PB@Au Core–Satellite Multifunctional Nanotheranostics for Magnetic

Dec 28, 2016 - As revealed by both MR and CT imaging, CSNPs realized efficient tumor localization by passively ... as long blood circulation time X-ra...
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PB@Au Core−Satellite Multifunctional Nanotheranostics for Magnetic Resonance and Computed Tomography Imaging in Vivo and Synergetic Photothermal and Radiosensitive Therapy Yan Dou,†,‡ Xue Li,§ Weitao Yang,† Yanyan Guo,§ Menglin Wu,§ Yajuan Liu,† Xiaodong Li,§ Xuening Zhang,§ and Jin Chang*,† †

School of Life Sciences, School of Material Science and Engineering, Tianjin University, Tianjin Engineering Center for Micro-Nano Biomaterials and Detection-Treatment Technology, Tianjin 300072, PR China ‡ Department of Radiology, Tianjin Key Laboratory of Functional Imaging, Tianjin Medical University General Hospital, Tianjin 300052, PR China § Department of Radiation Oncology and Department of Radiology, The Second Hospital of Tianjin Medical University, Tianjin 300211, PR China S Supporting Information *

ABSTRACT: To integrate multiple diagnostic and therapeutic strategies on a single particle through simple and effective methods is still challenging for nanotheranostics. Herein, we develop multifunctional nanotheranostic PB@Au core−satellite nanoparticles (CSNPs) based on Prussian blue nanoparticles (PBNPs) and gold nanoparticles (AuNPs), which are two kinds of intrinsic theranostic nanomaterials, for magnetic resonance (MR)−computed tomography (CT) imaging and synergistic photothermal and radiosensitive therapy (PTT− RT). PBNPs as cores enable T1- and T2-weighted MR contrast and strong photothermal effect, while AuNPs as satellites offer CT enhancement and radiosensitization. As revealed by both MR and CT imaging, CSNPs realized efficient tumor localization by passively targeted accumulation after intravenous injection. In vivo studies showed that CSNPs resulted in synergistic PTT−RT action to achieve almost entirely suppression of tumor growth without observable recurrence. Moreover, no obvious systemic toxicity of mice confirmed good biocompatibility of CSNPs. These results raise new possibilities for clinical nanotheranostics with multimodal diagnostic and therapeutic coalescent design. KEYWORDS: nanotheranostics, prussian blue nanoparticles, gold nanoparticles, multimodal imaging, synergistic therapy

1. INTRODUCTION

nanomaterials. Prussian blue is a clinical U.S. Federal Drug Administration (FDA)-approved drug, and PBNPs have emerged as the latest generation of agents for both magnetic resonance (MR) imaging and photothermal tumor ablation. 18−20 Compared to Gd(III)-based T 1-weighted or magnetic iron oxide nanoparticles-based T2-weighted MR contrast agents,21−25 PBNPs could have advantages for simultaneously T1-weighted and T2-weighted MR imaging due to their special crystal structure.18,26 Also for this reason, PBNPs possess better stability than gold nanostructures except for the strong photothermal conversion efficiency of nearinfrared (NIR, 700−1100 nm) light.27−29 To date, X-rayexcited theranostics based on high-Z atoms (rare earth, bismuth, gold, etc.) have attracted more attentions to improve the ability of combining clinical technologies such as X-ray computed tomography (CT) or radiotherapy (RT).30−32

Nanotheranostics offer efficient strategies with which to achieve personalized cancer therapy, which could realize precise diagnosis, optimize therapeutic efficacy, and mitigate side effects.1−4 For constructing nanotheranostic systems, recent developments focused on assembling more materials on single particle to simultaneously obtain various functions.5−10 Many aspects such as physical properties (solubility, etc.), function exertion affected by each other, and biocompatibility need to be considered, such that complicated designs have always been carried out to complete the desired combination.1,11−13 Despite inspiring results, simplified strategies still deserve further study for future industrialization and clinical applications.1,7,14,15 At present, intrinsic theranostic nanomaterials, which possess at least one kind of diagnostic and therapeutic function in themselves, received more attention in regard to new opportunities for function integration by simple manufacture protocols.16,17 Prussian blue nanoparticles (PBNPs) and gold nanoparticles (AuNPs) are two noteworthy kinds of intrinsic theranostic © XXXX American Chemical Society

Received: October 22, 2016 Accepted: December 28, 2016 Published: December 28, 2016 A

DOI: 10.1021/acsami.6b13493 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

water (18.2 M < OMEGA> cm) was used by Milli-Q ultrapure water system. 2.2. Synthesis of PBNPs, AuNPs, and CSNPs. Citric acid capped PBNPs were prepared according to typical procedure of previous reports,19,51 and AuNPs sized at 13 nm was chosen to be prepared for the superior X-ray properties according to our previous work.37 To prepare CSNPs, 2 mL of 1 mM PBNPs were added dropwise into 4 mL of 0.5 mg/mL PEI solution (MW = 20 000) under ultrasonication for 2 min.51 After 2 h of continuous stirring at 25 °C, they were washed with water at least three times with centrifugation at 13 000 rpm to remove excess PEI and obtain PEI−PBNPs. After we adjusted the pH to 6.5, sodium citrate-capped AuNPs were added into PEI− PBNPs solutions to coat AuNPs on the surface of PBNPs under gentle stirring for 4 h at 25 °C. Then, 1 mL of 2 mM SH−PEG−COOH was added into above CSNPs solutions under incubation for 12 h 25 °C. Then, PEG-modified CSNPs were purified by centrifugation for 10 min at 10 000 rpm and redispersed in deionized water for future use. 2.3. Characterization. Transmission electron microscopy (TEM) was carried out by JEOL 2010F electron microscope at 200 kV operating voltage. UV−vis−near-infrared (NIR) spectra were measured by UV−vis−NIR spectrophotometer (PerkinElmer). The dynamic light scattering (DLS) and ζ potentials at different fabrication stages were evaluated by a Malvern Zetasizer Nano ZS model ZEN 3600 (Worcestershire, UK). The content of Fe and Au in CSNPs solutions was acquired by inductively coupled plasma mass spectrometry (ICP-MS, DRCII, PerkinElmer). Bio-Rad FTS 6000 spectrometer (Bio-Rad Company, Hercules, CA) was used to acquire Fourier transform infrared (FTIR) spectra. X-ray diffraction (XRD) was measured with an X-ray diffractometer (Bruker AXS, D8-Focus). 2.4. MR and CT Contrast Measurements. CSNPs with iron concentrations (0−0.8 mM) compared with PBNPs were scanned by a clinical 3.0 T MR clinical scanner (Magnetom Trio with Tim, Siemens, Germany). T1-weighted MR images were obtained under the following parameters: TE, 2.5 ms; TR, 350 ms; matrix size, 128 × 128; FOV, 3 × 3, and slice thickness, 0.5 mm. T2-weighted MR images were acquired under the following parameters: TE = 81 ms and TR = 7500 ms with the same other parameters. T1 and T2 relaxation time were evaluated by a magnetic resonance imaging system (HT/MRSI60-60KY, Huantong Science and Education Equipment Co. Ltd., Shanghai, China). CT images of CSNPs and iohexol at the same concentrations (0− 50 mM) of gold or iodine were obtained by being scanned using a clinical Light Speed VCT CT imaging system (GE Medical Systems, Milwaukee, WI). CT scanning was performed under the following parameters: 120 kV, 200 mA; slice thickness, 2.5 mm; pitch, 1:1; field of view, 512 × 512; gantry rotation time, 1 s. Hounsfield units (HU) were obtained from region of interest for each sample. Image postprocessing was performed using the sygon.via VA20 software (Siemens, Germany). Data analysis was performed by fitting to relaxivity curves and CT values curves using OriginPro 9.0 programs. 2.5. Photothermal Effect Measurements. CSNPs solutions with different mass concentrations (0.1−0.8 mg/mL) were irradiated by NIR laser exposure (808 nm, 1.5 W/cm2) in quartz cuvettes of 1.5 mL. PBNPs and AuNPs were also exposed to the same NIR laser for comparison to DI water as control. The different output power (0.8, 1.5, and 2.5 W/cm2) were performed to select the appropriate one. The temperature was monitored by digital thermometer for 0.5 min. IR thermal imaging camera (TiS55, Fluke) was used to obtain images. Furthermore, photothermal stability of CSNPs solutions was also investigated by 5 minof continuous irradiation followed by another 5 min of irradiation after dropping to the original temperature for a total of five cycles. UV−vis−NIR spectra were also measured before and after irradiation. 2.6. Cytotoxicity Test. 4T1 cells were cultured at 1 × 104 cells per well in 96 well plates at 37 °C for 24 h under 5% CO2 with RPMI1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin. Next, 100 μL of PBNPs (0−0.4 mg/mL), PBNPs−PEI (0−0.4 mg/mL), AuNPs (0−0.4 μg/mL), and CSNPs (0−0.4 mg/mL) at various mass concentrations were added for another 24 h. 3-(4,5-Dimethylthiazol-2-yl)-2,15-diphenyltetrazolium

AuNPs with high amounts of X-ray photons capture a crosssection of gold (Z = 79, k-edge value of 80.7 keV) have been widely investigated as CT contrast agents or radiosensitizers that could greatly increase the contrast difference in tissues or enhance the irradiation damage in tumors.16,23,33−37 To combine PBNPs with AuNPs could be undoubtedly of great significance for cancer nanotheranostics. For diagnosis, CT, as one of noninvasive commonly used diagnostic tools, possess high resolution and excellent three-dimensional (3D) reconstruction for lesions, while its ability to distinguish the details of soft tissues is far from satisfactory.23,34,38 The introduction of MR imaging, another clinical imaging technique, could bring excellent soft-tissue contrast and high sensitivity to afford nondestructive details of soft tissues and functional information on the lesions.39 Therefore, CT−MR imaging is advantageous to realize precise diagnosis. For therapy, photothermal therapy (PTT) has achieved developments due to low attenuation of tissues in NIR transparency window,40−43 while deep-located tumors are difficult to be treated resulting from the limited penetration depth of NIR laser.5 Remarkably, high-energy X-rays from RT could kill cancer cells by directly breaking DNA strands without depth restriction.44,45 Moreover, some reports have showed that PTT could effectively increase sensitivity of hypoxic cells to RT or prevent self-repair of tissues after RT.46,47 Therefore, PTT integrated with RT would bring a great synergistic action by compensating with each other to obtain greater antitumor effectiveness than two strategies, respectively. Although most of the studies have focused on the combination of PBNPs with AuNPs for the ultrasensitive detection of tumor markers,48−50 Jing’s groups29 have recently developed Prussian blue coated AuNPs to combine photoacoustic imaging and PTT of PB with CT imaging of AuNPs. However, the intrinsic theranostic properties of PBNPs and AuNPs have not been fully achieved on a single particle, which still need further studies. In this work, we have successfully fabricated PB@Au core− satellite nanoparticles (CSNPs) based on two kinds of intrinsic theranostic nanomaterials (PBNPs and AuNPs) to their fullest potentials through electrostatic adsorption and specific combination of amidogen and gold, which has not been investigated to date. In this system, PBNPs as cores endow CSNPs with strong T1- and T2-weighted MR contrasts as well as significant cellular damage and tumor destruction under NIR laser excitation, while AuNPs as satellites provide great CT contrast and radiosensitization. We found that tumor growth could be almost completely inhibited by synergistic PTT−RT therapy under MR−CT imaging in living mice. Furthermore, no obvious systemic toxicity has been observed by histological analysis in major organs in a mouse model after systemic administration within 14 days, revealing good biocompatibility. Our results suggest that PB@Au core−satellite nanoparticles (CSNPs) could potentially open new avenues for clinical tumorous nanotheranostics with precise diagnosis and imagingguided therapeutic coalescent design.

2. EXPERIMENTAL SECTION 2.1. Chemicals. K4[Fe(CN)6]·3H2O, FeCl3·6H2O, citric acid, and trisodium citrate were of chromatographic reagent grade and obtained from Guangfu Fine Chemical Research Institute (Tianjin, China). Tetrachloroauric acid trihydrate (HAuCl 4 , ≥ 99.9%), poly(ethylenimine) (PEI, Mw = 20 000) and SH−PEG−COOH (Mn = 2000) from Sigma-Aldrich were purchased. Other chemicals and reagents were all of analytical grade without purification. Deionized B

DOI: 10.1021/acsami.6b13493 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Design and characterization of the CSNPs. (a) Core−satellite nanostructure of CSNPs for tumor theranostics. PBNP cores are used for MR imaging and convert the 808 nm laser into heat for PTT, and AuNP satellites are used for CT imaging and enhanced RT. (b−e) TEM images of (b) PBNPs, (c) PBNPs−PEI, (d) AuNPs, and (e) PEGylated CSNPs at same magnifications. Inset: the corresponding digital photos of aqueous solutions. (f) DLS particle sizes and surface potentials after each step of synthesis. (g) UV−vis−NIR absorption spectra of nanoparticles after each step of synthesis (hereafter, the final product PEGylated CSNPs are referred to as CSNPs for short). nm, 1.5 W/cm2) for 5 min, the cells were cultured for another 30 min and then radiated by 4 Gy X-rays with continued incubation for 12 h. Next, MTT assay was performed to determine cell viability and to calculate projected value.5 2.10. Animals and Tumor Xenograft Models. Beijing Cancer Hospital provided female Balb/c mice, which were cared for under institutional committee protocols for animal care (SYXK 2011-008). To set up 4T1 tumor xenograft models, mice were injected subcutaneously in their right thighs with 2 × 106 cells per mouse. When tumor volume reached about 80 mm3, in vivo experiments were carried out. 2.11. In Vivo MRI and CT Imaging. Mice bearing 4T1 xenograft tumors were injected intravenously with CSNPs solutions (100 μL, 10 mg/mL mass concentrations of CSNPs) prior to imaging. After 24 h, the mice were anesthetized by intraperitoneal injection of 0.5% pentobarbital sodium (5 mL/kg). MR imaging was performed under a 3.0 T clinical MRI scanner equipped with a small-animal coil, and CT images were obtained from GE Light Speed VCT clinical imaging system, respectively. All of the experimental parameters were in accordance with 1.4 MR and CT Contrast Measurements. 2.12. In Vivo PTT−RT Synergistic Therapy. After being weighed, mice bearing 4T1 xenograft tumors were divided into various groups as follows: (1) PBS; (2) NIR alone; (3) RT alone; (4) CSNPs alone; (5) CSNPs + NIR; (6) CSNPs + RT; and (7) CSNPs + NIR + RT. Each group included five mice. Mice were injected intravenously with CSNPs solutions (100 μL, 10 mg/mL mass concentrations of CSNPs) for 24 h. Group 2 and group 5 were performed under NIR laser exposure (5 min, 1.5 W/cm2). After the mice were fixed on the board, IR images and mean temperature at tumor sites before and after laser irradiation was recorded by IR− thermal camera every minute. Groups 6 and 3 received 4 Gy X-ray radiation on Siemens Primus clinical linear accelerator (6 MeV) using a 1.5 cm × 1.5 cm radiation-field at a source-to-skin distance (SSD) of 100 cm. The irradiation treatments were given only once for each

bromide (MTT) assay was performed to evaluate cytotoxicity at various mass concentrations of samples. A total of 20 μL of 5 mg/mL MTT solution was added for a further 4 h of incubation, and then the medium was removed carefully and followed by the addition of 200 μL of dimethyl sulfoxide. The absorbance of 560 nm was detected using a multifunctional microplate reader (PerkinElmer). 2.7. In Vitro PTT Test. For the in vitro PTT test, 4T1 cells were seeded in 96 well plates at 1 × 104 cells per well. Cells were then incubated with different mass concentrations of CSNPs (0.025−0.4 mg/mL) for 4 h. After NIR laser exposure (808 nm, 1.5 W/cm2) for 5 min, the cells had another 12 h of incubation, which was then followed by MTT analysis. In addition, the cells after treatment were dualstained by calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI), and images were obtained using inverted fluorescence microscope (Olympus, IX73). 2.8. In Vitro Enhanced RT Test. 4T1 cells were seeded into 96 well plates at 1 × 104 cells per well. After 24 h of incubation, CSNPs with different mass concentrations (0.025−0.4 mg/mL) were added for another 4 h of incubation. PBNPs, AuNPs, and PBS was used as control. After X-ray radiation of 0 (control), 2, 4, 6, or 8 Gy, the cells were continued for another 12 h incubation and then followed by MTT analysis. The irradiation treatments were given only once for each sample. CSNPs, PBNPs, and AuNPs at 0.2 mg/mL were added to cells for 24 h, which received X-ray radiation for 0, 2, 4, 6, and 8 Gy under 400 mGy/s dose rates. All radiations were given only once for each sample. Then colony formation assay was performed after radiation for calculation of the surviving fraction (SF), as previously reported.16,37 Moreover, Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) was used in accordance with standard protocol,37 and then cell apoptosis was analyzed by flow cytometry (BD FACSCalibur). 2.9. In Vitro PTT−RT Synergistic Therapy. For the in vitro PTT−RT synergistic action, CSNPs at the concentrations of 0.2 mg/ mL were coincubated with 4T1 cells. After NIR laser exposure (808 C

DOI: 10.1021/acsami.6b13493 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. MR and CT contrast abilities of CSNPs. (a) T1-weighted (upper panel) and T2-weighted MR images (lower panel) for CSNPs at various Fe concentrations. (b) CT phantom images of CSNPs (upper panel) and Iohexol (lower panel) at different Au or I concentrations. (c) Linear relationship of T1 relaxation rate and T2 relaxation rates with various Fe concentrations. (d) CT values of CSNPs and Iohexol with different Au or I concentrations. sample. For group 7, NIR laser exposure was implemented, and after 30 min, X-ray radiation was implemented. Body weight and tumor volume of each mouse were measured every 2 days for 14 days (n = 5), while survival rates were also measured as the percentages of surviving mice compared to total mice for each group (n = 5). 2.13. Histology Analysis. At 14 days, major tissues including liver, heart, lung, kidney, spleen, and tumors were harvested from euthanized mice and weighed. After being fixed in 10% neutral formalin, paraffin-embedded tissues were sectioned for hematoxylin and eosin (H&E) staining and then observed under an optical microscope (DM5500B, Leica). 2.14. Data Analysis. Error bars were presented with mean and standard deviation for experiments repeated at least three times. Data analysis was performed using OriginPro 9.0 and Microsoft Excel. The significance between the groups were analyzed using a two-tailed t test (*, p < 0.05; **, p < 0.001; ***, p < 0.0001). Significance was indicated by p values of 0.05.

phase information from XRD data (Figure S3). PEI-modified PBNPs resulted in larger sizes with better size distribution (Figures 1c and S2b) with surface potentials increased from −21 to +10 mV, indicating successful amination of PBNPs (Figure 1f). Trisodium citrate stabilized AuNPs (Figure 1d) with mean diameters of 13.2 ± 0.8 nm (Figure S2c) were chosen to be synthesized as CT contrast agents and radiosensitizers according to our previous work for superior X-ray properties.37 Core−satellite structure of CSNPs (Figure S1d) indicated a good distribution of AuNPs satellites on the surface of the PBNP core. Finally after PEGylation, ultimate average diameter of CSNPs increased to 138.8 nm with better uniformity and distribution (Figures 1e,f, S1e, and S2d), while the surface potential of −10 mV ensured good colloidal stability53 (Figure 1e,f) (hereafter, the final product referred to as CSNPs for short). FTIR spectra measurements (Figure S4) also confirmed PEI and PEG modification by the appearance of the variations of characteristic functional groups. Small changes in DLS measured sizes and the maximum absorption wavelength of UV−vis−NIR spectra supported the idea that CSNPs could prevent aggregations in various physiological solutions until 28 days (Figure S5), indicating suitability for further use in clinical application.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of CSNPs. CSNPs were successfully fabricated by electrostatic adsorption strategy with specific reactions of amidogen and gold (Figure 1a).37,51,52 As-prepared PBNPs showed cube shape (Figures 1b and S1) with an average 87.3 of nm (Figure S2a), whose face-centered cubic structure was confirmed through the crystallography and D

DOI: 10.1021/acsami.6b13493 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 3. Photothermal performance of CSNPs. Thermographs (a) and corresponding temperature-changes curves (b) of CSNPs with various mass concentrations and pure water. (c) Temperature curves of CSNP solutions at 0.2 mg/mL under NIR laser exposure for 5five cycles (5 min for each cycle). (d) UV−vis−NIR absorption spectra of CSNPs solutions before and five cycles after radiation. All of the experiments were conducted under the same conditions (808 nm, 5 min, 1.5 W/cm2).

Strong absorbance in NIR regions is essential for PTT.19,41 UV−vis−NIR spectra exhibited wide absorption with a high absorption peak at about 735 nm for both PBNPs and PEI− PBNPs (Figure 1g). The color of the solutions changed from blue to lavender (Figure 1b−e) after AuNPs satellites attached, while the characteristic absorption peak at 525 nm of AuNPs was obviously observed without destroying the NIR absorbance of PBNPs (Figure 1g). In addition, the actual mass ratio of Fe and Au in CSNPs solutions was determined to be 7:4 by ICPMS. 3.2. CT and MR Contrast Capabilities. Next, we performed MR scanning for CSNPs solutions under five increasing concentrations. Concentration-dependent brightening and darkening effects were, respectively, observed by T1-weighted and T2-weighted MR images (Figure 2a). Concentration-normalized r1 and r2 relativity values were derived from the slope of inverse relaxation time versus the molar concentration of Fe. CSNPs showed r1 relativity values of 7.48 mM−1 s−1 and r2 relativity values of 8.02 mM−1 s−1 (Figure 2c). As reported previously, CSNPs may serve as T1-weighted MR contrast agent, which possessed a relatively high r1 value and a low r2-to-r1 ratio.52,54 In addition, a remarkably enhanced T2-weighted signal (Figure 2a) also suggested that CSNPs would also be used as a promising T2-weighted MR agent candidate. CSNPs compared to clinical-used contrast agent (iohexol) at different molar concentrations (gold or iodine) were monitored, and CT contrast phantom images indicated that CSNPs possessed stronger contrast enhancement than iohexol (Figure 2b), which were further determined by linear

relationships between CT values and concentrations. The slope of CSNPs (3.75 HU mM−1 [Au]) was higher than iohexol (2.69 HU mM−1 [I]) (Figure 2d), indicating superior CT contrast capabilities of CSNPs. Moreover, MR contrast of CSNPs compared to PBNPs were studied and showed no obvious changes of r1 or r2 relativity values (Figure S6), indicating that AuNPs satellites could not affect the MR contrast abilities of PBNP cores. 3.3. Photothermal Effect. The photothermal conversion properties of CSNPs were investigated and temperature change curves presented that temperature of CSNPs solutions and PBNP core increased quickly to about 50 °C, while the temperature of pure water or AuNPs solutions only increased to less than 35 °C during NIR laser irradiated time (Figure S7a), suggesting excellent photothermal efficiency of CSNPs mainly attributed to PBNP cores. Among frequently used NIR laser power densities, a power density of 1.5 W/cm2 was selected, at which the temperature of CSNPs could smoothly rise to 50 °C within 5 min, less than the 60 °C that is much higher for in vivo application (Figure S7b). The obvious temperature increases with concentrations of CSNPs were shown during NIR laser exposure compared to water with little change (Figure 3a,b). It was found that the photothermal conversion ability remained strong without dropping during NIR laser exposure for five cycles (Figure 3c). No significant change was observed in NIR absorption spectrum for five cycles irradiation (Figures 3d and S8) and in DLS measured size of CSNPs after 5 min of NIR laser exposure (Figure S9), demonstrating the excellent photothermal stability of CSNPs. E

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Figure 4. In vitro analysis of PTT−RT synergistic therapy. (a) Fluorescence images for 4T1 cells with different treatments by calcein AM (live cells, green) and PI (dead cells, red) staining after NIR laser exposure. (b) Flow cytometry plots for cellular apoptosis and necrosis after different treatments. (c) Cell viability of 4T1 cells incubated with CSNPs at different mass concentrations after different treatments. (d) Cellular PTT−RT synergistic therapy for 4T1 cells incubated with CSNPs (0.2 mg/mL). All of the experiments were conducted under the same conditions (PTT: 808 nm, 5 min, 1.5 W/cm2; RT: 4 Gy). Error bars: standard deviation of mean for at least three replicates. P values: *, P < 0.05.

3.4. In Vitro PTT−RT Synergistic Therapy. Encouraged by the above results, we further assessed CSNPs for in vitro 4T1 cancer cell destruction, and there was no obvious cytotoxicity for CSNPs (Figure S10). Calcein AM and propidium iodide (PI) dual staining was carried out to visualize the PTT effect. The cells treated with CSNPs upon NIR laser exposure were considerably killed, while those either only under NIR laser exposure or treated by CSNPs alone were not affected with PBS treatment as the control (Figure 4a). Moreover, with the increase of CSNPs concentrations, more cells demonstrated much-lower cell viability after PTT treatments by MTT results ( Figure S11). Clonogenic assays were performed to estimate an enhanced RT effect, and significant separations of CSNPs and AuNPs with PBNPs and radiation alone from surviving fraction curves (Figure S12) revealed that AuNPs satellites contributed significantly to the radiosensitization of CSNPs. Cell viability greatly decreased with X-ray radiation doses and concentrations of CSNPs (Figure S13). Flow cytometry experiments were implemented to quantitatively analyze the CSNPs-caused apoptotic or necrotic cell death.55,56 Only PBS or CSNPs could not produce significant cell death. Apoptosis and necrotic ratios were 31% for CSNPs radiosensitization, which were much higher than 6% for RT alone (Figure 4b). The capabilities of CSNPs as photothermal agents and radiosensitizers simultaneously were then quantitatively evaluated by MTT assays. More than 88% of the cells treated with CSNPs alone are still alive at up to 0.4 mg/mL. However, cell

viability greatly dropped to 63% after NIR laser exposures, while the percentage declines to 42% after X-ray radiations, much lower than NIR or X-ray radiation alone without CSNPs (Figure 4c,d). The results of PTT−RT synergistic action demonstrated that only less than 11% of cells remained viable after these combinational treatments, which presented 51% and 31% below CSNPs-induced PTT and RT and even 15% below their projected values (Figure 4d).5 All of these results strongly demonstrated a great potential of CSNPs for synergetic PTT− RT therapy in the future. 3.5. In Vivo Multimodal Imaging. Then, MR and CT imaging in vivo was performed using 4T1 xenograft tumors bearing mice before and after a 24 h injection of CSNPs (10 mg/mL, 100 μL) intravenously (Figure 5a). Enhanced T1weighted (Figure 5b) and T2-weighted (Figure 5d) MR signal intensity at tumor sites was observed, which increased from 3298.2 to 4933.7 (Figure 5c) and decreased from 6183.5 to 4364.9 (Figure 5e), respectively. In the meantime, CT images showed the strong contrast at tumors (Figure 5f), and CT values increased from 32.6 HU to 72.4 HU (Figure 5g), indicating the efficient passive accumulation of CSNPs by the enhanced permeability and retention effect (EPR) of tumors. Therefore, CSNPs could realize precise location of tumor sites and the combination of CT and MR imaging, which could be used as a promising MR−CT diagnostic agent for clinical multimodal-imaging-guided therapy in the future. 3.6. In Vivo PTT−RT Synergistic Therapy. After validating the above favorable results, we further examined F

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Figure 5. In vivo analysis of MR−CT imaging. (a) Scheme of CSNPs (10 mg/mL, 100 μL) intravenously injected into mice bearing 4T1 xenograft tumors for in vivo imaging and then aggregated in tumors through EPR effect followed by X-ray and NIR irradiations. (b,c) T1-weighted MR images and quantitative analysis of signals in tumors. (d,e) T2-weighted MR images and quantitative analysis of signals in tumors. (f,g) CT images and quantitative analysis of CT signals in tumors.

The results revealed the fact that RT could kill deeply located cancer cells, and PTT could make the radio-resistant hypoxic cells more sensitive to X-rays and easily killed.47,58−60 No significant weight loss was observed for all groups (Figure 6e). The survival time obviously showed that the synergistic treatments by CSNPs + NIR laser + RT could remarkably delay the mice death (Figure S14), which were further identified by H&E staining results. The more obvious tumor destruction was noticed in group 7 (CSNP + NIR laser + RT) than in groups 4 (CSNPs + NIR laser) or 6 (CSNPs + RT), retaining more tumor cells with the normal morphology, further demonstrating the inhibition results of tumor growth rates (Figures 6f and S15). In vivo toxicity of CSNPs was analyzed by H&E staining, although PEG and PB had been approved by the FDA, and AuNPs have been verified for good biocompatibility.18−20,61

the efficacy of PTT−RT synergetic therapy in vivo using 4T1 xenograft tumor bearing mice by intravenous injection with CSNPs (10 mg/mL, 100 μL) for different treatments. Tumor temperature images showed that during the PTT process, the tumors without any treatment showed less change in temperature (below 40 °C), while the temperature of the CSNPs-treated tumors rapidly increased to ∼55 °C (Figure 6a,b). CSNPs-treated tumors under NIR irradiation or X-ray radiation alone could partially suppress tumor growth, confirmed, respectively, by a considerable tumor growth inhibition of 39.06% and 47.21% (TGI) (Figure 6c,d).5,16,57 More effectively suppressed tumor growth of 64.27% (TGI) was attributed to the radiosensitization induced by CSNPs. The near-eradication of the tumors was shown in several days (TGI ≈ 94.35%; Figure 6c,d), which proved the synergistic action between CSNPs-induced RT and PTT in vivo. G

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Figure 6. In vivo analysis of PTT−RT synergistic therapy. Thermographs (a) and corresponding temperature changes (b) of mice bearing 4T1 xenograft tumors during NIR laser exposure after intravenous injection of CSNPs (10 mg/mL, 100 μL) for 24 h. (c) Representative photos of harvested tumors from mice at day 14 after different treatments (n = 5). Tumor growth inhibition curves (d) and body weight changes (e) for various treatments (n = 5). (f) H&E staining results at day 14 after different treatments. All of the experiments were conducted under the same conditions (PTT: 808 nm, 5 min, 1.5 W/cm2; RT: 4 Gy). Error bars: standard deviation of mean for at least three replicates. P values: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

sensitization from AuNP satellites, revealing remarkably efficient therapeutic effects compared to any individual treatment and completely tumor eradication without regrowth over our studied therapeutic period of 14 days by the intravenous injection of CSNPs. More importantly, CSNPs could exhibit no significant toxicity observed in our studies, which would have much fewer barriers toward future clinical application on patients because of FDA-approved component PB and PEG with the already studied excellent biocompatibility of AuNPs. Our preliminary results clearly demonstrated that PBNPs and AuNPs possess intrinsic theranostic properties, and integrating both could achieve precise diagnostic guidance and significant therapeutic efficiency. We also anticipate that our integration strategy could be further used to attach photoacoustic imaging or other imaging modalities with photodynamic therapy, chemotherapy, or other therapeutic protocols based on red-emissive carbon dots or other intrinsic theranostic nanomaterials. Therefore, multifunctional nanotheranostics based on the combination of intrinsic theranostic nanomaterials could demonstrate fascinating strategies and prospects in the future.

There were no obvious organ damages or toxic side effects, such as inflammatory lesions observed at our tested dose in all major organs (Figure S16). To quantitatively detect the amount of CSNPs accumulated in different tissues after the treatments, the Fe and Au contents were measured by ICP. The high Fe and Au contents in tumor sites suggested that the size and in vivo circulation time of CSNPs were appropriate for EPR effects of tumors, although CSNPs were uptaken by other organs, especially liver and spleen. It was also found that more than 90% of CSNPs could be cleared from the body after 7 days after intravenous injection (Figure S17). All of these results convincingly guarantee the in vivo stability and biocompatibility of our synthesized CSNPs as nanotheranostic agents for future biomedical researches and applications.

4. CONCLUSIONS In this work, a multifunctional nanotheranostic system was successfully constructed based on PB@Au core−satellite nanoparticles (CSNPs) by integrating PBNP cores and AuNP satellites together with PEG modification for achieving MR− CT multimodal imaging and synergetic PTT−RT therapy. Results of our work demonstrated that effective MR and CT imaging could be both achieved by CSNPs as an excellent T1and T2-weighted MR and CT contrast agent for acquiring accurate location of the tumors in vivo by passively targeted accumulation. Moreover, upon exposure to NIR and X-ray irradiation, multimodal imaging-guided synergetic PTT−RT therapy can be achieved by CSNPs with high photothermal conversion efficiency from PBNP cores and great radio-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b13493. H

DOI: 10.1021/acsami.6b13493 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

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Images showing TEM results, size distributions, Fourier transform infrared spectra, the colloidal stability of CSNPs, MR contrast abilities of CSNPs and PBNPs, the photothermal effects of different nanoparticles and power densities, UV−vis−NIR absorption spectra, DLS measured size change of CSNPs after irradiation, cell viability, clonogenic survival assays, survival rates of 4T1 tumor-bearing mice after different treatments, H&E staining results, and in vivo biodistribution of iron and gold element in different organs. (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jin Chang: 0000-0002-6752-8526 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (grant nos. 51373117 and 51573128), Key Project of Tianjin Natural Science Foundation (grant no. 16JCZDJC35100), and the Key Project of Tianjin Health and Family Planning Committee Foundation (grant no. 15KG137)

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