to screen for the optical imaging and phototherapy. Indocyanine green (ICG) as a fluorescence dye, is authorized by Food and Drug Administration (FDA) and employed for combined imaging (NIRF14 and PA imaging15) and synergistic therapy (PDT14 and PTT16) in clinical applications. However, ICG with free structure has high photo-quenching, low fluorescence intensity and fast


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clearance from body. Generally, ICG has been encapsulated by some of drug carriers like inorganic nanoparticles,17 macromolecules,18 liposome19 and protein.20 Albumin as a common protein carrier was utilized to delivery ICG by the non-covalently binding process, which increased the fluorescent intensity and stability of ICG.21 Moreover, albumin as a protein template was also employed to synthesize nanoclusters with low bio-toxicity and high biocompatibility, for instance, bovine serum albumin (BSA) assisted gold nanoclusters (Au NCs) with excellent biomedical imaging22–26 and therapeutic potentials.27,28 Thus, BSA-stabilized Au NCs was also devoted to loading ICG or its derivative for medical imaging and cancer treatment.29,30 Recently, the strategy for evaluating treatment response has explored. Ding et al. believed that the gene therapeutic efficacy was assessed by monitoring hypoxic districts of tumor.31 The chemotherapeutic response was evaluated for monitoring the metabolic alteration by nuclear magnetic resonance (NMR) spectroscopy.32 Moreover, optical imaging as a real-time method could also monitor the protein expression33,34 and the fluorescent alteration35–37 after cancer treatment. However, the fluorescent dyes in the theranostic system showed high photo bleaching under laser irradiation. Thus, the therapeutic response was not completely evaluated by monitoring the fluorescence alteration. Dual fluorescent emission based on fluorescence resonance energy transfer (FRET) could limitedly improve this shortcoming when the distance of them closed 1 nm to 10 nm. FRET has a wide variety of potential applications in monitoring filed, like molecular changes,38,39 DNA synthesis,40 enzyme activity41 and drug release.42 More importantly, based on FRET real-time imaging, the apoptosis process of tumor cells was monitored by the functional gold nanoparticles,43 which revealed a novel strategy of


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fluorescent imaging for evaluating therapeutic response. To avoiding the bleaching of dyes, Au NCs as one of fluorescent groups were investigated. the dyes-encapsulated Au NCs were employed for molecular detection,44,45 biological imaging46 and cancer treatment.30 Therefore, the non-covalent assemblies by the conjugation of Au NCs with ICG also were devoted in monitoring the phototherapeutic process based on FRET properties. In this work, we designed a novel probe based on the non-covalently binding of Au NCs assemblies with ICG for applications in dual-modal imaging guided synergetic phototherapy and therapeutic real-time monitoring of cancer. Herein, the Au NCs was self-assembled in the presence of L-Glutathione reduced (GSH) based on the protein cross-linking approach.20 Meanwhile, ICG was encapsulated to Au NCs assemblies by the non-covalently binding,21 which was significantly different from gold nanoparticles conjugated with ICG.47 The obtained ICG loaded Au NCs assemblies, named as Au NCsICG nanoprobes (Au NCs-INPs), with the well-controlled size and non-toxicity, exhibited highly effective cellular uptake and precise tumor targeting, which was attributed to the enhanced permeability and retention (EPR) effect and the gp60-mediated SPARC (secreted protein acidic and rich in cysteine) combined transport pathway from albumin-drug complex.48 Furthermore, ICG largely accumulated in cancer cells and tumor demonstrated the applicants of dual-modal NIRF/PA imaging and synchronous PDT-PTT upon NIR irradiation. Additionally, with the advancement of cancer treatment, the fluorescent intensity was gradually decreasing of ICG with supersession and simultaneously recovering of Au NCs, which indicated the real-time monitoring of therapeutic process. In summary, the well-defined Au NCs-INPs as a theranostic agent


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had the important applications in dual-modal imaging, simultaneous phototherapy of cancer and imaging-guided therapeutic monitoring in real time. RESULTS AND DISCUSSION Preparation and characterization of Au NCs-INPs BSA as reducers and stabilizers had been utilized in the preparation of Au NCs.22 The as-prepared fluorescent Au NCs showed the slight changes of size and zeta potentials compared to BSA, suggesting that Au NCs also was employed as carriers to directly encapsulate ICG by the non-covalent process.21 As shown in Figure S1, Au NCs-ICG was synthesized by the non-covalently conjugation of Au NCs with ICG, which showed the similar hydrodynamic size (5.79±0.09 nm) compared to Au NCs (3.57±0.09 nm). However, the higher renal clearance significantly hampered the clinical applications. Therefore, a well-defined Au NCs-INPs was constructed by the conjugation of Au NCs assemblies with ICG. The schematic illustration of the synthesis process of Au NCs-INPs is shown in Figure 1A. Au NCs assemblies were prepared according to the literature of protein nanoparticles.49 Specifically, GSH were employed for the reduction of Au NCs by the breakage of intramolecular disulfide bonds, and then the reduced Au NCs was assembled by the generation of intermolecular disulfide bonds via the disulfidesulfhydryl interchange, followed by the encapsulation of Au NCs assemblies with ICG to prepare Au NCs-INPs. Unlike the preparation of BSA nanoparticles, the structure of Au NCs had changed with the increase of GSH concentration, which limitedly impacted on the fluorescence property of Au NCs assemblies. But with the suitable reaction time and GSH concentration, the size-controlled Au NCs assemblies were prepared with limitedly decreasing of fluorescence intensity and slightly blue-shift. The as-synthesized Au NCs-


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INPs showed the well-defined spherical shape with average diameters of 31.97±1.49 nm in TEM images (Figure 1B). To further investigate the stability and dispersity of Au NCs-INPs, the diameter and surface potential in water were measured by dynamic light scattering (DLS). The average hydrodynamic size indicated Au NCs-INPs with 118.3±7.85 nm (Figure 1C), which was different from TEM results due to the dehydration or hydration morphology of materials. And the average surface potential of Au NCs-INPs had a little change compared to Au NCs-ICG from -43.8±0.43 mV to 46.7±1.56 mV (Figure S2), which indicated the good mono-dispersity and colloid stability in aqueous solution (Figure S3A). Table S1 showed the loading capacity (LC) and encapsulation efficiency (EE) of ICG. The LC and EE of Au NCs-INPs were calculated at 10.32% and 78.05% in comparison to Au NCs-ICG with 11.84% and 81.4%. The results revealed that the values of LC and EE of Au NCs-INPs showed slightly lower than Au NCs-ICG, suggesting that some binding sites of Au NCs was occupied for the formation of the intermolecular disulfide of Au NCs assemblies. The absorption and fluorescence spectra were investigated compared with Au NCs and ICG. After the encapsulation of ICG, the absorption peak had not changed in Au NCs-ICG (Figure S4A) and Au NCs-INPs (Figure 1D). But the fluorescence emission peak of Au NCs-INPs exhibited a blue shift from 660 nm to 600 nm compared to Au NCs, and increasing in comparison to ICG at 476 nm excitation (Figure 1E), which also was indicated at 760 nm excitation (Figure S4B). It suggested that the decomposition of ICG was delayed after the encapsulation in Au NCs-INPs, which further improved the fluorescence stability of ICG (Figure S3B). Moreover, the fluorescence quantum yields (QY) was calculated by the emission spectra of rhodamine B in ethanol (corrected for absorbance at 476 nm) and


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ICG in DMSO (corrected for absorbance at 760 nm). Table S2 indicated that the QY increased from 0.066 of Au NCs to 0.074 of Au NCs-ICG and 0.08 of Au NCs-INPs. And after the encapsulation of ICG (0.033), the QY also increased to 0.04 of Au NCsICG and 0.069 of Au NCs-INPs. The higher QY of Au NCs-INPs improved the applicants in bio-imaging of ICG. To quantitatively determine the concentration of Au NCs-INPs, the absorbance and fluorescence spectra of ICG were measured (Figure S5), and indicated the mean concentration of 316 µg/mL in all ICG-composited materials (Table S3). Furthermore, the gold contents were detected by ICP-MS and showed the mean concentration of 176 µg/mL in all Au NCs-composited materials, suggesting that Au NCs exhibited slightly damaged in the Au NCs-INPs formulation. Compare to ICG and Au NCs-ICG, the as-prepared Au NCs-INPs demonstrated the enhanced quantum efficiency, the available size and the improved stability for the intensive studies in vitro and vivo. In vitro NIRF and PA imaging To determine in vitro imaging of Au NCs-INPs, cellular viability and uptake were performed. As shown in Figure S6, MTT assays revealed that all ICG-composited materials had the non-cytotoxicity after 24 h incubation in 293T normal cells and 4T1 cancer cells at the concentration even to 63.2 µg/mL of ICG. To explore cellular uptake, Au NCs, ICG, Au NCs-ICG and Au NCs-INPs suspensions (35.2 µg/mL of Au NCs and 63.2 µg/mL of ICG) were incubated in 4T1 cells for 4 h, followed by the observation with blue emission of nuclei, green emission of Au NCs and red emission of ICG in confocal fluorescence microscope. As shown in Figure 2A, Au NCs-INPs presented dual fluorescence imaging and higher cellular uptake via endocytosis compared to Au NCs


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and ICG. The low uptake and fluorescence of Au NCs were ascribed to the etching in tumor cells, where BSA was replaced by GSH to form GSH coated Au NCs with blueshift emission.50 After ICG encapsulated to Au NCs, the Au NCs-ICG limitedly prevented the etching process from high levels of GSH, which was verified by 2.4 times and 3.5 folds increasing of fluorescence signals compared to ICG and Au NCs (Figure S7). However, the Au NCs-INPs presented higher fluorescence intensity than that of Au NCs-ICG for the suitable size and protection from hydrated layer. The fluorescence intensity was determined by region of interest (ROI) for 3.7 times and 24 folds increasing compared to ICG and Au NCs. To evaluate the mechanism of cellular uptake of Au NCsINPs, 4T1 cells were pretreated with low temperature for 0.5 h and showed lower NIRF signal than that of control (no pretreatment) (Figure S8A), suggesting the Au NCs-INPs was uptaken via endocytosis. The endocytosis inhibitors, methyl-β-cyclodextrin (MβCD), sucrose and amiloride, were employed to verify the caveolae-mediated endocytosis, clathrin-mediated endocytosis, and macropinocytosis, respectively. As shown in Figure S8B, the intracellular fluorescent intensity was significantly decreased when the cells were treated with MβCD and amiloride, suggesting that the endocytosis process of Au NCs-INPs through caveolae-mediated endocytosis and micropinocytosis. Moreover, the higher uptake of Au NCs-INPs also was further demonstrated by flow cytometry analysis. In comparison to ICG and Au NCs-ICG, Au NCs-INPs exhibited 1.4 and 3.8 folds increasing in FL3-A (Au NCs, Figure 2B), and 7.7 and 2.2 times increasing in FL4-A (ICG, Figure 2D), which consistently revealed that Au NCs-INPs significantly improved the uptake ability of Au NCs and ICG. Moreover, as a dual-modal contrast agent, the Au NCs-INPs had excellent PA property, which was gradually enhanced with the increase of


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ICG concentration (Figure S9A-B). To evaluate the intracellular PA imaging, 4T1 cells treated with all suspensions (35.2 µg/mL of Au NCs and 63.2 µg/mL of ICG) for 4 h incubation, and then collected and dispersed in PBS. As shown in Figure 2C, the Au NCs-INPs showed higher PA intensity compared to Au NCs-ICG, which was determined by ROI for 1.6 times increasing (Figure S9C). It further demonstrated that Au NCs-INPs with highly effective cellular uptake was devoted to dual-modal NIRF and PA imaging in vitro. In vivo NIRF and PA imaging The 4T1 tumor-bearing nude mice were employed for imaging in vivo. The mice were intravenously injected all ICG-composited suspensions (2.37 mg/kg) when the volume of tumor attached to ~150 mm3. NIRF images were acquired at the different time intervals on IVIS spectrum imaging system by using a 710 nm excitation wavelength and a 730 nm filter. Mice with pre-injection were studied as control. As shown in Figure 3A, mice treated with Au NCs-INPs exhibited the gradual increase of fluorescence signals on tumor location with the prolonging of time after intravenous administration and a peak at 24 h post-injection. On the contrary, the fluorescence signals on tumor location showed slightly increasing of mice treated with Au NCs-ICG and completely disappeared of ICGtreated mice after 24 h post-injection. The semi-quantitative analysis by ROI indicated 3.3 times increasing of Au NCs-INPs treated mice on tumor location compared to Au NCs-ICG at 24 h post-injection (Figure 3B), suggesting Au NCs-INPs with the suitable size for the improved tumor targeting. To further investigate the bio-distribution of Au NCs-INPs, the major organs and tumors after 24 h post-injection were placed with the cushion for NIRF imaging ex vivo. As shown in Figure 3C, the fluorescence signals of


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Au NCs-ICG and Au NCs-INPs were mainly located in the liver, lung, kidney and tumor compared to ICG (without distribution). The two ICG-composited materials (Au NCsICG and Au NCs-INPs) were partly accumulated in liver and lung for the size of nanomaterials, and located in the kidney due to some of Au NCs-ICG depolymerized from Au NCs-INPs at the high concentration of GSH in tissues. But on tumor location, the Au NCs-INPs exhibited higher NIRF signals than that of Au NCs-ICG, which was semi-qualified by ROI for 7.4 times increasing (Figure 3D). In addition, the Au NCs-INPs as a dual-modal contrast agent showed excellent PA signals at 800 nm wavelength. To explore PA imaging in vivo, the mice were chose by the suitable volume of tumor with ~80 mm3, followed by the intravenously injection of ICG-composited materials (2.37 mg/kg). As shown in Figure 3E, before intravenous administration, all groups showed indistinct and unsharpened blood vessels from an endogenous contrast agent, hemoglobin. After 4 h post-injection, the PA signals exhibited slightly increasing of Au NCs-ICG and gradually decreasing of ICG. Comparatively, the Au NCs-INPs presented stronger PA signal than that of Au NCs-ICG and kept substantially increased intensity over time, which was verified by ROI determination (Figure 3F). The strongest PA intensity of Au NCs-INPs was attributed to the longcirculation time in blood and effective accumulation within tumor by EPR (enhanced permeability and retention) effect, which was consistent with NIRF imaging. Therefore, in vivo NIRF and PA imaging by Au NCs-INPs provided the identification of the location and morphology of tumor, which was sufficient to guide synergistic treatment and monitor the therapeutic approach in real time. ROS generation and photothermal efficiency of Au NCs-INPs


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Upon the NIR laser irradiation, the Au NCs-INPs presented ROS generation and heat radiation production for the combined PDT and PTT for synergistic phototherapy (Figure 4A). ROS levels were evaluated by the changes in fluorescence intensity of hydrogen peroxide and singlet oxygen probes. With prolonging the laser irradiation time, the fluorescence intensity of hydrogen peroxide (Figure 4B) and singlet oxygen probes (Figure 4C) generation from Au NCs-INPs gradually increased compare to Au NCs and ICG alone, suggesting that ROS was continuously generated from Au NCs-INPs. It demonstrated that the conjugation of Au NCs with ICG was beneficial to generate ROS. However, the Au NCs-INPs showed slightly increasing of singlet oxygen signals (Figure S10A) but significantly enhancement of hydrogen peroxide signals (Figure S10B) compared to Au NCs-ICG at 4 min post-irradiation. The ROS signals of Au NCs assemblies was measured under 808 nm laser and exhibited stronger intensity than that of Au NCs, indicating that the Au NCs assemblies was in favor of hydrogen peroxide generation compared to Au NCs for the electric structure and well-defined activity sites (Figure S11). It revealed that Au NCs-INPs had improved ROS levels under NIR irradiation for PDT of tumor. Additionally, upon 808 nm laser irradiation, the temperature of ICG-composited materials gradually increased with the prolonging of irradiation time, which was performed by an infrared thermal imaging camera (Figure S12). As shown in Figure 4D, the Au NCs-INPs exhibited higher temperature than that of Au NCs-ICG and ICG after 4 min irradiation. The temperature of Au NCs-INPs increased to 68.2 °C, while PBS only increased to 25.1 °C (Figure 4E). It suggested that Au NCs-INPs with high photothermal response was employed for PTT of tumor due to the generation of irreversible damages of cancer cells and even tumor. Accompanied with


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ROS and heat radiation generated from Au NCs-INPs, it demonstrated the therapeutic strategy of synergistic PDT-PTT for enhanced treatment efficiency. In vitro simultaneous phototherapy To visually assess the phototherapeutic response of Au NCs-INPs in vitro, 4T1 cells were performed by staining calcein-AM and propidium iodide (PI) to distinguish viable cells (green fluorescence) and dead cells (red fluorescence), respectively. As shown in Figure 5A, upon the NIR laser irradiation, 4T1 cells treated with Au NCs-INPs (63.2 µg/mL of ICG) showed significantly red fluorescence while PBS with green fluorescence, suggesting that NIR laser alone could not kill cancer cells. To explore PDT alone, 4T1 cells were placed on ice in the NIR laser irradiation process to maintain a constant temperature for avoiding the effects from PTT. Compared to ICG, the Au NCs-ICG and Au NCs-INPs had a wide of red fluorescence, which indicated the cell apoptosis triggered by PDT. The similar treatment effect of Au NCs-ICG and Au NCs-INPs was attributed to the higher killing efficiency of singlet oxygen than that of hydrogen peroxide. Moreover, NaN3 (100 mM) as a specific ROS inhibitor was employed to investigate the PTT alone. 4T1 cells treated with ICG-composited materials showed more robust red fluorescence than that of PTT alone, suggesting that the high temperature easily caused cells death compared to ROS. 4T1 cells treated with ICG-composited materials were directly irradiated to explore the combined PDT-PTT. After 4 min irradiation, Au NCs-INPs treated cells all displayed red fluorescence, suggesting that the synergistic phototherapy (PDT-PTT) exhibited more noble cells killing efficiency than that of PDT or PTT alone. In addition, MTT assays also demonstrated the consistent phototherapy efficiency of cancer cells. 4T1 cells treated all materials showed non-toxicity without laser irradiation.


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After 4 min irradiation, the cells treated with Au NCs-INPs had 95% cells killing efficiency of PDT-PTT (Figure 5D), which was higher than that of PDT alone (40%) (Figure 5B) or PTT alone (70 %) (Figure 5C), suggesting that the synergetic PDT-PTT had excellent killing effects on tumor cells. In all treatment process, the Au NCs-INPs exhibited the higher cells killing than that of Au NCs-ICG, which was attributed to the highly effective cellular uptake, significantly ROS generation and obviously heat production, which indicated that the synergistic PDT-PTT of Au NCs-INPs exhibited the improved treatment efficiency of 4T1 cells. In vivo synergetic phototherapy of PDT-PTT To directly visualize the phototherapeutic process of cancer, thermal images were captured with an infrared thermal imaging camera. 4T1 tumor-bearing mice were treated intravenously injected with ICG-composited materials (3.16 mg/kg), followed by recording the temperature changes at 30 s intervals under 808 nm laser irradiation (0.8 W/cm2) after 24 h post-injection. As shown in Figure 6A, the temperature located on tumor location of ICG-composited materials treated mice showed obviously increasing with the prolonging of irradiation time. On the contrary, the control group (PBS treated mice) had slightly enhanced of temperature. And the temperature of Au NCs-INPs treated mice increased to 53.1 °C at 5 min irradiation while Au NCs-ICG to 48.1 °C (Figure 6B), suggesting Au NCs-INPs upon NIR irradiation generated the obvious temperature increment for tumor removal. Tumor sections also demonstrated that Au NCs-INPs treated mice exhibited largely tissues necrotic and karyolysis (marked as black arrows) in comparison to Au NCs-ICG (Figure 6C), suggesting that Au NCs-ICG had insufficient to cause tumor ablation. During the treatment process, the body weight and tumor volume


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were measured at 2-3 days intervals. After post-treatment, the weight of mice treated with Au NCs-INPs gradually decreased from day 1 post-treatment to day 10 post-treatment and recovered due to the tumor suppression and autoimmunity, which was different from the control groups without irradiation. But on day 23 post-treatment, the weight increased about 4% of Au NCs-ICG treated mice and 2% of Au NCs-INPs treated mice compared to 6% of control groups. There was not significantly difference for Au NCs-INPs with or without treatment, suggesting the mice treated with Au NCs-INPs had well-tolerated with NIR laser irradiation (Figure 6D). Moreover, H&E stained results revealed that the sections from the major tissue of mice treated with PBS and Au NCs-INPs had not significantly tissues damages and inflammation, suggesting that Au NCs-INPs showed low cytotoxicity and good biosafety (Figure S13). When the volume of tumor attached to 500 cm3, mice were settled as death. As shown in Figure 6E, the mice treated with Au NCs-INPs exhibited significantly tumor removal effects after 5 min irradiation. On the contrary, Au NCs-ICG-treated mice showed decreasing of tumor volume on day 5 posttreatment, but continuously growing in the following days. As shown in Figure 6F, the mice treated with Au NCs-INPs showed 100% survival rate, which revealed that the tumor growth after treatment was completely suppressed and not relapsed. However, the survival rate of Au NCs-ICG and ICG-treated mice on day 23 post-treatment decreased to 80% and 40%, respectively. At this time point (day 23 post-treatment), the tumor was completely removed of Au NCs-INPs group, limitedly suppressed of Au NCs-ICG group and existed of ICG and control groups (Figure S14). It suggested that Au NCs-INPs presented the synchronous therapy from the enhanced PTT from the PDT-triggered vasoconstriction and the increased PDT from the PTT-improved drug penetration. The


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results demonstrated that Au NCs-INPs with low toxicity exhibited the improved treatment efficiency by guiding with imaging. FRET process between Au NCs and ICG As one of fluorescent resonance energy transfer (FRET) probes, Au NCs-INPs exhibited two completely separated fluorescent emission wavelengths at 476 nm excitation, where Au NCs was employed as a donor to ICG (an acceptor) (Figure 7A). To explore the FRET in Au NCs-INPs, ICG loaded Au NCs was prepared by the directly conjugation of Au NCs with ICG, and showed slightly increasing compared to ICG (Figure S15A). To verify the mechanism of FRET, the Au NCs emission and ICG absorption spectra were normalized, where Au NCs emission significantly overlapped with the ICG absorption (oblique line parts). It revealed high possibility of energy transfer between Au NCs and ICG. Compared to Au NCs and ICG alone, quenching of the Au NCs fluorescence and enhancement of the ICG fluorescence occurred (~50% change), as shown in Figure S15B. The mean fluorescent lifetime was measured for evaluating the FRET efficiency. All materials were excited at 476 nm and emission was observed at 660 nm. The average lifetime was 15.4 µs of Au NCs alone and decreased to 11.7 µs at the presence of ICG (Figure 7C). The FRET efficiency is 24% as calculated by using average lifetime data. The fluorescence alteration could be visualized by NIRF imaging with CRi MaestroTM imaging system. With adding ICG to Au NCs suspension, the fluorescence gradually increased of ICG (brown) and decreased of Au NCs (red) (Figure 7D). After that, to further assess the fluorescence varying in the treatment process, ICG-loaded Au NCs was exposed at 808 nm laser (0.2 W/cm2). With the prolonging of irradiation time, the ICG fluorescence quenched while Au NCs recovered


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(Figure 7E). The presence and deficiency of ICG resulted in the Au NCs fluorescence decreasing and recovery, suggesting the tragedy for monitoring the therapeutic process by the observation of changes in fluorescence intensity. In vitro and vivo therapeutic monitoring To further explore the therapeutic monitoring in vitro and vivo, NIRF imaging was investigated and performed by irradiating with 808 nm laser (0.2 W/cm2). As shown in Figure 8A, the green and red fluorescence were observed in 4T1 cells after 4 h incubation of Au NCs-ICG suspension. After 10 min irradiation, the red fluorescence (ICG) gradually decreased and green fluorescence (Au NCs) increased, suggesting the Au NCs fluorescence was recovered with ICG fluorescence quenching. The mean intensity showed 96% decreasing of ICG and 1.5 times increasing of Au NCs (Figure 8B). And the fluorescence of Au NCs exhibited slightly change at the presence of ROS, heating and laser irradiation (Figure S16), which eliminated the effects of photochemistry and photothermal reaction of ICG when exposed on 808 nm laser and verified the fluorescence changes from FRET between them. Moreover, the FRET efficiency also was performed by confocal fluorescence microscopy, which indicated the fluorescence alteration located into the same region of treatment (Figure 8C). The fluorescence increased 3.3 times of Au NCs accompanied with 60% decreasing of ICG (Figure 8D). It demonstrated that the synergistic fluorescence varying directly attributed to FRET between Au NCs and ICG, suggesting the therapeutic monitoring in vitro. After that, 4T1 tumor-bearing nude mice (~150 mm3) were employed to evaluate the therapeutic monitoring in vivo by NIRF imaging. After 24 h post-injection, Au NCs-INPs treated mice were irradiated with 808 nm laser for 30 min (0.2 W/cm2). Compared to pre-


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treatment, the fluorescence signals on tumor location decreased of ICG and recovered of Au NCs after 30 min treatment (Figure 8E). The fluorescence intensity was determined by ROI for 2.2 folds increasing of Au NCs and 1.6 times decreasing of ICG (Figure 8F). Thus, ICG contents could be monitored by the observation of changes in Au NCs fluorescence signals, which further revealed the process of therapeutic response. In vitro and vivo FRET imaging indicated that Au NCs-INPs as a detected agent could be employed to monitor the therapeutic process in real time. CONCLUSION The real-time monitoring strategy has been developed by using FRET properties between Au NCs and ICG. To improve the fluorescence of ICG, the Au NCs assemblies were employed for the encapsulation of ICG by the non-covalently bonding process. The as-synthesized Au NCs-INPs with low toxicity highly effective cellular uptake and precise tumor targeting due to the EPR effect and the gp60-mediated SPARC combined transport pathway, which further realized dual-modal NIRF/PA imaging in vitro and vivo. Compared the efficiency of PDT or PTT alone, the synergistic PDT-PTT of Au NCsINPs resulted in 95% cell killing and completely tumor removal. It demonstrated that ICG encapsulated into Au NCs-INPs make it possible for imaging-guided therapy. To monitor the therapeutic response, we investigated the strategy by the observation of fluorescence alteration based on 24% FRET efficiency between Au NCs and ICG. With the advancement of therapy, the fluorescence intensity of ICG was gradually decreased together with suspension. At this moment, the fluorescence of Au NCs recovered and showed 1.5 times increasing accompanied with 96% decreasing of ICG. Thus, the fluorescence of Au NCs quenched before treatment and then gradually recovered with the


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advancement of treatment, suggesting that the FRET properties realized the real-time monitoring of phototherapy in vitro and vivo. In summary, the biocompatible Au NCsINPs exhibited great potentials in dual-modal NIRF/PA imaging, synchronous cancer treatment and real-time therapeutic monitoring based on FRET. MATERIALS AND METHODS Materials Bovine serum albumin (BSA) and L-glutathione reduced (GSH) were purchased from BIOSHARP. Propidium iodide (PI), Phosphotungstic acid hydrate, 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Hoechst 33258, Sodium azide (NaN3), Hematoxylin, Indocyanine green (ICG) and 2’,7’-Dichlorofluorescin diacetate (DCFDA) were obtained from Sigma-Aldrich. Deuterium oxide (D2O) was bought from J&K Scientific. Singlet Oxygen Sensor Green (SOSG) was purchased from Thermo Scientific. Pentobarbital sodium was bought from Merck (Germany). Chloroauric acid hydrated (HAuCl4)，Sodium hydroxide (NaOH), Sucrose, Methanol, Xylene, Dimethyl sulfoxide (DMSO) and Ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Methyl-β-cyclodextrin (MβCD) and amiloride were obtained from Aladdin Industrial Corporation (Shanghai, China). Phosphate-buffered saline (PBS, pH 7.4), Fetal bovine serum (FBS), DMEM, Penicillin-streptomycin and trypsin-EDTA were purchased from Gibco Life Technologies (Switzerland). Isoflurane was obtained from RWD life science (Shenzhen, China). 7,000 MW dialysis bag was bought from Huashi science (Shenzhen, China). 4% parafoemaldehyde (4% PFA) was obtained from Boster (Wuhan, China). Neutral balsam was purchased from Solarbio life science (Beijing, China). Ultrathin Carbon Film on Lacey Formvar/Carbon on 200 Mesh (Copper)


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was obtained from Beijing Zhongjingkeyi Technology Co., Ltd (Beijing, China). Tissue OCT-freeze medium was bought from Leica (Germany). All other chemicals used in this study were of analytical reagent grade and used without further purification. Ultrapure water (18.25 MΩ.cm, 25 °C) was used to prepare all solutions. Preparation of Au NCs-ICG and Au NCs-INPs Gold nanoclusters (Au NCs) were prepared by a typical protocol in the previous research.22 Briefly, 5 mL 10 mM HAucl4 solution was added into 5 mL 50 mg/ml BSA under vigorously stirring at room temperature for 2 min. 0.5 mL 1 mM NaOH was introduced with vigorously stirring 1 min followed with lightproof package for 12 hours at 37 °C. After that, the prepared suspension would be preserved at 4 °C for the following experiments. ICG was encapsulated to Au NCs by the non-covalently binding process.21 Au NCsICG was prepared by adding 2 mL 2 mg/mL ICG aqueous solution to 1 mL Au NCs suspensions under vigorous magnetic stirring at room temperature for 1 h. After that, to remove the excessive ICG, the solution was dialyzed by ultrapure water using dialysis membrane (7,000 MW) for 3 hours. The as-synthesized Au NCs-ICG solution was stored at 4 °C with lightproof package. In the same way, Au NCs-INPs was constructed by referring to the preparation method of ICG loaded albumin nanoparticles.20 1 mL 200 mM GSH aqueous solution was added to 1 mL Au NCs suspensions under vigorous magnetic stirring at room temperature for 1 h, followed by introducing 2 mL 2 mg/mL ICG ethanol solution to keeping reaction 1 h. The excessive ICG was dialyzed against ultrapure water using dialysis membrane (7,000 MW) for 9 hours prior to remove partial


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ethanol under the vacuum condition. The final solution was also persevered into 4 °C with lightproof package. Characterization of Au NCs-ICG and Au NCs-INPs The as-synthesized Au NCs-ICG and Au NCs-INPs were characterized with size, morphology and optical properties. The morphology was measured with transmission electron microscope (TEM) (Tecnai G2 F20, USA). TEM samples were negatively stained with 2% (W/V) phosphotungstic acid hydrate due to BSA scaffolds. One droplet of solution was dropped onto an ultrathin carbon film on a 200 mesh copper. After air drying of the droplet on mesh, the phosphotungstic acid hydrate solution was dropped onto it for fewer seconds followed by penetrating from mesh and natural drying. The hydrodynamic diameter and zeta potentials were measured at room temperature with Zetasizer (Malvern Zetasizer Nano ZS, UK). To evaluate the stability of materials, the materials dispersed in ultrapure water were measured at the determined time interval. The photographs of materials were obtained by a digital camera under visible light. The optical properties of materials were evaluated by the fluorescent spectra with fluorescence spectroscopy (Edinburgh Instruments F900, UK) (excitation 476 nm for Au NCs and 760 nm for ICG) and UV-Vis spectrophotometer (Pekin Elmer Lambda25, USA), respectively. All optical measurements were performed at room temperature. And fluorescence quantum yields (QY) was detected by the formula (1). ∅ = ∅ ∙ ⁄ ∙ ⁄ ∙ ⁄ 1 where S is the comparison standard and X is the samples need to test, ∅ is the fluorescence quantum yields, n is the refractive indices of media, A is the absorbance at


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the excitation wavelength and F is the integrated fluorescence intensity. The comparison standard was settled as rhodamine B in ethanol for Au NCs (476 nm excitation) and ICG in DMSO for ICG (760 nm excitation). To confirm the concentration of Au NCs-ICG and Au NCs-INPs, gold contents were measured by inductively coupled plasma mass spectrometry (ICP-MS) (Pekin Elmer OPTIMA 7000DV, USA). Moreover, ICG loading capacity (LC) was detected by using the formula (2), and encapsulation efficiency (EE) was measured by using the formula (3). LC% = ⁄ × 100 2 EE% = ⁄ × 100 3 where WICG is the weight of ICG in the nanoprobes, WT is the total weight of the nanoprobes and WI is the weight of ICG initially adding. Measurement of ROS generation ROS generation was detected by using DCFDA probe and SOSG probe, respectively. ROS was generated by photochemical reaction triggered by 808 nm laser (Laserwave LWIRL808nm, Beijing). The power density was tested by an optical power meter (Laserwave LI-P20W, Beijing) to be 0.8 W/cm2 for irradiation. DCFDA and SOSG as fluorescent probes were highly sensitive for hydrogen peroxide and singlet oxygen, respectively. 4.85 mg of DCFDA was dissolved in 1 mL DMSO to make a stock solution of approximately 10 mM, which stored at -20 °C. In H2O2-sensing experiment, 1 µM DCFDA was added into 600 µL of all suspensions (17.6 µg/mL as Au and 31.6 µg/mL as ICG). The generated hydrogen peroxide was determined by detecting the recovered fluorescence of DCFDA with the prolonging of laser irradiation by using fluorescence spectrometer (Pekin Elmer LS55, USA) (494 nm excitation). Similarly, the stock solution


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of SOSG (5 mM) was prepared by dissolving one 100 µg vial of SOSG in 33 µL of methanol. 6 µM SOSG was dispersed in D2O solution containing all suspensions for monitoring the level of singlet oxygen when exposed to 808 nm laser, which determined by the enhanced SOSG intensity (393 nm excitation). Evaluation of photothermal efficiency The photothermal efficiency was assessed by the temperature changes at 808 nm laser irradiation. 700 µL of all tested agents (17.6 µg/mL as Au and 31.6 µg/mL as ICG) was placed in 1 mL cuvettes for thermal imaging, which was recorded at 30 s intervals by an infrared thermal imaging camera (Fluke Ti27, USA). PBS was settled as control groups. The output power was calibrated using an optical power meter (Laserwave LI-P20W, Beijing) to be 0.8 W/cm2. FRET properties After ICG non-covalently binding to Au NCs, FRET effect between them brought about the fluorescent alternation. To evaluate the FRET efficacy of Au NCs and ICG, 0.5 mL 1 mg/mL ICG aqueous solution was added into Au NCs solution with vigorous stirring 1 h at room temperature. The obtained ICG-loaded Au NCs solution was stored at 4 °C with lightproof package for the following experiment. To calculate FRET efficiency, the mean fluorescence time of Au NCs, ICG and ICG-loaded Au NCs was measured by fluorescence spectroscopy (Edinburgh Instruments F900, UK). ICG-loaded Au NCs was detected with 476 nm excitation and 660 nm observation wavelength. FRET efficiency was calculated by using the formula (4). FRET efficiency = 1 − &' ⁄&( × 100 4


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where τA is the mean fluorescent lifetime of donor at the presence of acceptor and τB is the mean fluorescent lifetime of donor at the absence of acceptor. The fluorescence alteration of ICG-loaded Au NCs was imaged by fluorescence imaging system (CRi MaestroTM, USA) with different concentration radio. Furthermore, when exposed to 808 nm laser (0.2 W/cm2), the fluorescent intensity of ICG was changed with the consumption and supersession, which further led to the fluorescent varying of Au NCs. According to this fluorescent alteration, the new strategy to monitor the therapeutic efficacy was performed by FRET between Au NCs and ICG. Cellular culture The mouse cell lines 4T1 (breast tumor) and the human cell lines 293T (embryonic kidney) were purchased from Cellbank (Shanghai, China). All were cultured at 37 °C 5% carbon dioxide cell incubator with Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cellular viability MTT assays were employed to calculate cellular viability. These cells were cultured on 96 well plates (5×103 cells per well) overnight. Subsequently, 200 µL of Au NCs, ICG, Au NCs-ICG and Au NCs-INPs suspensions (35.2 µg/mL as Au and 63.2 µg/mL as ICG) in 10% FBS DMEM prior to 0.45 µm filtration were diluted with different concentration and then incubated on 96 well plates for 24 h. After washing with PBS, 200 µL 0.5 mg/mL MTT solution in 10% FBS DMEM was added into each well. After 4 h incubation, MTT solution was removed and the cells were washed by PBS followed by adding 200 µL of DMSO for 10 min shaking. The value of OD490nm was measured with


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ELISA Reader (Thermo Scientific Multiskan GO, USA) for calculating cellular viability by the formula (5). Cell viability% = / − 0 ⁄ − 0 × 100 5 where AE is the mean absorbance of experiment group, AC is the mean absorbance of control group and AB is the mean absorbance of black group. On the whole steps, the blank (only cells) and control well (cells with PBS) need to settle. In vitro NIRF/ PA imaging In vitro NIRF images were acquired by confocal laser scanning microscope (Leica TCS SP5-II, Germany), which also was employed to evaluate cellular uptake of Au NCsINPs. 4T1 cells (5×103 per well) were inoculated into 8 well plates and then cultured overnight at 37 °C. And then, 200 µL of Au NCs, ICG, Au NCs-ICG and Au NCs-INPs suspensions (35.2 µg/mL as Au and 63.2 µg/mL as ICG) in 10% FBS DMEM with 0.45 µm filtration was placed with each well for 4 h incubation at 37 °C. After washing by PBS, 4% PFA was added in each well for 10 min incubation at 37 °C, followed by washing with PBS and staining nucleus dyes (hoechst 33258, 10 µg/mL) about 5 min at 37 °C. After incubation, the stained solution was removed and the cells were washed 2-3 times in PBS. Three channels were chose for NIRF imaging with 405 nm of hoechst 33258, 488 nm of Au NCs and 633 nm of ICG, respectively. Additionally, the cellular uptake was also quantitatively determined by flow cytometry (BD Accuir C6, USA). 4T1 cells were cultured on a 48 well plate (1×104 cells per well) at 37 °C overnight. The same concentration of all suspensions was placed with each well for 4 h incubation. After that, cells were rinsed with PBS three times and digested with trypsin–EDTA solution, followed by resuspension with 1 mL PBS and the measurement of flow cytometry (FL-


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3A for Au NCs and FL-4A for ICG). All data were analysed by using BD CSampler Software. Moreover, the mechanism of cellular uptake was probed by pre-treating of methyl-β-cyclodextrin (MβCD), sucrose, amiloride and low temperature for 0.5 h, followed by adding all suspensions (63.2 µg/mL) for 2 h incubation to take the fluorescence images. To further quantify the intracellular fluorescence intensity, all cells were collected and lysed with RIPA lysis buffer for 0.5 h on ice, and the fluorescence intensity was measured on fluorescence spectrophotometer. The endocytosis percentage was calculated according to the formula (6). Endocytosis% = F⁄ 5 × 100 6 where F is the fluorescence intensity in the presence of inhibitors, and F0 is the fluorescence intensity of the control group. To further assess PA imaging of Au NCs-INPs in vitro 4T1 cells were inoculated and cultured on a 6 well cellular plate (1×104 cells per well) overnight. After that, 500 µL of all suspensions as the same concentration as previous experiment were incubated at 37 °C 5% carbon dioxide cell incubator. After 4 h incubation, the cells were treated with trypsin–EDTA solution followed by centrifugation with 1,000 rpm for 4 min. The cells were collected and re-dispersed by 50 µL PBS solution. PA images were acquired by PA imaging system (Endra Life Sciences NEXUS 128, USA) (800 nm wavelength, 10 angles, 20 pulses, ~8 mJ/pulse). The suspension of cells was dropped into a thin membrane and floated in a dimple (filled with water) of a hemi-spherical bowl. The PA signals of materials were also measured by the same way. The computed tomography system was employed for calculating the intensities of PA signals at 800 nm. In vitro simultaneous phototherapy


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To evaluate the phtotherapeutic efficiency in vitro, cellular viability under NIR irradiation was performed by MTT assays. All suspensions were incubated for 24 h at 37 °C. After that, the cells were exposed to 808 nm laser at the power intensity of 0.8 W/cm2. With different irradiated time, the fresh 10% FBS DMEM was placed with each well for further 4 h incubation, followed by MTT assays to calculating cellular viability. In the process of laser irradiation, PDT alone was verified by keeping 4 °C for avoiding heat production, and PTT alone was performed by treating cells with 100 mM NaN3 to avoid ROS generation. Moreover, calcein-AM/PI staining also displayed the phototherapy efficiency. 4T1 cells were cultured on 48 well plates (1×104 cells per well) overnight. All suspensions were incubated 24 h at 37 °C. After 4 h incubation prior to 4 min irradiation by using 808 nm laser, the cells was rinsed with PBS and stained with calcein-AM/PI dyes (2 µg/mL calcein-AM, 3 µg/mL PI) about 8-10 min at 37 °C. The supernatant was removed and washed by PBS. The viable or dead cells were observed by fluorescence inverted microscope (OlympusIX71, Japan) with 488 nm excitation of calcin-AM and 535 nm of PI. The efficiency of PDT or PTT alone was also performed by previous experiment. On the whole steps, the control wells (without irradiation) were settled. In vitro FRET efficiency FRET in vitro was illustrated by fluorescent imaging. 4T1 cells (5×104 per well) was inoculated into 1×1 cm2 cover glass fixed into 6 well plates and cultured at 37 °C overnight followed by the incubation of Au NCs-ICG suspension 4 h. After that, the cover glass with cells was transferred into the slide for observing with fluorescence inverted microscope (OlympusIX71, Japan). And then, the cells were irradiated by 808


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nm laser (0.2 W/cm2) for 10 min for detecting the fluorescence alteration of Au NCs and ICG. Moreover, confocal laser scanning microscope (Leica TCS SP5-II, Germany) was employed to clearly observe the fluorescence varying of Au NCs-ICG after NIR irradiation. 4T1 cells (5×103 per well) were inoculated into 8 well plates and then cultured overnight at 37 °C. After that, 200 µL Au NCs-ICG suspension was placed with each well. After 4 h incubation, the cells was rinsed by PBS and further observed the fluorescence of Au NCs and ICG. Subsequently, the cells were exposed to 808 nm laser (0.2 W/cm2) for 10 min to monitoring the fluorescence varying of Au NCs and ICG. Mouse modal Female BALB/c mice and athymic nude mice were obtained from Charles River Laboratories (Beijing, China) and maintained under Specific Pathogen Free (SPF) conditions in a small animal isolator. All food, water, bedding and cages were autoclaved before use. We used 4T1 tumor-bearing nude mice for in vivo imaging, cancer treatment and therapeutic monitoring. 4T1 cells were magnified incubating at 37 °C 5% carbon dioxide cell incubator. After that, cells (about 1×106 cells) were rinsed with PBS three times and digested with trypsin–EDTA solution, followed by resuspension in 100 µL icecold PBS solution, which was subcutaneously injected into on the flank region of mice. Tumor volume was calculated as the formula (7). Tumor volume = : × ⁄2 7 where The tumor volume was estimated using the formula, LT is the length of tumor and WT is the width of tumor. In vivo NIRF/PA imaging


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4T1 tumor-bearing nude mice were randomly divided into three groups (two of each group) when the volume of tumor attached to ~150 mm3. 150 µL of ICG, Au NCs-ICG and Au NCs-INPs suspensions (2.37 mg/kg) were injected via caudal vein after anaesthetizing mice with 1% (W/V) pentobarbital sodium (6-8 µL/g). NIRF images were detected at 0, 1, 4, 7, 24 h after injection by using an optical Imaging System (IVIS Spectrum, USA) with 710 nm excitation and 730 nm filter. After that, the mice after 24 h post-injection were sacrificed and collected the isolated mouse organs for the evaluation of bio-distribution of ICG-composited materials. The mean intensity of fluorescence signals were calculated by using the ROI of each image. To assess PA imaging in vivo, 4T1 tumor-bearing nude mice were chose with the volume of tumor about ~80 mm3, which was suitable in PA imaging system (Endra Life Sciences NEXUS 128, USA). As the same as previous NIRF imaging, 150 µL of ICG, Au NCs-ICG and Au NCs-INPs suspensions (2.37 mg/kg) were injected via caudal vein after anaesthetizing mice with 1% (W/V) pentobarbital sodium (6-8 µL/g). PA images were taken at 0, 1, 4, 7, 24 h after injection with 800 nm wavelength at 10 angles, 20 pulses and approximately 8 mJ/pulse. When the temperature of water in transducer bowl was attached to 38 °C, the tumor was put into a dimple (filled with water) of a hemispherical bowl to acquire PA images by using the computed tomography system. The mean PA intensity was measured by using the ROI of each image. In vivo synergetic phototherapy Mice bearing 4T1 tumors were randomly divided into five groups (six per group) when the volume of tumor attached to ~80 mm3. 200 µL of ICG, Au NCs-ICG and Au NCs-INPs suspensions (3.16 mg/kg) were injected via caudal vein after anaesthetizing


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mice with 1% (W/V) pentobarbital sodium (6-8 µL/g). Mice treated with 200 µL PBS were studied as control group. After 24 h post-injection, the tumor location was exposed to 808 nm laser at the power intensity of 0.8 W/cm2 for 5 min. Meanwhile, the increased temperature and thermal imaging were detected 30 s time intervals by an infrared thermal imaging camera (Fluke Ti27, USA). To assess the phototherapy efficiency and bio-safety, one mouse in each group was euthanized for preparing tissue frozen sections from tumors and other major organs, which were observed by using hematoxylin and eosin (H&E) staining. The steps of H&E staining were as follows: fresh tissue (excluding lung and tumor) was embedded into tissue OCT-freeze medium at -80 °C for 10-20 min, which immediately was prepared as frozen sections about 6-8 µm followed by fixing with methanol for 10 s. Tumor and lung were firstly fixed by 4% PFA (overnight) and dehydrating with 10%-30% (W/V) Sucrose (disperse in PBS), which further was embedded into tissue OCT-freeze medium at -80 °C overnight for preparing the slices. The H&E stained slices were re-dehydrated with 75% ethanol, followed by covering with 50-100% (V/V) Xylene (disperse in ethanol) and sealing with neutral balsam for putting blank condition overnight. The photograph of tissue slice was captured with upright microscope (Olympus BX56, Japan). The other mice were employed to evaluate the phototherapeautic effects by the alteration of body weight (Mettler Toledo AB265-S analytical balance, Switzerland) and the tumor volume. In vivo therapeutic monitoring based on FRET The therapeutic response based FRET was monitored by using NIRF imaging. 4T1 tumor-bearing nude mice with the volume of tumor about ~150 mm3 were treated with 150 µL of Au NCs-INPs suspensions (2.37 mg/kg) via caudal vein. After 24 h post-


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injection, NIRF images were captured by using an optical Imaging System (IVIS Spectrum, USA) with 465 nm excitation of Au NCs and 710 nm of ICG. Subsequently, the tumor location of mice was exposed to 808 nm laser at the power intensity of 0.2 W/cm2 for 30 min to monitoring the fluorescence alteration after treatment.

ASSOCIATED CONTENT Supporting information (TEM images of Au NCs, quantum yields, ICP-MS, size and fluorescence stability, cell viability, the mechanism of cellular uptake, ROS and thermal measurement, H&E staining and Tumor photographs of Au NCs-ICG and Au NCs-INPs ) is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION *Correspondence and requests for materials should be addressed to Z. Sheng ([email protected]) and L. Cai ([email protected])

ACKNOWLEDGEMENTS The authors gratefully acknowledge support for this research from the Major State Basic Research Development Program of China (973 Program) (2015CB755500), Natural Science Foundation of China (81571745, 81401521, 81327801), the Science and Technology

Innovation

Fund

of

Shenzhen

(JCYJ20150401145529015,

JCYJ20160229200902680).

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FIGURE CAPTIONS Figure 1. Synthesis and characterization of Au NCs-INPs. (A) Schematic illustration of the prepared process of Au NCs-INPs. Au NCs, disulfide bonds and ICG were represented by red, black and brown respectively. (B) TEM images of Au NCs-INPs by negatively staining 2% phosphotungstic acid. The inset shows the typical enlarged TEM images of separated Au NCs-INPs. The scale bar indicates 25 nm. (C) Size distribution of Au NCs-INPs (orange). The inset indicates the photograph of Au NCs-INPs aqueous solution. (D) Absorbance spectra of Au NCs (black), ICG (orange) and Au NCs-INPs (purple). (E) The fluorescent emission spectra of Au NCs (black), ICG (orange) and Au NCs-INPs (purple) (λex = 476 nm). Figure 2. In vitro NIRF and PA imaging. (A) Confocal fluorescence imaging of 4T1 cells treated with Au NCs, ICG, Au NCs-ICG and Au NCs-INPs for 4 h incubation. The scale indicates 10 µm. Blue, green and red fluorescence images show nuclear staining with Hoechst 33258, Au NCs and ICG, respectively. (B, D) Flow cytometry analysis of fluorescence intensity detected under FL3-A (Au NCs) and FL4-A (ICG), respectively. 4T1 cells treated with media (black), Au NCs (red), ICG (purple), Au NCs-ICG (green) and Au NCs-INPs (wine) for 4 h. (C) In vitro PA imaging of 4T1 cell treated with Au NCs, ICG, Au NCs-ICG and Au NCs-INPs for 4 h. (CAu NCs = 35.2 µg/mL, CICG = 63.2 µg/mL) Figure 3. In vivo NIRF and PA imaging. (A) In vivo fluorescence images at different time intervals in 4T1 tumor-bearing nude mice intravenously injected with ICG, Au NCsICG and Au NCs-INPs. (B) The mean fluorescence intensity on tumor location quantified at indicated time points. (C) Ex vivo fluorescence images of major organs and tumors


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after 24 h post-injection. (D) Semi-quantitative bio-distribution of ICG, Au NCs-ICG and Au NCs-INPs in nude mice determined by the mean fluorescence intensity of major organs and tumor. (E) PA images of 4T1 tumor-bearing nude mice intravenously treated with ICG, Au NCs-ICG and Au NCs-INPs, respectively. (F) The mean PA intensity of tumor at 1, 4, 7, 24 post-injection, respectively. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 (IDICG = 2.37 mg/kg) Figure 4. ROS and local hyperthermia generation of Au NCs-INPs. (A) Schematic photographs of ROS generation and heat radiation of Au NCs-INPs exposed to 808 nm laser (0.8 W/cm2). (B-C)Time-dependent hydrogen peroxide (DCFDA probes, λex = 494 nm) and singlet oxygen (SOSG sensors, λex = 393 nm) generation under 808 nm laser irradiation of ICG (orange), Au NCs (magenta), Au NCs-ICG (purple) and Au NCs-INPs (gray), respectively (CICG = 31.6 µg/mL). (D) Thermal images of different materials irradiated with 808 nm laser for 4 min. (E) Time-dependent temperature changes of PBS (black), Au NCs (orange), ICG (magenta), Au NCs-ICG (purple) and Au NCs-INPs (gray) exposed to 808 nm laser. (CICG = 63.2 µg/mL) The data are shown as mean ± SD (n=3). (**) p < 0.01, (***) p < 0.001 Figure 5. In vitro phototherapy of 4T1 cancer cells. (A) Fluorescence images of 4T1 cells after PDT, PTT and simultaneous PDT-PTT treatments. 4T1 cells were exposed to 808 nm laser (0.8 W/cm2) for 4 min by treating with PBS, ICG Au NCs-ICG and Au NCsINPs for 24 h. Viable cells were stained green with calcein-AM, and dead cells were stained red with PI, respectively. The scale bar indicates 50 µm. (B-D) Quantitative detection of 4T1 cells viability following PDT alone, PTT alone and simultaneous PDT/PTT treatments. 4T1 cells were irradiated with 808 nm laser (0.8 W/cm2) at


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different time prior to 24 h incubating with PBS (orange), ICG (magenta), Au NCs-ICG (purple) and Au NCs-INPs (gray). The data are shown as mean ± SD (n=3). (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 (CICG = 63.2 µg/mL) Figure 6. In vivo cancer phototherapy of mice bearing 4T1 tumors. (A) Thermal images of 4T1 tumor-bearing mice irradiated with 808 nm laser for 5 min (0.8 W/cm2). (B) In vivo time-dependent temperature of 4T1 tumor-bearing mice treated with PBS (orange), ICG (magenta), Au NCs-ICG (purple) and Au NCs-INPs (gray), respectively. (C) H&E stained images of tumor sections collected from different groups of mice 24 h post treatment. (D) Body weights measured during the 24 day evaluation period in mice under different conditions. (E) Tumor growth curves of different groups in mice bearing 4T1 tumors. (F) Survival rates of 4T1 tumor-bearing mice after different treatments. The data are shown as mean ± SD (n=5). (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 (IDICG = 3.16 mg/kg) Figure 7. FRET between Au NCs and ICG. (A) Schematic illustration of FRET between Au NCs and ICG. (B) The normalized absorbance and fluorescence spectra of Au NCs and ICG. The oblique line parts indicate the FRET efficiency of Au NCs-ICG. (C) Time resolved fluorescence decay curves of Au NCs and ICG-loaded Au NCs (λex = 476 nm, λobs = 660 nm). (D) Fluorescence images of ICG (brown) and Au NCs (red) with different concentration ratio. (E) Fluorescence images of ICG and Au NCs with NIR laser irradiation at different time (0.2 W/cm2). (CAu NCs = 17.8 µg/mL, CICG = 31.6 µg/mL). Figure 8. In vitro and vivo real-time monitoring treatment process based on FRET. (A) Fluorescence microscopy imaging of 4T1 cells treated with Au NCs-ICG before and after therapy. The scale indicates 30 µm. (B) The fluorescence alteration of Au NCs-ICG in


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Hoechst 33258, ICG and Au NCs at pre-therapy and post-treatment. (C) Confocal fluorescence imaging of 4T1 cells treated with Au NCs-ICG before and after therapy. The scale indicates 7.5 µm. (D) The fluorescence alteration of Au NCs-ICG in Hoechst 33258, ICG and Au NCs at pre-therapy and post-treatment. (CAu NCs = 35.2 µg/mL, CICG = 63.2 µg/mL) (E) In vivo NIRF imaging of 4T1 tumor-bearing nude mice with intravenous injection of Au NCs-INPs after 24 h post-injection before and after therapy. (F) The mean fluorescence intensity around tumor quantified at pre-therapy and posttreatment. The photo treatment with 808 nm laser irradiation (0.2 W/cm2) was settled 10 min for cells and 30 min for tumor of mice, respectively. (IDICG = 2.37 mg/kg) (*) p < 0.05, (**) p < 0.01


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.


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Figure 6.


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Figure 7.


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Figure 8.


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Table of contents (TOC) graphic


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Gold Nanoclusters-Indocyanine Green Nanoprobes for Synchronous

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