Formation of Gold Nanostar-Coated Hollow Mesoporous Silica for

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Formation of Gold Nanostar-Coated Hollow Mesoporous Silica for Tumor Multi-Modality Imaging and Photothermal Therapy Xin Li, Lingxi Xing, Kailiang Zheng, Ping Wei, Lianfang Du, Mingwu Shen, and Xiangyang Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15185 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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ACS Applied Materials & Interfaces

Formation of Gold Nanostar-Coated Hollow Mesoporous Silica for Tumor Multi-Modality Imaging and Photothermal Therapy

Xin Lia,§, Lingxi Xingb,§, Kailiang Zhengc, Ping Weia, Lianfang Dub*, Mingwu Shena*, Xiangyang Shia*

a

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of

Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, P. R. China b

Department of Ultrasound, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong

University, Shanghai 200080, P. R. China c

Engineering Department, Crop Science Division of Bayer, Institute, WV 25112, USA

________________________________________________________ * To whom correspondence should be addressed. E-mail addresses: [email protected] (L. Du), [email protected] (M. Shen), and [email protected] (X. Shi) §

Authors contributed equally to this work.

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ACS Paragon Plus Environment


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ABSTRACT Development of multifunctional nanoplatforms for tumor multi-mode imaging and therapy is of great necessity. Herein, we report a new type of Au nanostar (NS)-coated, perflurohexane (PFH)-encapsulated hollow mesoporous silica nanocapsules (HMSs) modified with polyethylene glycol (PEG) for tumor multi-mode ultrasonic (US)/computed tomography (CT)/photoacoustic (PA)/thermal imaging and photothermal therapy (PTT). HMSs were first synthesized, silanized to have thiol surface groups, and coated with gold nanoparticles via Au-S bond. Followed by growth of Au NSs on the surface of the HMSs, encapsulation of PFH in the interior of the HMSs, and surface conjugation of thiolated PEG, multifunctional HMSs@Au-PFH-PEG NSs (for short HAPP) were formed and well characterized. We show that the HAPP are stable in a colloidal manner and non-cytotoxic in the studied range of concentrations, possess multi-mode US/CT/PA/thermal imaging ability, and can be applied for multi-mode US/CT/PA/thermal imaging of tumors in vivo after intravenous or intratumoral injection. Additionally, the near infrared absorption property of the HAPP enables the use of the HAPP for photothermal ablation of cancer cells in vitro and a tumor model in vivo after intratumoral injection. The developed multifunctional HAPP may be used as a novel multifunctional theranostic nanoplatform for tumor multi-mode imaging and PTT.

Keywords: Hollow mesoporous silica; gold nanostars; tumors; US/CT/PA/thermal imaging; photothermal therapy

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INTRODUCTION The past decades have witnessed rapid developments in the application of nanotechnology for different biomedical purposes, including diagnostic imaging,1-3 disease treatment,4-6 gene transfection,7-8 and drug delivery.9-11 Various organic or inorganic nanoparticles (NPs) or nanocapsules (NCs) have been used as theranostic nanoplatforms for cancer imaging and therapy due to their unique structure and properties.12-18 The key to construct a theranostic nanoplatform is to integrate both therapeutic and imaging elements together within one nanoscale agent. For diagnosis, many imaging modalities have been achieved by using multifunctional nanoplatforms, such as computed tomography (CT),19-20 magnetic resonance (MR),21-22 single-photon emission computed tomography,23-24 positron emission tomography (PET),25 photoacoustic (PA) imaging,26-27 ultrasound (US) sonography,28-29 and thermal imaging.30-31 Each imaging mode possesses its own merits and drawbacks, while multi-mode imaging can circumvent the limitations and combine their advantages, consequently allowing for more accurate diagnosis than single-mode imaging.32-34 Therefore, a wide variety of dual-mode or multi-mode imaging agents have been prepared. For instance, Xiao et al.35 synthesized FeWO4@PPy core/shell composites for MR, CT and thermal imaging of tumors. Cai et al.36 fabricated Fe3O4/Au composite NPs via a dendrimer-assisted approach for specific dual mode tumor CT/MR imaging. Ma et al.37 prepared magnetic core/mesoporous silica shell nanoellipsoids with Au nanorods capped on their surface for in vivo MR, thermal and optical imaging applications. For cancer treatment, among the used different therapy methods,38-41 chemotherapy and radiotherapy are always accompanied with toxic and adverse effects to normal tissues, while photothermal therapy (PTT) has been attractive due to its superior local treatment efficacy and slight 3



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systemic side effects.42-44 PTT of cancer can be realized by NPs with near infrared (NIR) absorption, such as Au nanostars (Au NSs),45 Au nanorods,46 tungsten oxide (WO2.9) nanorods,47 and Cu7.2S4 nanocrystals.48 Besides, PTT can also be combined with chemotherapy via incorporation of anticancer drug within NPs having an NIR absorption feature. In a recent study, Li et al.49 prepared hollow Au nanoflowers physically loaded with anticancer drug doxorubicin for synergistic chemo-photothermal therapy of cancer. To build up a theranostic nanoplatform, one has to optimize the approaches or strategies for facile combination of imaging and therapeutic elements within one nanoparticulate system. Due to the high atomic number of Au, Au NP-based nanosystem can be employed for CT imaging.50-51 Likewise, due to the NIR absorption feature, Au NPs with particular shapes such as NSs,52 nanoflowers,53 nanorods,54 nanoshells,55 and nanocages56 enable tumor PA/thermal imaging and PTT. For instance, Tian et al.57 synthesized Au NSs modified with aminated polyethylene glycol (PEG) that have increased cellular uptake, allowing for effective X-ray/CT imaging and PTT of tumors. Liang et al.58 used Au NSs with polyethylene glycol (PEG) and CD44v6 modified on their surface for multifunctional PA imaging and plasmonic PTT of gastric cancer stem-like cells. For tumor US imaging and therapy applications, hollow mesoporous silica (HMS) nanostructs59 have been synthesized and used for both US imaging and drug delivery for cancer treatment.60 In addition, PEGylated HMS loaded with doxorubicin (DOX) have been constructed as a pH-triggered delivery system for tumor chemotherapy.61 Furthermore, hollow magnetic mesoporous silica filled with phase change material/DOX mixture can be used for MR/thermal imaging-guided thermo-chemo combinational cancer therapy.62 Our previous work has demonstrated that due to the synergistic photothermal property of polydopamine and Au NSs, polydopamine-coated Au NSs enable CT imaging and enhanced PTT of 4


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tumors.63 In another work, hyaluronic acid- or folic acid-modified Fe3O4@Au core/shell NSs64-65 have been constructed for targeted CT/MR/thermal imaging and PTT of tumors. Logically, we hypothesize that the merits of Au NSs and HMS NCs may be combined to create a unique platform for US/CT/PA/thermal imaging and PTT of tumors. In this present study, a unique nanoconstruct of HMS NCs coated with Au NSs, encapsulated with PFH, and surface modified with mPEG-SH (for short, HAPP) was designed and synthesized. HMSs were first synthesized, modified with thiol groups on their surface (HMSs-SH), and then attached with the Au NPs via strong Au-S bond. By exposing the formed HMSs@Au seed to an Au growth solution, Au NSs were formed. The generated HMSs@Au NSs were used to encapsulate liquid PFH, followed by surface modification with mPEG-SH via Au-S bond (Figure 1). The synthesized HAPP were thoroughly characterized with different techniques in terms of structure, morphology, composition, stability, imaging properties, and cytocompatibility. The use of this multifunctional nanoplatform for multi-modality US/CT/PA/thermal imaging and PTT of tumors was systematically investigated. To the best of our knowledge, we are the first to create Au NS-coated HMS NCs as a theranostic platform for tumor multi-mode imaging and PTT.

EXPERIMENTAL SECTION Materials. All chemicals and materials were from commercial resources and were used as received. Water used in all experiments was purified according to our previous work.50 Synthesis of Au NPs, HMSs-SH, HMSs@Au seed and HMSs@Au NSs with different Au amounts. Au NPs having a diameter of approximately 10 nm were prepared according to the literature.66 HMSs with a size of 200 nm and a shell thickness of 25 nm were prepared according to a selective etching method described in the literature.67 HMSs-SH and HMSs@Au seed were also 5



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prepared according to the previous work with a slight modification.68 The different amount Au NPs were coated on the surface of the HMSs-SH by Au-S bond, and the mass ratio of Au NPs/HMSs-SH was set at 1:57 or 5:57, respectively. HMSs@Au NSs with different Au loadings were prepared using a seed-mediated method with a slight modification.64-65 Synthesis of HAPP. PFH (150 µL) was infused dropwise into a 5 mL Eppendorf tube containing 50 mg dry powder of the HMSs@Au NSs. Then, the tube cap was tightly closed and sealed with parafilm to prevent evaporation of PFH, followed by ultrasound sonication in ice bath for 2 min to obtain the HMSs@Au-PFH NSs. Then the HMSs@Au-PFH NSs were dispersed in 25 mL water and reacted with mPEG-SH solution (15 mg/mL, 4 mL in water) under gentle magnetic stirring for 3 h at room temperature. The formed HMSs@Au-PFH-mPEG NSs (HAPP) were obtained after centrifugation (8500 rpm, 20 min) and redispersion in water for 3 times, then dispersed in 6 mL of water or PBS and stored at 4

o

C before use. For comparison,

HMSs@Au-mPEG NSs (HAP) without PFH were also obtained under the same experimental conditions. Characterization Techniques. TEM imaging, ζ-potential measurements, dynamic light scattering (DLS), thermal gravimetric analysis (TGA), nitrogen adsorption-desorption isotherms, Fourier transform infrared (FTIR) spectrometry, UV-vis spectrometry, X-ray attenuation property evaluation, and inductively coupled plasma-optical emission spectroscopy (ICP-OES) were performed to chracterize the synthesized materials according to standard procedures reported in the literature.42, 64, 68 The photothermal property and stability of the HAPP were tested according to the literature.42, 69 In Vitro Cytotoxicity Assay. CCK8 viability assay and morphology observation of C6 cells were used to evaluate the cytotoxicity of the HAPP at different concentrations without and with 6


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ultrasound sonication (1 MHz, 1.2 W/cm2, 50% duty cycle) for 30 s according to protocols described in the literature.42 US/CT/PA Imaging Performance of the HAPP. HAPP or HAP were dispersed in water at two different concentrations (5 and 20 mg/mL). Degassed water, HAP and HAPP solutions were filled in 2-mL Eppendorf tubes and placed in the degassed water sink before scanning by a Philips IU-Elite US imaging system (Eindhoven, Netherland) under contrast mode with a frequency at 15 MHz and a mechanical index at 0.07. The average gray values were recorded in the uniform round region of interest

on

the

resultant

US

image

using

an

Image

J

software

(http://rsb.info.nih.gov/ij/download.html). The US contrast enhancement of HAP and HAPP solutions at different concentrations was measured by the average gray values. HAPP or Omnipaque (300 mg/mL, GE Healthcare, used as control) solutions with the same Au or I concentrations (5-40 mM) were prepared for CT phantom studies using a CT imaging system and operation parameters similar to those reported in our previous work.70 HAPP solutions with different Au concentrations (0.25-4.0 mM) were injected into the thin hose which was buried in an ultrasonic special glue and then irradiated under an 808 nm laser. The PA images were measured using the Vevo LAZR PA Zmaging System (Visualsonics Inc., Toronto, Canada). The intensity of PA signal was evaluated in arbitrary units (a.u.) by selecting a region of interest on the digital PA images. Cellular Uptake Assay. Specific cellular uptake of the HAPP within C6 cells was tested by quantitative ICP-OES analysis according to the literature.64 In Vitro Photothermal Killing of C6 Cells. C6 cells were seeded into a 96-well plate (1 × 104 cells/well) with fresh Dulbecco's modiﬁed eagle medium (DMEM). After overnight incubation, the medium was replaced with 100 µL of fresh medium containing 10 µL of HAPP solution (in PBS) 7



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with the final Au concentrations ranging from 0 to 0.8 mM. After cultured for another 8 h, the cells were irradiated by an 808 nm laser (1.2 W/cm2) for 5 min. In comparison, the control group received no laser irradiation. CCK8 assay was used to analyze the cell viability as described above. To further prove the PTT efficacy, the morphology of cells after different treatments was observed using a Leica DM IL LED inverted phase contrast microscope (Wetzlar, Germany). A magnification of 200× was applied for each sample. In Vivo US/CT/PA/Thermal Imaging of Tumors. We performed animal experiments following the protocols approved by the institutional committee for animal care and the policy of the National Ministry of Health. For in vivo US imaging, the 5-week-old male nude mice (22-25 g, Shanghai Slac Laboratory Animal Center, Shanghai, China) were subcutaneously injected with 5 × 106 C6 cells/mouse in the right hind leg. The tumor nodules had a volume of 0.08-0.1 cm3 at 3 weeks postinjection. The HAPP dispersed in 200 µL PBS (10 mg/mL) were intravenously injected to each mouse via tail vein. US imaging was performed by a Philips IU-Elite US imaging system at a frequency of 15 MHz under B-mode, contrast mode and color Doppler mode at the corresponding mechanical index of 0.6, 0.07 and 0.6, respectively. Images were captured before injection and at 0.5 h, 1.0 h, 2.0 h, and 3.0 h postinjection. For in vivo CT imaging, the HAPP dispersed in PBS ([Au] = 0.04 M, 200 µL) were injected into each mouse via tail vein. CT images of tumors were collected at different time points (0 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, and 6.0 h, respectively) post intravenous injection by a GE Discovery STE PET/CT system according to the literature.71 The tumor CT values were quantified. In vivo PA imaging of tumors using the HAPP as a contrast agent was investigated by a Vevo LAZR PA Zmaging System under 808 nm laser irradiation. HAPP dispersed in PBS ([Au] = 0.04 M, 200 µL) were injected to each mouse via the tail vein. The PA images and signal intensity of tumor 8


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site were obtained using the PA imaging system at the predetermined time intervals postinjection (0 h, 0.5 h, 1.0 h, 2.0 h, 3.0 h, and 4.0 h, respectively). For in vivo thermal imaging, the HAPP were dispersed in PBS ([Au] = 32 mM, 50 µL) and intratumorally injected. In the control group, intratumoral injection of 50 µL PBS was performed. Then, the tumor region was irradiated by an 808 nm NIR laser for 300 s and the thermal images were recorded according to the literature protocols.64 In Vivo PTT of Tumors. The C6 tumor-bearing nude mice with a tumor volume of 0.05-0.06 cm3 were randomly allocated into four groups (n =5): PBS control, PBS + Laser, HAPP, and HAPP + Laser. HAPP dispersed in PBS ([Au] = 32 mM, 50 µL) or PBS (50 µL) were intratumorally injected, and laser irradiation was performed using an 808 nm NIR laser (1.2 W/cm2) for 10 min. At critical points in the treatment course, the tumor volume, body weight and survival rate of all mice were recorded and the pictures of mice were obtained by digital camera. The computational formula of tumor volumes and the survival rate was evaluated by literature protocols.64 Standard TdT-mediated dUTP Nick-End Labeling (TUNEL) staining tests were performed according to our previous work64 to confirm the tumor cell apoptosis efficacy. Furthermore, the biodistribution of HAPP in the tumor-bearing mice was investigated by ICP-OES.50

RESULT AND DISCUSSION Synthesis and Characterization of mSiO2@sSiO2, HMSs, HMSs@Au seed and HMSs@Au NSs. The detailed steps used to synthesize the HAPP are described in Figure 1. In this study, the mSiO2@sSiO2 and HMSs were first synthesized according to the literature67 and characterized by TEM (Figure 2a-b). It can be seen that HMS NCs display a diameter of about 200 nm with a typical inner cavity having a size of 180 nm (Figure 2b), which were successfully prepared from 9



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mSiO2@sSiO2 (Figure 2a) according to the selective etching method. The Brunauer-Emmett-Teller (BET) surface area, pore volume and average pore diameter of HMSs were determined to be 283.98 m2/g, 0.34 cm3/g and 3.8 nm, respectively from N2 adsorption/desorption isotherms (Figure S1a, Support Information) and the corresponding pore size distribution curve (Figure S1b, Support Information). Then, the HMSs were modified with thiol group (-SH) on their surface to form the HMSs-SH. Based on the results of TGA and derivative thermogravimetry data (Figure S2, Support Information), it can be deduced that HMSs-SH have been successfully prepared because the DTG peak at 450 oC is related to the oxidation temperature of thiol group. The modification of -SH group on the HMSs surface was further validated by zeta-potential measurements of the HMSs and HMSs-SH (Table S1, Support Information). It is notable that the surface potential of the HMSs-SH is much higher than that of the HMSs, which is due to the shielding effect of the -SH that can partially cover the negatively charged silanol groups. The HMSs-SH were next coated with Au NPs via strong Au-S bond formation64 to form the HMSs@Au seed, which were subsequently exposed to an Au growth solution to form the HMSs@Au NSs according to the literature.64-65 The HMSs@Au seed and HMSs@Au NSs were characterized by TEM (Figure 2c-d). Clearly, Au NPs (~ 10 nm) and Au NSs (~ 60 nm) are successfully coated on the surface of the HMSs-SH. The formation of Au NPs and Au NSs onto the surface of the HMSs were further confirmed by UV-vis spectra, where a strong localized surface plasmon resonance (LSPR) peak at 520 nm and 795 nm emerges for the HMSs@Au seed and HMSs@Au NSs, respectively (Figure 3a). The NIR absorption of the HMSs@Au NSs may allow for their uses in PA imaging and PTT applications. In addition, the amount of Au NSs loaded onto the HMSs can be tuned by varying the amount 10


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of Au NPs deposited onto the HMSs. The HMSs@Au seed formed using an Au NPs/HMSs-SH mass ratio of 5:57 has increased density of Au NPs loaded onto the HMSs (Figure S3a, Support Information), thus resulting in the formation of HMSs@Au NSs with increased amount of Au NSs (Fig. S3b). The increased amount of Au NPs or Au NSs loaded onto the HMSs can be further confirmed by UV-vis spectroscopy (Figure S4, Support Information). Although the HMSs@Au seed with a higher loading amount of Au NPs has a more or less similar LSPR absorption band to the one with a lower Au NP loading amount, the HMSs@Au NSs prepared using HMSs@Au seed with a higher loading amount of Au NPs have a slight red shift of the LSPR band (810 nm versus 795 nm). To avoid the use of more Au NPs, the HMSs@Au NSs prepared using the HMSs@Au seed with a lower Au NPs/HMSs-SH mass ratio of 1:57 were selected for subsequent studies. Synthesis and Characterization of the HAPP. The formed HMSs@Au NSs were used to encapsulate PFH, followed by surface modification with mPEG-SH via Au-S bond formation. An apparent phase-separation in phosphate buffered saline (PBS) solution of the PFH (Figure S5a, marked by circle, Support Information) was observed due to its insolubility in PBS. In contrast, after the PFH encapsulation within the HMSs@Au NSs, the solution became homogeneous and transparent (Figure S5a, right navy blue). Further, both PFH and HAPP in PBS were shaken for 10 s and left still for 30 s. It was observed that the droplets of free PFH were attached to the tube wall (Figure S5b) and precipitated at the bottom of tube after 5 min due to its super hydrophobicity (Figure S5c). In comparison, HAPP solution was stable and well-dispersed in PBS even after 5 min (Figure S5c). Meanwhile, the modification of mPEG-SH was qualitatively confirmed by FTIR spectrum, where the absorption peak of C-H vibration at 2886 cm-1 for the HAPP can be clearly seen (Figure 3b). After the whole modification, the loading of Au NSs onto the HAPP was quantified by ICP-OES to be 89.86 µg/mg. 11



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US/CT/PA Imaging and Photothermal Properties of the HAPP. The contrast-intensified US imaging of degassing water, the HMSs@Au-mPEG NSs (HAP) without PFH and HAPP at different concentrations under contrast mode were investigated (Figure 3d). The US imaging in the region of interest was captured and the corresponding average gray value was calculated (Figure 3d1). Compared to the water control, the brightness of US images and average gray value of HAP and HAPP are enhanced and increases with the particle concentration. Importantly, the average gray value of HAPP at a concentration of 5 and 20 mg/mL is 1.8 and 1.6 times higher than that of HAP at the same concentration (p < 0.05). This should be ascribed to the PFH-induced US contrast enhancement. Overall, the formed HAPP are able to enhance US imaging under a contrast mode. The CT imaging property of the HAPP was investigated by comparing with Omnipaque. The HAPP solution and Omnipaque at the same concentrations of Au or I were used to generate CT images (Figure 3e). Clearly, the brightness of CT images and X-ray attenuation intensity increase with the Au or I concentration (Figure 3e1). However, at the same Au or I concentrations the CT value of HAPP is significantly higher than that of Omnipaque. Thus, the formed HAPP exhibit a better CT imaging property than Omnipaque. PA imaging and the corresponding PA signal intensity were studied at different concentrations of HAPP (Figure 3f). Clearly, with the Au concentration, the brightness of PA images and PA signal intensity of the HAPP gradually enhances (Fig. 3f1). A linear relationship between the PA signal intensity and the HAPP Au concentration was observed, implying that the HAPP could serve as an excellent PA imaging agent. The NIR absorption feature not only renders the HAPP with PA imaging property, but also with photothermal property. The temperature changes of water, and HMSs-SH, HMSs@Au seed and HAPP solution at different Au concentrations (1.0-20.0 mM) under 808 nm laser irradiation were 12


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monitored for 350 s (Figure 4a). Clearly, the temperature of HAPP solution significantly increases as a function of time, and the increasing tendency is enhanced with the increase of Au concentration. The temperature can reach 70.2 oC at an Au concentration of 20 mM. The temperature change (∆T) of HAPP solution at different Au concentrations from 1.0 to 20.0 mM was quantified to be 25.0, 27.5, 31.2, 34.6 and 40.2 oC, respectively (Figure S6, Support Information). In comparison, the temperature of the water, HMSs-SH and HMSs@Au seed solution (at the same Au concentration of 1 mM) just slightly increases under the same experimental conditions, and their maximum ∆Ts are all less than 8 oC. The photothermal conversion efficiency (η) of the HAPP solution was further measured under 808 nm laser irradiation (1.2 W/cm2). The aqueous solution of HAPP ([Au] = 1 mM) was first irradiated for 220 s, then the laser was turned off (Figure 4b). The rapid cooling of the HAPP solution suggests the excellent thermal conversion performance of the HAPP, which can be shown in the typical photothermal profile (Figure 4c). According to the methods reported previously,65 the

η of HAPP was calculated to be 67.1%. In addition, the photothermal stability of the HAPP was evaluated by repetitive laser irradiation and cooling down of their aqueous suspension. The HAPP solution is able to reach the same peak temperature (52.4 oC) from the initial temperature (29.1 oC) under laser irradiation and cool down to the initial temperature (29.1

o

C) for at least 4 times (Figure S7, Support Information).

These results indicate that the HAPP have an excellent photothermal stability. Cytotoxicity and Cellular Uptake Assays. It is necessary to test the cytocompatibility of the HAPP with/without ultrasonic irradiation by CCK8 cell viability assay (Figure 3c) before biomedical applications. The results show that the viability of C6 cells treated with both HAPP alone and with US irradiation for 30 s do not have any significant changes in the concentration ranging from 10 to 13



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500 µg/mL when compared to the PBS control. This implies that the developed HAPP are quite cytocompatible in the studied concentration range. Further, we tested the cytocompatibility of the HAPP with/without US irradiation by observation of the morphology of C6 cells after treatments for 24 h (Figure S8b-d and S8f-h, Support Information). It is clear that the treated C6 cells do not have any appreciable morphological changes when compared to those treated with PBS (Figure S8a and S8e, Support Information). To investigate if the HAPP can be uptaken by cells, ICP-OES was carried to quantify the Au uptake in C6 cells after incubation with the HAPP at different concentrations (25, 50 and 100 µg/mL, respectively) for 12 h (Figure S9, Support Information). It can be seen that the Au uptake in the C6 cells follows a concentration-dependent manner with higher Au uptake at a higher HAPP concentration. At the HAPP concentration of 100 µg/mL, the cellular Au uptake can reach 38.7 pg/cell, implying that the developed HAPP are able to be taken up by cells, likely through two mechanisms of phogocytosis and diffusion via cell walls. In Vitro Photothermal Killing of Cancer Cells. The photothermal ablation effect of cancer cells in vitro was evaluated by detecting the viability of C6 cells treated with the HAPP at different Au concentrations (0.05-0.8 mM) under 808 nm laser irradiation for 5 min (Figure 4d). It can be found that the cell viability significantly decreases when the Au concentration exceeds 0.2 mM. Especially at the Au concentration of 0.8 mM, the viability of C6 cells decreases to 55.7%, which is much lower than that treated with HAPP alone without laser irradiation (87.9%, p < 0.001). In addition, the PTT efficacy of the HAPP with/without laser irradiation was assessed by observing the morphology of C6 cells treated for 8 h (Figure S10, Support Information). Compared with C6 cells treated with PBS, a significant portion of cells treated with higher HAPP under laser irradiation are rounded and detached, indicating the cell death. These results reasonably confirmed the 14


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photothermal ablation effect of cancer cells in vitro using the HAPP. In Vivo US/CT/PA/Thermal Imaging of a Xenograft Tumor Model. We next investigated the use of the HAPP for US/CT/PA/thermal imaging of a xenograft tumor model. US imaging carried out using different modes show that the HAPP are able to generate increasing contrast enhancement in the tumor region with the time postinjection and the US signal intensity reaches the highest at 2 h postinjection and starts to decline at 3 h postinjection (Figure 5a). The US signal intensity quantified by measurements of average gray value of tumor region under either B mode (Figure 5b) or contrast mode (Figure 5c) show that peak value is reached at 2.0 h, which is much higher than that before injection (0 h). This implies that the developed HAPP may be accumulated within the tumor region possibly through the passive enhanced permeability and retention (EPR) effect. For CT imaging (Figure 5d), the tumor CT images collected at different time points post intravenous injection of the HAPP were quantified to calculate the CT value of the tumors (Figure 5d1). Clearly, tumor region has a peak CT value of 51.8 HU at 2.0 h postinjection, which is 1.6 times higher than that before injection (32.8 HU). For PA imaging, the tumor PA images show that the PA signal intensity increases with the time postinjection (Figure 5e), and reaches a peak value at 2 h postinjection. This can be confirmed by quantitative PA signal intensity analysis (Figure 5e1), in consistence with the peak time point for the US and CT imaging. It is interesting to note that the concentration of HAPP used in US imaging is higher than that of CT/PA imaging. This should be due to the fact that the sensitivity of US imaging is dependent on the PFH concentration in the HAPP, while that of CT/PA imaging is solely dependent on the concentration of Au NSs of the HAPP. Since different imaging modalities requires different levels of the imaging elements, for the effective US imaging, a higher concentration of HAPP is required. 15



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Due to the NIR absorption property, the developed HAPP can also be used for thermal imaging of tumors. After intratumoral injection of the HAPP, infrared camera was used to monitor the temperature of the tumor region (a-Ar1 and b-Ar1) under 808 nm laser irradiation (Figure 6a). The temperature changes slightly in the tumor after treated with PBS (a-Ar1 in Figure 6a). In sharp contrast, the temperature rapidly increases to 70 oC in the tumor treated with the HAPP after laser irradiation for 200 s (b-Ar1 in Figure 6a and Figure 6b). Overall, our results showed that the developed HAPP enabled effective tumor US/CT/PA/thermal imaging. In Vivo Photothermal Ablation of a Xenograft Tumor Model. Next, we explored the use of HAPP for PTT of tumors in vivo (Figure 6c). Amazingly, the tumors in the HAPP + Laser group are able to be completely ablated after 2 days, while the tumors in all the other groups (PBS, PBS + Laser, and HAPP) are growing gradually with the time postinjection (Figure 6c and Figure S11, Support Information). Furthermore, we observed that the scar produced by PTT in the HAPP + Laser group was almost healed on the 12th day (Figure S11) and the tumor did not recur during the experimental time period. In addition, the body weight of the tumor mice in the HAPP + Laser group maintained in a certain range due to the cured tumor, while increased as the tumor continued to grow in other groups (Figure 6d). In order to further study the PTT efficacy of the HAPP, we measured the mouse survival rate in the four groups. It is clear that the mice treated with the HAPP + Laser display a 100% survival rate after 60 days, while mice in the other three groups are all dead on day 60 (Figure 6e). The PTT of tumors using the HAPP was further confirmed by histological examinations of the tumor sections after different treatments. The tumor sections after treated with PBS, PBS + Laser, HAPP, and HAPP + Laser were TUNEL stained (Figure S12, Support Information). Obviously, the tumors treated with PBS, PBS + Laser, and HAPP alone display just sparse positive staining of 16


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apoptotic cells. However, a large amount of positive stained apoptotic cells can be seen in the tumors treated with HAPP + Laser, and the apoptosis rate reaches 84.0% by the quantitative analysis of TUNEL staining (data not shown). Our results suggest that the developed HAPP are able to be used as an efficient nanoplatform for PTT of tumors in vivo. In Vivo Biodistribution. In order to assess the in vivo behavior of the HAPP more accurately, ICP-OES analysis was used to evaluate the biodistribution of Au element within the heart, liver, spleen, lung, kidney and tumor at 24 h post intravenous injection of the HAPP solution (Figure S13, Support Information). It is clear that the liver, spleen and lung have a high Au uptake due to the clearance effect by the reticuloendothelial system (RES) located in these organs. The Au uptake in the tumor site is 39 µg/g, significantly higher than that of the control group (p < 0.001). This further suggests that the HAPP are able to escape from the RES and be accumulated to the tumor region via a passive EPR effect.

CONCLUSION In summary, we demonstrated a facile approach to preparing multifunctional HAPP as a nanoplatform for US/CT/PA/thermal multi-modality imaging and PTT of tumors. Our results show that the HAPP with Au NSs coated onto the surface of the HMS NCs and PFH encapsulated within the HMS NCs are able to be formed to have uniform size distribution, good colloidal stability, and good cytocompatibility in the given concentration range. With the excellent US/CT/PA/thermal imaging property and NIR absorption-resulted photothermal conversion efficiency (67.1%), the developed HAPP are able to be used for efficient US/CT/PA/thermal imaging and PTT of tumors. Overall, our study suggests that the HAPP developed may be used as a promising theranostic nanoplatform for multi-mode imaging and PTT of different tumors. 17



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ACKNOWLEDGEMENTS This research is financially supported by the National Natural Science Foundation of China (21273032, 81571679, and 81271596), the Science and Technology Commission of Shanghai Municipality (15520711400 for M. Shen), the Sino-German Center for Research Promotion (GZ899), and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. X. Li and M. Shen thank the support from the Fundamental Research Funds for the Central Universities.

Supporting Information: Additional experimental details and data of zeta potential, nitrogen adsorption-desorption isotherms, TGA, digital pictures of HAPP with PFH encapsulated, DLS assessment of colloidal stability of HAPP, photothermal conversion efficiency and stability, cell morphology after different treatments, ICP-OES analysis of Au uptake in cells, digital pictures of mice under different treatments at different time periods, TUNEL images of tumor sections, and in vivo biodistribution of the HAPP. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure captions Figure 1. Schematic illustration of the synthesis of HMSs, HMSs@Au NSs, and the HAPP. Figure 2. High-resolution TEM images of sSiO2@mSiO2 (a-a2), HMSs (b-b2), HMSs@Au seed (c-c2), and HMSs@Au NSs (d-d2). Figure 3. (a) UV-vis spectra of Au seed, HMSs-SH, HMSs@Au seed, and HMSs@Au NSs. (b) FTIR spectra of HMSs@Au-PFH NSs and HAPP. (c) CCK8 viability assay of C6 cells treated with the HAPP for 24 h without and with US irradiation for 30s (1 MHz, 1.2 W/cm2) and duty cycle (50%). The cells treated with PBS were used as control. The data were expressed as mean ± SD (n=3). (d) US images and (d1) the corresponding average gray values of water, HAP and HAPP with different concentrations under contrast-mode. (e) CT images and (e1) X-ray attenuation intensity (HU) of (1) HAPP and (2) Omnipaque with different concentrations of radiodense element (Au or I). In (e1), the numbers on the left represent the Au or I concentrations (M) for the HAPP or Omnipaque. (f) PA images and (f1) PA values of the HAPP with different Au concentrations. Figure 4. (a) Temperature elevation of water and the aqueous solution of the HMSs-SH, HMSs@Au seed and HAPP at different Au concentrations (1, 2, 5, 10 and 20 mM, respectively) under the irradiation of a 808 nm laser with a power density of 1.2 W/cm2 as a function of irradiation time. (b) Photothermal effect of an aqueous HAPP solution irradiated by an 808 nm laser at a power density of 1.2 W/cm2. The laser was turned off after irradiation for 300 s. (c) Plot of cooling time (after 220 s) versus negative natural logarithm of the driving force temperature obtained from cooling stage. (d) CCK8 viability assay of C6 cells after treatment with the HAPP at different Au concentrations for 8 h, followed by laser irradiation for 5 min or without laser irradiation (0 min). Figure 5. (a) B-mode, contrast mode and color Doppler mode US images and the corresponding average gray values of B-mode (b) and contrast mode (c) of the C6 tumor xenografts in nude mice before and at different time points post intravenous injection of the HAPP (10 mg/mL, in 200 µL 27



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PBS). (d) CT images and (d1) CT values of the C6 tumor xenografts in nude mice before and at different time points post intravenous injection of the HAPP ([Au] = 0.04 M, in 200 µL PBS). (e) PA images and (e1) PA values of tumor-bearing mice before and at different time points post intravenous injection of the HAPP ([Au] = 0.04 M, in 200 µL PBS) under 808 nm laser irradiation. The white star in (a), (d) and (e) indicates the location of the tumor. Figure 6. (a) Thermal images of tumor-bearing mice injected with PBS (0.05 mL) or HAPP ([Au] = 32 mM, in 0.05 mL PBS), respectively, followed by irradiation with a 808 nm laser (1.2 W/cm2) at a time point of 0 and 5 min, respectively. (b) The temperature profiles in a-Ar1 and b-Ar1 as a function of the laser irradiation time. The relative tumor volume (c), body weight (d), and survival rate (e) of C6 tumor-bearing mice as a function of time post treatment.

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Figure 1 Li et al.

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Figure 2 Li et al. 30


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Figure 3 Li et al.

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Figure 4 Li et al.

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Figure 5 Li et al.

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Figure 6 Li et al.

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Formation of Gold Nanostar-Coated Hollow Mesoporous Silica for

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