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Cancer Diagnosis and Imaging-guided Photothermal Therapy Using a Dual-modality Nanoparticle Chaoting Zeng, Wenting Shang, Xiaoyuan Liang, Xiao Liang, Qingshan Chen, Chongwei Chi, Yang Du, Chihua Fang, and Jie Tian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06883 • Publication Date (Web): 12 Oct 2016 Downloaded from http://pubs.acs.org on October 13, 2016

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ACS Applied Materials & Interfaces

Cancer Diagnosis and Imaging-guided Photothermal Therapy Using a Dual-modality Nanoparticle Chaoting Zeng1,2‡, Wenting Shang1,3‡, Xiaoyuan Liang1,2, Xiao Liang1,3, Qingshan Chen1,2, Chongwei Chi1,3, Yang Du1.3, Chihua Fang2*, and Jie Tian1,3* 1

Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of

Sciences, Beijing 100190, China. 2

Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical

University, Guangzhou 510280, China. 3

Beijing Key Laboratory of Molecular Imaging, Beijing 100190, China.

‡

The parallel first authors

Mr. Chaoting Zeng, Dr. Wenting Shang. *

The parallel corresponding authors

Prof. Jie Tian, Ph.D., Fellow of IEEE, SPIE, IAMBE, AIMBE, IAPR President of the Chinese Society for Molecular Imaging Professor and Director of the Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences Professor and Director of Beijing Key Laboratory of Molecular Imaging Zhongguancun East Road #95, Haidian Dist. Beijing 100190, P. R. China Phone: +86 10 82618465; Fax: +86 10 62527995 Email: [email protected]

Prof. Chihua Fang Director of the Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, No. 253, Gongye Avenue, Guangzhou 510280, P.R. China Phone: +86 20 61643208 Email: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT To improve patient outcome and decrease overall health-care costs, highly sensitive and precise detection of a tumor is required for its accurate diagnosis and efficient therapy; however, this remains a challenge when using conventional single mode imaging. Here, we successfully designed a near-infrared ray (NIR)-response photothermal therapy (PTT) platform (Au@MSNs-ICG) for the location, diagnosis, and NIR/computer tomography (CT) bimodal imaging-guided PTT of tumor tissues, using gold (Au) nanospheres coated with indocyanine green (ICG)-loaded mesoporous silica nanoparticles (MSNs), which would have high sensitivity and precision. The nanoparticles (NPs) exhibited good monodispersity, fluorescence stability, biocompatibility, and NIR/CT signaling, and had a preferable temperature response under NIR laser irradiation in vitro or in vivo. Using a combination of NIR/CT imaging and PTT treatment, the tumor could be accurately positioned and thoroughly

eradicated

in

vivo

by

Au@MSNs-ICG

injection.

Hence,

the

multifunctional NPs could play an important role in facilitating the accurate treatment of tumors in future clinical applications. KEYWORD: Gold nanospheres; Indocyanine green; Computed tomography; NIR fluorescence imaging; photothermal therapy; Imaging-guided therapy.

2


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INTRODUCTION Cancer has become one of the major threats to human health in today’s society, leading of morbidity and mortality worldwide1-3. Tumors can be detected by many advanced techniques4-7, such as computer tomography (CT), magnetic resonance imaging (MRI), ultrasound (US), and positron emission tomography (PET). Once detected, tumors can be treated by surgery8-9, radiotherapy10, chemotherapy11-12, interventional therapy, or other methods13-14. However, the patients’ poor outcome is inevitable for the following reasons: (1) ineffective detection of localized or metastatic tumors < 1 cm, (2) positive surgical margins, (3) limited efficacy of radiotherapy, or (4) severe systemic side effects and limited toxic effects of chemotherapy. The sensitive and precise detection of tumors for accurate diagnosis and efficient therapy is also a challenge. Currently, nanomedicine has been developed to integrate multiple imaging approaches and therapeutic modalities for the precise theranostics of cancer15-19. Combining multimodality imaging-guided methods with photothermal therapy (PTT) to treat tumors with greater specificity and sensitivity. has received significant attention20-21. Recently, near-infrared ray (NIR) fluorescence equipment has been a powerful tool for guided diagnosis and therapy of tumors22-23. Fluorescence imaging offers low-cost, portable, non-ionizing radiation and key advantages (including real-time imaging, superior resolution, and high sensitivity)24. NIR fluorescence imaging (650 to 900 nm) has certain advantages over conventional optical imaging: deeper tissue penetration, lowest tissue absorption, and a higher signal-to-background ratio18, 25-26. It enables surgeons to confirm the tumor’s location and margins in real-time26. However, even though NIR fluorescent imaging has high sensitivity for diagnosing tumor tissues, it cannot distinguish between anatomical structures and has limited penetration depth (< 10 mm), which remains a barrier for its widespread application25. Because no single imaging modality is either perfect or adequate to obtain all the necessary information, combining NIR fluorescence imaging with another imaging modality would help to avoid the shortcomings of both techniques. CT is one of the most widely used clinical diagnostic tools; its advantages include a high resolution 3



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ratio, no depth limitations, and three-dimensional reconstruction27-29. However, soft tissues, such as many types of tumors, cannot be imaged clearly because of the intrinsic limitations of CT imaging30-31. Furthermore, small iodinated molecules, the current contrast agents for CT, are be rapidly cleared by the kidneys and may cause renal toxicity32-34. Therefore, CT is not ideal as the sole technique for tumor imaging and angiography. Instead, given that typical anatomical structures are visible through CT, combining it with NIR fluorescence imaging would result in precise diagnosis. The combination method would have the high sensitivity of NIR fluorescence imaging and the accurate anatomical-structure visualization of CT, for the location and imaging-guided treatment of tumor tissues. Indocyanine green (ICG) is one of the clinical NIR fluorescent agents approved by the U.S. Food and Drug Administration (FDA) as a clinical imaging diagnostic agent. ICG, which performs in the NIR fluorescent spectrum,

has minimized

auto-fluorescence and the lowest tissue absorption and light scatter35-36. ICG has been widely applied to assess hepatic function, ophthalmic angiography, and surgical navigation25, 37. ICG is also a good photosensitizer and absorbs NIR fluorescence to generate heat efficiently38-40. However, ICG has poor photostability in aqueous solution and rapid blood clearance (its plasma half-life is 2–4 min)16,

41

. These

shortcomings limit ICG use in PTT. With the development of nanotechnology, ICG has been incorporated into various nanoplatforms, thus to compensating for its drawbacks and enabling its application in PTT and imaging42. The materials with large atomic numbers (Ta, 73; Au, 79; Bi, 83) and X-ray attenuation coefficients (Ta, 4.30; Au, 5.16; Bi, 5.74 cm2g-1 at 100 keV) can therefore be used as CT contrast agents31, 34, 43-44. Among these, gold (Au) is non-toxic and has low osmolality and viscosity, even at high concentrations. It has been used in many biomedical research studies27,

34

. Compared with the commercially available

iodine-based contrast agents, Au has a higher atomic number, absorption coefficient, longer circulation time, and is easily modified at the surface, which is helpful for improving detection sensitivity45. Recently, some researchers used Au nanorods coated with mesoporous silica nanoparticles (MSNs) for CT imaging46-48, this, 4


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however, could not replace the intrinsic limitations of CT imaging. In contrast, some researchers used Au nanorods coated with ICG-loaded MSNs for CT and NIR imaging49-50. The emission spectrum of ICG and the excitation spectra of Au nanorods almost overlap, resulting in light quenching inside the probes. To overcome these shortcomings, some researchers use Au nanospheres (NSs) coated with quantum dot (QD)-loaded MSNs for CT and NIR imaging43; however, QDs are toxic. Another multifunctional probe has also been synthesized, using an up-conversion luminescent nanoparticle (NP) as the core with Au-NP doping on the surface43, 51; however, the special requirement of the equipment for up-conversion fluorescence imaging limited its application. Additionally, the gold NPs adsorbed the outer layer, which would also negatively affect fluorescence imaging. In this study, we designed a theranostic platform (Au@MSNs-ICG) that could perform NIR/CT bimodal imaging for accurate visualization of anatomical structures and tumor location, as well as real-time monitoring and guiding of PTT in vivo. Our platform, which was verified both in vitro and in vivo, is based on ICG and gold nanospheres. These Au@MSNs-ICG probes also provided powerful PTT for ablating tumors effectively. Hence, these methods offer a powerful theranostic platform for achieving multimodality imaging-guided PTT for precision cancer treatment. 2. MATERIALS AND METHODS 2.1 Materials Chloroauric acid (HAuCl4·4H2O), tetraethylorthosilicate (TEOS, ≥ 98%), cetyltrimethylammonium bromide (CTAB, ≥ 98%), 3-aminopropyltriethoxysilane (APTES, 99%), polyethylene glycol (5,000 MW), and sodium borohydride (NaBH4) were purchased from Alfa-Aesar (USA). Silver nitrate (AgNO3), sulfuric acid (H2SO4, 98%), and ascorbic acid were purchased from Sinopharm (China). ICG was purchased from Dandong Medical and Pharmaceutical Co. Ltd. (Dandong, China), which has been approved by the Chinese FDA for the production of ICG for clinical applications. All reagents used were of analytical grade unless specified otherwise. Millipore Milli-Q grade deionized water was used throughout. 2.2 Synthesis of Au NSs 5



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Nano-Au core NPs were prepared according to methods in previous reports52. Seed solution: CTAB (1.5 mL, 0.20 M) and HAuCl4 (5 mL, 0.5 mM) solutions were mixed in a round-bottom flask. Ice-cold NaBH4 (0.60 mL, 0.01 M) was added to the stirred solution, turning it brownish-yellow; the resulting solution was stirred for 3 min and kept at 25 °C for 2 h. Growth solution: CTAB (200 mL, 0.1 M), AgNO3 (2 mL, 0.01 M), and HAuCl4 (10 mL, 0.01 M) were mixed in a 250-mL flask and allowed to react for 3 min. Ascorbic acid (1.6 mL, 17.6 mg/mL) was added to the solution and stirred for 5 s, turning it colorless. Approximately 4 mL of H2SO4 solution was then slowly added to the solution and allowed to react for 30 min. Finally, 0.5 mL of the seed solution was added to the growth solution and kept at 30 °C for 12 h. 2.3 Synthesis and Characterization of the Au@MSNs-ICG The prepared gold NS solution was washed with ultrapure water and then centrifuged (10,000 rpm for 20 min) to remove the excess CTAB surfactant. The supernatant was then discarded and the precipitate was dispersed in 10 mL ultrapure water. NaOH (100 µL, 0.1 M) solution was added to the stirred solution, and three doses of 20% TEOS in ethanol (20 µL) were injected drop-by-drop at 30-min intervals. The shell thickness of the Au@MSNs was adjusted by adding appropriate amounts of TEOS. Then, the reaction was observed for 48 h. Subsequently, ethanol was added to the solution for demulsifying, the resulting solution was centrifuged (10,000 rpm for 20 min), and the supernatant decanted. The precipitates were washed three times and purified with ethanol. The precipitates were finally dispersed in 30 mL of ethanol. The Au@MSNs-ICG probes were synthesized by adding Au@MSNs (10 mg) to ICG

solution

(1

mL,

10

mg/mL)

and

vibrating

overnight.

Then

3-aminopropyltriethoxysilane (100 µL) was added, and the system was maintained at 80 °C for 2 h. After the suspension was centrifuged at 12,000 rpm for 25 min, the supernatant was decanted. The Au@MSNs-ICG product was collected and then re-dispersed in polyethylene glycol (5 mL, 1 mg/mL) overnight. After washing several times with water, the product was finally dispersed in water and protected 6


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from light. The morphology and size of the Au@MSNs-ICG were observed using transmission electron microscopy (TEM, JEOL-1011, Japan) in a 100-kV acceleration voltage. Dynamic light scattering (DLS) was used to measure the hydrodynamic particle size distribution of the Au@MSNs-ICG using a Malvern Zetasizer (ZEN 3600, UK). The Au@MSNs-ICG were incubated with phosphate-buffered saline (PBS), fetal bovine serum (FBS), or cell culture media for 7 days, and then each sample’s DLS was measured to study the stability of Au@MSNs-ICG in various physiological solutions. The fluorescence excitation and emission spectra of ICG, Au@MSNs, and Au@MSNs-ICG were measured using a fluorescence spectrofluorometer (F-7000, Hitachi, Japan). To analyze its stability, Au@MSNs-ICG (50 µg) was added to FBS(1 mL; Gibco, Australia) and its optical absorbance at 780 nm was monitored using ultraviolet-visible spectroscopy (UV-vis, UV-2450, Shimadzu, Japan) at multiple time points for 24 h. A serum-only solution was also monitored as the control. To confirm the photothermal activity of the NPs, PEGylated Au@MSNs-ICG (1 mL ICG at 0.25 mg/mL), Au@MSNs (1 mL), free ICG (1 mL), and PBS (1mL) in an Eppendorf vial were irradiated by an 808-nm NIR laser with the intensity of 1 W/cm2 for 5 min, and the temperature change was measured by an infrared thermal imaging camera (FLUKE, Ti25, America). To compare the photostability of Au@MSNs-ICG with that of free ICG, the samples were irradiated by the laser for 8 min (LASER ON), followed by natural cooling (LASER OFF) to room temperature for 30 min. This cycle was repeated three times and then TEM images were obtained to characterize the Au@MSN-ICG morphologies. 2.4 CT and Fluorescence Imaging Using Au@MSNs-ICG The CT images were captured with the self-built CT system. Its spatial resolution in our laboratory was 100 µm. The tube voltage and current in the experiment were 40 kV and 0.8 mA, respectively. NIR fluorescence was visualized using a small-animal optical molecular imaging system (IVIS Spectrum, Caliper Life Science. USA). Different concentrations of Au@MSNs-ICG) were prepared and used in CT and NIR 7



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imaging (1.56, 3.12, 6.25, 12.5, 25 mg/mL and 0.33, 0.65, 1.33, 2.6, 5.2, 10.4, 20.8 µg/mL, respectively). 2.5 Cell Culture and Tumor Model Generation Cells from human hepatic cancer cell line (HepG2-Fluc)—procured from the Department of Radiology, Peking Union Medical College Hospital—were cultured (37 °C, 5% CO2) in 89% Dulbecco’s modified Eagle’s medium (Hycolone, Thermo Scientific) with 10% FBS (Gibco, Australia) and 1% penicillin/streptomycin (Hyclone, Thermo Scientific). All animals were purchased from the Beijing Weitong Lihua Experimental Animal Technical Co., LTD (China). All experimental protocols were approved by the Institutional Animal Care and Ethics Committee of Zhujiang Hospital of Southern Medical University, and all methods were carried out in accordance with the approved guidelines. The subcutaneous hepatic tumor model was established by injecting 2 × 106 HepG2-Fluc cells subcutaneously into the left upper elbow of 5-6-week-old BALB/c nu/nu male mice. 2.6 In Vitro Cytotoxicity Study and Cellular Uptake The cytotoxicities of free ICG, Au@SiO2, and Au@SiO2-ICG were assessed by avoiding light using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HepG2-Fluc cells were plated in a 96-well white plate (1 × 104 cells per well) and cultured for 24 h. The cells were incubated with different doses of the probe. The concentrations of free ICG, Au@SiO2, or Au@SiO2-ICG were tested from 0 to 400 µg/mL. The cells’ viability in the different treatments was assayed using MTT. Briefly, 10 µL of the MTT (5 mg/mL) reagent was added into each well and incubated for 4 h. Then, the formazan crystals were dissolved using DMSO. The absorbance at 490 nm was measured using a microplate absorbance reader (Biorad iMARK™, USA). For in vitro cell fluorescence imaging, HepG2-Fluc cells were incubated with Au@SiO2-ICG (0.2 mg/mL) for 1 h, and after being washed three times with the cell culture medium and twice with PBS, 2 mL of the cell culture medium was added to the cell culture dish. Then, the split type fluorescent microscope and NIR-fluorescence imaging-guided surgery system were applied to observe the cellular 8


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uptake of Au@SiO2-ICG (emission wavelength of 808 nm and excitation wavelength of 840 nm). 2.7 Photothermal Effects in Cells HepG2-Fluc cells were plated in 96-well plates (1 × 104 cells/well) and cultured for 24 h. The cells were then treated with PBS, ICG (7.5 µg/mL), Au@SiO2 (30 µg/mL), or Au@SiO2-ICG (30 µg/mL, containing ICG, 7.5 µg/mL) for another 24 h. For each well, the old medium was removed, the well was washed with PBS three times, and 100 µL of fresh cell culture media was added. Subsequently, each well was exposed to an 808-nm laser at 1 W/cm2 for 5 min. Following this, the cells were incubated for a further 24 h and cell viability was evaluated using the MTT assay. To further study the photothermal effects of Au@SiO2-ICG, HepG2-Fluc cells were plated in 96-well plates (1 × 104 cells/well) and cultured for 24 h. Then, the cells were

treated

with

ICG,

Au@SiO2-ICG,

ICG+808-nm

laser,

or

Au@SiO2-ICG+808-nm laser for a further 24 h. The concentrations of Au@SiO2-ICG used were 30, 60, 120, and 240 µg/mL (Equivalent containing ICG concentrations of 7.5, 15, 30, and 60 µg/mL, respectively) and the concentrations of ICG were 7.5, 15, 30 and 60µg/ mL in 200-µL cell culture media. For each well, the old medium was removed, the well was washed with PBS three times, and 100 µL of fresh medium was

added.

After

that,

each

well

of

the

ICG+808-nm

laser

and

Au@SiO2-ICG+808-nm laser groups were exposed to the laser at 1W/cm2 for 5 min, then the cells were processed and analyzed in the same way as for the previous experiment. 2.8 CT and NIR Fluorescence Imaging In Vivo For in vivo fluorescence imaging, 6 subcutaneous liver tumor models were chosen, each with tumors of 5 – 6 mm diameter. The mice were separated randomly into 2 groups (n = 3/group), the ICG group and the Au@SiO2-ICG group. Each mouse was anesthetized with 2% isoflurane. Free-ICG or Au@SiO2-ICG–PBS (150 µL; the reference dosage of ICG was 5 µg/g, so the dosage of Au@SiO2-ICG was 20 µg/g) solution was injected into a mouse via the tail vein. Approximately 10 min, 24 h, 48 h, 72 h, and 96 h after the injection, the region of interest was monitored using the IVIS 9



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Spectrum imaging system with excitation and emission wavelengths of 745 nm and 840 nm, respectively, and the mouse was scanned by the self-built CT system at 3 h, 6 h, and 12 h. The data were analyzed using IVIS Living Imaging 3.0 software (PerkinElmer, USA) and 3D-Med (Chinese Academy of Sciences Institute of Automation). 2.9 Tumor Xenografts and Antitumor Therapy When tumor diameters reached 5 – 6 mm, bioluminescnece imaging (BLI) was performed on the subcutaneous tumor-bearing mouse. According to the fluorescence intensity of the tumors, the tumor-bearing mice were randomly separated into four groups (n = 4/group). The mice in different groups were treated with PBS+808-nm laser, ICG+808-nm laser, Au@SiO2+808-nm laser, or Au@SiO2-ICG+808-nm laser. The different samples were injected into the mice via the tail vein. The tumor region was then irradiated with the laser 48 h after the injection. To avoid possible tissue damage by hyperthermia, laser treatment was carried out for 5 min at a power density of 1 W/cm2. The NIR-fluorescence imaging system was applied to locate the tumor and monitor the PTT. The mice were injected intraperitoneally with 80 mL D-luciferin solution (15 mg/mL) 8 min before BLI. BLI of the tumor region was performed using a small animal optical molecular imaging system (IVIS Spectrum) and analyzed using IVIS Living Imaging 3.0 software (PerkinElmer, USA) on day 0, 3, 6, 9, 12, and 15 after PTT treatment. The mice were anesthetized with 2% isoflurane during imaging. The parameters for the BLI imaging system were: binning = 4 and exposure time = 1 s. The mouse’s body weight was measured using an electronic balance on day 0, 3, 6, 9, 12, and 15 during treatment. The tumor volume was measured using a vernier caliper. 2.10 Statistical Analysis Statistical analysis was performed using a Statistical Package for Social Sciences (SPSS. 20). Data are expressed as mean ± SD. To test the differences between the optical densities and fluorescence intensities of the region of interest for the groups, a two independent samples t-test was used. All statistical tests were two-tailed and differences were considered significant at a p-value < 0.05. 10


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3. RESULTS AND DISCUSSION The Au@MSNs-ICG were first synthesized into gold nanoparticles, coated with MSNs, and then loaded with ICG. TEM indicated that the Au@MSNs-ICG showed a typical spherical morphology with an average diameter of approximately 80 nm. The silica coating had a uniform thickness of approximately 20 nm. The gold core was approximately 30 nm (Figure 1A - B). It also exhibited good monodispersity and the average hydrodynamic diameter of the Au@MSNs-ICG was approximately 90 nm as measured by DLS (Figure 1C). The DLS data of samples in various solutions (PBS, FBS, cell culture media) for 7 days suggested the Au@MSNs-ICG still have good monodispersity and the diameter of NPs resist in approximately 90 nm (Figure S1). The Au@MSNs-ICG probes performed well for both optical and long-term stability in plasma (Figure S2). And ICG loading efficiency is 24.8 wt %. Considering that the critical factors affecting tumor targeting in vivo are the size and morphology of a nanoprobe, particles with sizes between 30 and 150 nm are particularly favorable53. After PEG surface modification, the Au@MSNs-ICG could have good dispersibility and long in vivo residing time, which would offer an opportunity for NPs to target the tumor via the enhanced permeation and retention effect. The above results indicate that the size and morphology of Au@MSNs-ICG are favorable for tumor targeting in vivo. The absorption and emission spectra of Au@MSNs-ICG, gold NSs and ICG were measured by the UV-vis and fluorescence spectrofluorometer. The absorption peak of the gold NS was 515 nm (Figure 1D), which is consistent with that reported in a previous study52. Compared to that of free ICG, the absorption and emission peaks of Au@MSNs-ICG were found to be red-shifted approximately 4 nm and 10 nm, respectively (Figure 1D - E). These results suggest that ICG was loaded in MSNs efficiently. Au@MSNs-ICG showed a slight peak at 515 nm (Figure 1D), which indirectly confirms that it was made successfully. The absorption peak of the gold NS was at 515 nm, with almost no absorption above 800 nm (Figure 1D - E); this is very different from the emission spectrum of ICG or Au@MSNs-ICG. These data illustrate 11



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that NIR emission fluorescence of ICG or Au@MSNs-ICG would not be quenched by the core of the gold NS; hence, these features are good for NIR imaging. It is very different from other reported probes that fabricated the Au nanorod as the core and loading of ICG, which could not avoid NIR fluorescence quenched by the Au nanorod, since the absorption of the Au nanorod was in NIR fluorescence spectra50. In contrast, the Au@MSNs-ICG used in our study have a broad absorption spectrum in the NIR region (650 nm to 900 nm), which was a critical feature for its applications in NIR imaging and PTT. The Au@MSNs-ICG contained an Au NS core. Because gold has a favorable X-ray attenuation coefficient, the probe could be applied as a contrast agent in CT imaging. CT imaging of Au@MSNs-ICG diffused in PBS at different concentrations (25, 12.5, 6.25, 3.125, 1.56, and 0 mg/mL) showed different CT values (Figure 1F). The CT– Hounsfield-unit values (HU values) were linear with increasing concentration (Figure 1H). The slope of the line was 38, which was much higher than that of iopromide (16.38)54-55. Hence, X-ray attenuation of Au@MSNs-ICG was better than that of iopromide, the better X-ray attenuation would reduce the dosage of drug. The NIR fluorescence intensity, at different concentrations of the probe, gradually increased with increasing concentration (Figure 1G). A good linear correlation between the fluorescence signal and various concentrations of the probe demonstrated that the fluorescence was protected from the bleaching of the Au nanoparticles (Figure 1I). The above data indicate that the optical/CT probe has the potential to be functional in efficient dual-modality imaging. The photothermal efficiencies of PBS, free ICG, Au@MSNs, and Au@MSNs-ICG were measured using an infrared thermal imaging camera under 808-nm laser irradiation. The maximum temperatures of the Au@MSNs-ICG and free ICG were 58.6 and 54 °C, respectively, but PBS and Au@MSNs only increased to 26.3 and 33.1 °C, respectively (Figure 1J, 1K). The temperature of Au@MSNs-ICG could increase by 5–6 °C after 1 min of laser irradiation (Figure 1K), which was enough to irreversibly destroy tumor cells. However, free ICG should be irradiated for ~3 min to increase in temperature by 5–6 °C. This means that the encapsulation of ICG in the 12


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NPs enhanced its heat conversion efficiency, and the Au@MSNs-ICG could convert NIR energy rapidly and efficiently into thermal energy. Therefore, Au@MSNs-ICG has potential as a PTT agent for cancer therapy. To compare the NIR photostabilities of Au@MSNs-ICG and free ICG molecules, solutions of Au@MSNs-ICG and ICG were irradiated with NIR laser for 8 min (Figure S3A), followed by natural cooling to room temperature for 30 min (Figure S3A). This cycle was repeated three times to investigate the photostability of ICG molecules before and after loading into Au@MSNs-ICG. ICG (6 µg/mL) and equivalent Au@MSNs-ICG solutions have been chosen. The temperature elevation of 24.5, 19.6, and 17.08 °C for Au@MSNs-ICG and 20.05, 8.9, and 7.15 °C for free ICG was achieved after sequential LASER ON cycles(Figure S3A). The higher elevated temperature indicates that ICG molecules loaded into Au@MSNs-ICG show higher photostability than free ICG molecules do. Moreover, the Au@MSNs-ICG showed no morphology change, visualized under TEM (Figure S3B), indicating their good photostability after long periods of NIR laser irradiation. The HepG2-Fluc cellular uptake of the Au@MSNs-ICG was evaluated in vitro. The HepG2-Fluc cell showed significantly higher fluorescence intensity after 1 h incubation with Au@MSNs-ICG, which was stronger than that of free ICG (Figure 2B). Such results indicate the efficient cellular internalization of the Au@MSNs-ICG and demonstrate that Au@MSNs-ICG increase the endocytosis ability of ICG allowing for greater cellular uptake of ICG per unit of time. The safety of the nanomaterials is of utmost importance when they are used for biomedical applications. The cytotoxicities of free ICG, Au@MSNs, and Au@MSNs-ICG in HepG2-Fluc cells were assessed using the MTT assay. No significant cytotoxicities were observed for any of the molecules, even though they were at a high concentration (400 µg/mL) (Figure 2A). Au@MSNs-ICG exhibited excellent biocompatibility. For in vivo experiments, Au@MSNs-ICG had no obvious effects on mouse body weight, eating, drinking, or behavior/activity. The PTT efficiencies of Au@MSNs-ICG (120 µg/mL, containing 30 µg/mL ICG), Au@MSNs (120 µg/mL), free ICG (30 µg/mL), and PBS were evaluated in 13



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HepG2-Fluc cells. Cell viability for different treatments was estimated using the MTT assay. PBS, with light irradiation, had no obvious impact on cell viability (Figure S4). The viability of cells treated with Au@MSNs+808-nm laser was 92% (Figure S4), which suggested that Au@MSNs had almost no PTT efficiency with the 808-nm laser. The Au NS had almost no absorption above 800 nm. Compared with the cells treated with ICG+808-nm laser showing approximately 70% cell viability, the viability of the cells treated with Au@MSNs-ICG+808-nm laser was reduced to 20% - 26% (Figure S4). The further study is about the photothermal therapeutic efficiency of Au@MSNs-ICG. With laser irradiation, incubation with free ICG (containing 60 µg/mL of ICG) caused the death of ∼57.6% of cells (Figure 2C). the death of the Au@MSNs-ICG+808–nm laser group (with 60 µg/mL ICG) was up to 93.6% of cells, suggesting that Au@MSNs-ICG could effectively enhance the ICG concentration in cells (Figure 2C). This is consistent with the result that the Au@MSNs-ICG increased the endocytosis ability of ICG (Figure 2B). The free ICG and Au@MSNs-ICG groups showed that no toxic effect was exhibited in the cells, which was confirmed by the MTT assay. This indicates that cell death was solely due to PTT efficiency. This also indicated Au@MSNs-ICG had better photothermal toxicity than free ICG. Au@MSNs-ICG could be effectively absorbed by the cells, which is beneficial. Therefore, Au@MSNs-ICG has great potential for PTT of cancer with NIR irradiation. Au@MSNs-ICG showed good biocompatibility, high NIR absorption, good thermal conversion efficiency, and effective NIR/CT imaging; this encouraged us to evaluate their potential as NIR/CT bimodal imaging and PTT in vivo. In vivo metabolism of Au@MSNs-ICG was traced by ICG NIR fluorescence imaging by IVIS spectra imaging. In the free ICG group, ICG was distributed throughout the body of the mice 10 min after injection (Figure 3A - B). Free ICG was almost metabolized in the intestines with some residual NIR fluorescence signal in the tumor region after 12 h (Figure 3B). NIR signals were not detected at 24, 48, or 72 h (Figure 3B). In contrast, ICG signals of Au@MSNs-ICG in the tumor region gradually increased over time and largely accumulated in the tumor after 24 h (Figure 14


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3A). The tumor region in nude mice retained strong NIR fluorescence signals at 72 h (Figure 3A). Taken together, these data indicated that Au@MSNs-ICG could significantly reduce clearance from the body. Furthermore, we clearly demonstrated that Au@MSNs-ICG present in the tumor and was efficient in passively targeting the tumor site. An ex vivo biodistribution of the Au@MSNs-ICG was performed on major organs 24 h after i.v. injection, which again demonstrated the higher tumour uptake of Au@MSNs-ICG (Figure S5). These data confirmed the tumor-homing effect of Au@MSNs-ICG. High fluorescence from the livers and low fluorescence from the kidneys revealed that the probes were excreted through the liver and digestive system (Figure S5). Based on our observations, 48 h was chosen as the time point for PTT to achieve maximum efficacy, because the tumor to background rate (TBR) (1.94 ± 0.12) was the highest at 48 h, indicating the probe accumulated in the tumor region; however, probe accumulation in normal tissues was limited (Figure 3C). Importantly, PTT performed at these time intervals caused almost no damage to the normal tissues (such as ambustion, the blood vessel seal, solidify and shrink, necrosis). To achieve 3D distribution of the dual-labeled NP, we performed in vivo tomographic imaging of the liver xenograft mouse model using both fluorescent molecular tomograph (FMT) and CT. We also analyzed the CT imaging capability of Au@MSNs-ICG in vivo. The tumor-bearing mice were imaged before and after Au@MSNs-ICG administration. White dots were clearly visible in the tumor area 3 h post NP-injection, indicating the remarkable CT contrast signals in tumors, which confirmed the accumulation of Au@MSNs-ICG in tumors (Figure 4). However, signal changes in the tumor region were not uniform, thereby indicating a heterogeneous intra-tumoral distribution of the NPs. Using the CT signal level of water as a baseline, paired t-tests demonstrated that there was a significant change in the relative CT signal intensity. Furthermore, 6 h after injection, the whole tumor area exhibited higher signal intensity than before suggesting a large amount of Au@MSNs-ICG accumulation in the tumor area (Figure 4). Tumor sites showed higher CT signal intensity, indicating that Au@MSNs-ICG NPs remained in the the tumor sites for at 15



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least 6 h, providing adequate time for treatment. Compared to the iodine contrast medium, Au NPs remained at the tumor region for longer allowing for satisfactory imaging. In other words, iodine has a certain amount of renal toxicity, and X-rays cause the iodine contrast medium to ionize, producing toxic iodine ions. However, Au NPs showed no release and have good biocompatibility. 3D FMT reconstruction showed the distribution of the NPs in the tumor region 3 hours after the injection. Figure 4 shows the axial CT images as well as the corresponding FMT images in the same mouse. As indicated previously, NP distribution in the fluorescence tomographic images corresponded well with the CT images, suggesting the further application of NPs in PTT. The tumor-homing effects shown in CT were consistent with that of FMT, which validated the efficient passive targeting ability of Au@MSNs-ICG in tumors. In addition, we observed clear contrast enhancement of the liver and spleen, suggesting that NRs accumulated via reticuloendothelial system absorption and was further metabolized in the liver and spleen. Thus, NRs have remarkable NIR/CT imaging capabilities and fairly obvious tumor-homing effects. The above data indicated the Au@MSNs-ICG serve as an efficient contrast agent for NIR and CT imaging. Moreover, its precise spatial-temporal resolution for monitoring PTT agents in vivo would shed new light on the therapeutic process and help avoid destruction of surrounding healthy tissue induced by external laser radiation. This, in turn, would reduce the side effects on normal tissue and would set a new gold standard for highly sensitive and precise detection of tumors for accurate diagnosis and efficient therapy. Having precisely monitored the trace and tumor-homing effect of Au@MSNs-ICG, we next investigated their PTT performance in vivo. When the tumor volume grew to ~200 mm3, 12 Hep-G2 tumor bearing nude mice were divided into four groups: (a) PBS+808

nm;

(b)

ICG+808

nm;

(c)

Au@MSNs+808

nm;

and

(d)

Au@MSNs-ICG+808 nm. The relative tumor volume changes, biofluorescence intensities of the tumor region, and body weights of mice in all of groups were recorded. Approximately 48 h after Au@MSNs-ICG injection, NIR-imaging guided 16


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photothermal therapy was performed in vivo (Figure 5). The tumor location and tumor margin could be clearly identified with the NIR fluorescent imaging system under continuous 808 nm laser irradiation. With NIR fluorescent imaging monitoring, laser orientation, and tumor margin, the doctor could adjust the laser orientation in real time for accurate photothermal therapy (Figure 5). In the free ICG group, after intravenous injection of free ICG at 48 h, the doctor was unable to detect the tumor location or margin due to the metabolization of ICG (Figure 5). In the Au@MSNs-ICG+808 nm group, the IR thermal camera showed that the temperatures of the tumor area increased remarkably to >42 °C after being irradiated with an 808 nm laser for 1 min and to ~55 °C for 5 min, which was capable of killing the cancer cells (Figure 6A). On the contrary, the temperatures of tumors in mice injected with PBS, free ICG, and Au@MSNs increased to ~39.8 °C, ~40.1 °C and ~39.6 °C (Figure 6A). These results also confirmed the potential of Au@MSNs-ICG as an efficient PTT agent for curing tumors. After 15 days of treatment, relative tumor volumes (V/V0) in groups a, b, and c were 10.47, 6.44, and 6.58, respectively (Figure 6C - D). Moreover, tumors grew rapidly in these groups. These results demonstrated that free ICG, Au@MSNs, or administration of the 808 nm laser treatment alone had negligible effects on tumor growth. However, the mice administered both the 808 nm laser and Au@MSNs-ICG showed efficient tumor growth inhibition of 0.002 (V/V0=0.002) (Figure 6C - D). To examine the anti-tumor effects, the BLI strategy was adopted to continuously monitor the therapeutic effects of PTT. With the aid of BLI, the liver tumor-bearing mice displayed a strong fluorescence signal in the tumor regions. In general, BLI fluorescence signal intensities of the tumor regions continued to increase through the 12 days of treatment with PBS+PTT, ICG+PTT, and Au@MSNs+PTT (Figure 6B), and the groups of mice exhibited a similar trend. Au@MSNs-ICG+PTT-treated mice showed slight fluorescence enhancement after 15 days, and as early as 3 days after treatment with Au@MSNs-ICG (Figure 6B). Furthermore, the average value of fluorescence light intensities of the Au@MSNs-ICG+PTT treatment group (1.1 × 108) was even lesser than that of ICG+PTT (1.72 × 1010) and the Au@MSNs+PTT group 17



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(1.84 × 1010) (*P < 0.05) on day 15 (Figure 6B). These results suggested that Au@MSNs-ICG+PTT facilitated the PTT anti-tumor treatment efficacy of ICG in vivo. Moreover, body weight increased in a time-dependent manner, illustrating that there were no adverse side effects owing to our theranostic agents (Figure 6F). In this regard, our findings are concordant with the results of the cell cytotoxicity assay in vitro. In contrast, representative images of tumor tissues excised from mice treated with PBS+PTT, ICG+PTT, Au@MSNs+PTT, and Au@MSNs-ICG+PTT on the 15th day were provided, which directly demonstrated this high inhibition effect. To further determine the efficacy of PTT, the hematoxylin and eosin (H&E) staining assay was introduced to study the morphology and apoptosis of tumor cells (Figure 6G). We then compared the control group with other treatment groups. The tumor in the control group was essentially intact and the tumors in the treated groups consequently appeared to have smaller cells with a uniform color and wider intracellular spaces (Figure 6G); furthermore, even karyolysis emerged, especially in the Au@MSNs-ICG+PTT group, indicating that there was some apoptosis in these superficial regions of tumor slices under various treatments. Therefore, the H&E staining assay also supported the superiority and anti-tumor efficiency of PTT in vivo.

4. CONCLUSIONS In summary, we have successfully synthesized mesoporous silica-coated gold NSs loaded with an organic cyanine dye (ICG) to be used as an ideal dual-modal anti-cancer platform due to the excellent properties of the multifunctional composite. First, the superior surface area and the satisfactory structure of mesoporous silica as well as the hollow cavity endow the composite with large drug storage capability. Additionally, its low toxicity as well as biocompatibility in vitro and in vivo allows its use in biological applications. Second, the gold NSs can be used for CT imaging, which would not be limited by depth, to provide accurate visualization of anatomical structures. In particular, with ICG-loading of the MSNs, the platform not only presents NIR imaging but also high PTT efficacy against tumor cells. The anti-tumor efficacy of the combination was higher than that of PTT with free ICG. These results 18


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indicate that this composite is a powerful theranostic candidate for precision nanomedicine that could achieve sensitive and precise tumor detection for accurate diagnosis and imaging-guided destruction of the tumors.

19



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Figure 1. Characteristics of Au@MSNs-ICG. (A) and (B) TEM images of Au@MSNs-ICG, showing the typical spherical structure and silica-coated 30-nm gold nanospheres, with an average particle size of 80 nm diameter. Scale bar, 100 nm. (C) Size distribution of Au@MSNs-ICG. (D) Excitation spectra of gold nanospheres, ICG, 20


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and Au@MSNs-ICG; (E) Emission spectra of gold nanospheres, ICG, and Au@MSNs-ICG; (F) CT images of Au@MSNs-ICG suspended in water. (G) Fluorescent images of Au@MSNs-ICG suspended in water; (H) CT Hounsfield unit plotted against various concentrations of Au@MSNs-ICG ranging from 0 to 25 mg/mL. (I) Fluorescent intensity plotted against various concentrations of Au@MSNs-ICG ranging from 0 to 20.8 mg/mL. (J) Infrared thermographic maps of the tubes with PBS, free ICG, Au@MSNs, and Au@MSNs-ICG were measured (K) Photothermal effect of PBS, free ICG, Au@MSNs, and Au@MSNs-ICG under continuous 808-nm laser irradiation at 1 W/cm2 power intensity.

21



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Figure 2. In vitro cytotoxicity, endocytosis evaluation, and photothermal effect of the nanoparticles on HepG2-Fluc cells. (A) Cell-viability assays (HepG2-Fluc cells) that compared ICG, Au@MSNs, and Au@MSNs-ICG over various concentrations. (B) NIR images of HepG2-Fluc cells after incubation with free ICG or Au@MSNs-ICG. Scale bar 100 µm. (C) Photothermal effect on Hep-G2 cells under different treatments (808-nm laser irradiation at 1 W/cm2 power intensity).

22


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Figure 3. In vivo comparison of the biodistribution of Au@MSNs-ICG and ICG. (A) and (B) In vivo continuous observations (96 h) of liver cancer xenografts administered with Au@MSNs-ICG (A) and ICG (B), using FMI. (C) Comparison of TBR profiles of the two probes. The peak and maximum differences were both at 48 h post-injection, which suggests an optimal surgical window.

23



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Figure 4. CT images of the tumor before and after IV administration of the Au@MSNs-ICG solution at different time points. FMT included the fluorescence tomographic images of the selected slice. Fluorescence reconstructed values of each image were normalized to the respective maximum value.

24


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Figure 5. In vivo NIR-imaging guided photothermal therapy with ICG or Au@MSNs-ICG. (A) and (B) 48 h after IV administration of Au@MSNs-ICG, the clinical doctor could see the tumor margin in real time, using the NIR fluorescent imaging system for guiding photothermal therapy under continuous 808-nm laser irradiation at 1 W/cm2 power intensity of, so that accurate photothermal therapy could be done. (C) and (D) 48 h after IV administration of free ICG, the clinical doctor could not define the tumor location or margin, using the same conditions for NIR fluorescent imaging, due to the inability of ICG to produce a good signal in the tumor region. Scal bar 5 mm.

25



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Figure 6. In vivo photothermal therapy properties. (A) Photothermal images and temperature plot of HepG2-Fluc liver tumor-bearing mice with IV administration of PBS, ICG, Au@MSNs, or Au@MSNs-ICG irradiated with an 808-nm laser at 1 W/cm2 power density for 5 min. PBS was used as the negative control group. (B) Biofluorescence intensity data analysis for liver tumor-bearing mice in PBS, ICG, Au@MSNs, or Au@MSNs-ICG for 15 days. (C) and (E) Representative images of different groups of HepG2-Fluc liver tumor-bearing mice, after different administration doses, 15 days after photothermal therapy. Scal bar 5mm. (D) Relative tumor volume profiles of different groups of mice after different administration doses. (F) Body weight of different groups of HepG2-Fluc liver tumor-bearing mice after various treatments. (G) H&E stained images of tumors collected from different groups of mice. Scal bar: 100 µm.

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ASSOCIATED CONTENT Supporting Information: The DLS data of the Au@MSNs-ICG in various solutions; The optical stability of the Au@MSNs-ICG; The temperature character of Au@MSNs-ICG; The cell viability rate of the HepG2-fLuc cells treated by different probes; Ex vivo fluorescence images of the organs from Hep-G2 xenografts. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected] Notes The authors declare no competing financial interest. Author Contributions: Jie Tian and Chihua Fang initiated the project and contributed to the experimental design as the principal investigators. Chaoting Zeng and Wenting Shang conceived the experiments. Wenting Shang designed and synthesized the probe. Chaoting Zeng and Wenting Shang performed the experiments and prepared figures. Xiaoyuan Liang, Xiao Liang, Yang Du, Qingshan Chen and Chongwei Chi assisted with the in vivo image-guided PTT. Xiaoyuan Liang and Xiao Liang assisted with the in vivo CT imaging and FMT imaging. Chaoting Zeng and Wenting Shang analysed data and wrote the manuscript. All authors reviewed the manuscript. Acknowledgments: The authors thank Adjunct Assistant Professor Dr. Karen M. von Deneen from the University of Florida and Professor Jintang Xia from the department of Hepatobiliary Surgery in the third hospital affiliated to guangzhou medical university for helping with the modifications to this paper. This work was supported by the National Natural Science Foundation of China under Grant Nos. 81227901, 81501540, 61231004, U1401254, 61401462, 61501462, 81527805, and 81470083, the National High Technology Research and Development Program of China (863 Program) under Grant No. 2012AA021105 and Guangdong provincial science and 27



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technology plan projects No. 2014A020212647. Special Funds from the Cultivation of Guangdong College Students' Scientific and Technological Innovation project (“Climbing Program” Special Funds) Nos. pdjh2016b0103 and pdjh2016a0090 were also used.

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Delivery,

Magnetic

Resonance

and

Optical

Dual-Model

Imaging-Guided

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