An Extendable Star-Like Nanoplatform for Functional and Anatomical

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An Extendable Star-Like Nanoplatform for Functional and Anatomical Imaging-Guided Photothermal Oncotherapy Xuexiang Han, Ying Xu, Yiye Li, Xiao Zhao, Yinlong Zhang, Huan Min, Yingqiu Qi, Gregory J. Anderson, Linhao You, Yuliang Zhao, and Guangjun Nie ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09607 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

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An Extendable Star-Like Nanoplatform for Functional and Anatomical Imaging-Guided Photothermal Oncotherapy Xuexiang Han1,2,3#, Ying Xu1,4,5#, Yiye Li1,3*, Xiao Zhao1,3, Yinlong Zhang1,3, Huan Min1,3, Yingqiu Qi6, Gregory J. Anderson7, Linhao You8, Yuliang Zhao1,3* and Guangjun Nie1,3* 1 CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, P. R. China 2 Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China 3 Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, P. R. China 4 Sino-Danish College, Sino-Danish Center for Education and Research, University of Chinese Academy of Sciences, Beijing 100049, P. R. China 5 Department of Pharmacy, Copenhagen University, Universitetsparken 2, DK-2100 Copenhagen, Denmark 6 School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China 7 QIMR Berghofer Medical Research Institute, Royal Brisbane Hospital, QLD 4029, Australia 8 Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University, Shijiazhuang, Hebei Province 050024, P. R. China #These

authors contributed equally to this work.

*To whom correspondence may be addressed: [email protected] (Yiye Li), [email protected] (Yuliang Zhao), [email protected] (Guangjun Nie). 1 ACS Paragon Plus Environment

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ABSTRACT

Combining informative imaging methodologies with effective treatments to destroy tumor is of great importance for oncotherapy. Versatile nanotheranostic agents that inherently possess both diagnostic imaging and therapeutic capabilities are highly desirable to meet these requirements. Here, a simple but powerful nanoplatform based on polydopamine-coated gold nanostar (GNS@PDA), which can be easily diversified to achieve various function extensions, is designed to realize functional and anatomical

imaging-guided

intrinsically

enables

photothermal computed

oncotherapy.

This

nanoplatform

tomography/photoacoustic/two-photon

luminescence/infrared thermal tetramodal imaging, and can further incorporate fibroblast activation protein (FAP, a protease highly expressed in most of tumors) activatable near-infrared fluorescence imaging and Fe3+-based magnetic resonance imaging for comprehensive diagnosis. Moreover, GNS@PDA exhibits excellent photothermal performance and efficient tumor accumulation. Under the precise guidance of multimodal imaging, GNS@PDA conducts homogeneous photothermal ablation of bulky solid tumors (∼200 mm3) in a xenograft mouse model. These results suggest great promise of this extendable nanoplatform for cancer theranostics.

KEYWORDS:

gold

nanostar,

polydopamine,

fibroblast

activation

protein,

photothermal oncotherapy, multimodal imaging, theranostics

2 ACS Paragon Plus Environment

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Malignant tumor is a major public health threat and is the second leading cause of death worldwide.1 Late diagnosis and profound resistance to existing therapies are two important factors that lead to poor prognosis of tumor patients, which underlines the urgent need for effective detection and treatment of tumor.

Multimodal imaging that can provide complementary information, such as anatomic, pathological and molecular information of tumor characteristics, holds great promise for accurate diagnosis, guiding therapeutic intervention, trafficking drug distribution and monitoring therapeutic response.2,3 X-ray computed tomography (CT) is a radiographic imaging system possessing the advantages of high resolution, no depth limitation, and three-dimensional (3D) reconstruction.4-6 Magnetic resonance imaging (MRI) can provide clear soft tissue structure with excellent spatial resolution.2 Photoacoustic imaging (PAI), an emerging imaging modality based on the photoacoustic effect, can offer high spatial resolution with real-time monitoring.7-9 Compared with these anatomical imaging modalities, activatable near-infrared fluorescence (NIRF) imaging, a highly desirable functional imaging approach, can be rationally designed to visualize the expression and activity of specific molecules for pathological detection with low cost, high specificity and high sensitivity.10-14 Since single modality is insufficient to provide all the necessary information, the combination of CT, MRI, PAI and NIRF imaging, which can integrate respective advantages and provide complementary functional and anatomical information, will be highly preferable for accurate diagnosis of tumor.15-17


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Currently, nanomaterial-based platforms have drawn considerable attention for multimodal imaging.3,18 In addition, inherent therapeutic properties upon stimulation make some imageable nanomaterials more attractive.19-22 One example is the nanomaterials with additional property of photothermal conversion, which can further conduct photothermal oncotherapy (PTT) under precise guidance and have demonstrated great potential in theranostic applications due to its precise spatial-temporal selectivity, efficient tumor ablation and minimum adverse effects on collateral tissues.23-27 However, current nanotheranostics integrate all ingredients together following “all-in-one” strategy, which largely restrict their biomedical applications due to complex compositions, difficult synthesis, poor stability and potential safety concerns.28,29 Herein, we develop an extendable nanoplatform based on a polydopamine-coated gold nanostar (GNS@PDA), which can further integrate different imaging modalities as needed through facile building process, for multimodal imaging-guided PTT (Scheme 1). The inner GNS, an ideal photothermal conversion agent with tunable plasmon peak in the NIR region (600-1100 nm),30-32 can serve as not only an excellent contrast agent for two-photon luminescence (TPL), CT and PA imaging, but also an efficient NIRF quencher. The versatile PDA shell allows further function extensions of this theranostic nanoplatform: (i) Quinone groups in PDA can react with thiol-containing peptide via Michael addition,33-35 which can be utilized to design tumor-associated

protease-activatable

fluorescent

probe.

As

a

paradigm,

Cyanine7-labeled peptide (Cy7-KTSGPNQC) was linked to GNS@PDA to build a 4 ACS Paragon Plus Environment

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fibroblast activation protein (FAP, a protease highly expressed in most of tumors) specific NIRF nanoprobe;36-39 (ii) PDA shows natural binding ability with various metal ions to serve as contrast agents.40-43 Fe3+-chelated GNS@PDA was demonstrated here for T1-weighted MRI. This nanoplatform exhibited high photothermal stability and photothermal conversion ability, and achieved efficient cellular uptake as well as tumor accumulation. During laser irradiation, the photothermal performance of GNS@PDA and the therapeutic response were monitored by infrared thermal (IRT) imaging. Our results show that this extendable and versatile nanotheranostics not only allows multimodal imaging for visualizing tumor structures and behaviors, but also realizes homogeneous photothermal ablation of tumor under precise guidance. RESULTS AND DISCUSSION

Synthesis and Characterization of GNS@PDA-based Nanoplaform. The construction of GNS@PDA-based extendable nanoplatform for functional and anatomical imaging-guided PTT is schematically illustrated in Scheme 1. GNS@PDA was easily prepared within one hour in a two-step, one-pot fashion. First, GNS was synthesized following a seed-mediated surfactant-free method through reduction of HAuCl4 by ascorbic acid.30 Subsequently, the direct coating of polydopamine onto the GNS was conducted using a one-pot procedure through self-polymerization of dopamine monomer, resulting in the formation of GNS@PDA. Transmission electron microscopy (TEM) showed that the average size of GNS@PDA was approximately 5 ACS Paragon Plus Environment

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90 nm with a star-shaped core and a 4 nm thick PDA shell (Figure 1a). GNS@PDA exhibited good dispersity (PDI = 0.19) with a hydrodynamic diameter of 91.2 ± 2.6 nm and zeta potential of 7.2 ± 0.2 mV. PEGylated GNS (GNS-PEG), with similar physical parameters (Table S1 and Figure S1,2), was synthesized as control according to a previous report.44 Notably, GNS-PEG displayed obviously increased hydrodynamic diameter in DMEM medium, indicating its severe aggregation under physiological conditions (Figure S3). In contrast, GNS@PDA maintained its excellent colloidal stability due to PDA shell-mediated good protection.45,46

As an extendable nanoplatform, GNS@PDA can be further diversified to achieve functional imaging, which was illustrated here by endowing GNS@PDA with FAP-activatable NIRF imaging modality. Cy7 with maximum emission wavelength at 774 nm was chosen to label the FAP specific peptide substrate (KTSGPNQC).36-38 A scrambled peptide sequence (KTGSPQNC) was also labeled with Cy7 to serve as an uncleavable control (Figure S4). Successful conjugation was indicated by a Cy7-peptide peak (m/z = 1539) on the mass spectrum of the purified product (Figure S5). As expected, the Cy7-KTSGPNQC conjugate could be cleaved by FAP at the site between Pro and Asn to give the fragment Cy7-KTSGP,37 but the scrambled sequence could not (Figure S6). Afterwards, the thiol groups in the cysteine of the Cy7-peptide reacted with the quinones in the PDA shell via the Michael addition reaction, forming an irreversible covalent bond under mild alkaline conditions,33,34 and thereby anchoring the Cy7-peptide on the surface of the GNS@PDA to built GNS@PDA-Cy7 or GNS@PDA-Cy7/scr (Figure S7). Successful conjugation was confirmed by 6 ACS Paragon Plus Environment

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UV-vis spectroscopy, as GNS@PDA displayed a strong absorption band at ca. 808 nm, while GNS@PDA-Cy7 spectrum showed an additional shoulder band at ca. 740 nm, which originated from the characteristic Cy7 absorption peak (Figure 1b). Moreover, the hydrodynamic size of GNS@PDA-Cy7 increased slightly to 95.6 ± 3.1 nm, and the zeta potential reversed to -29.4 ± 0.5 due to the anionic Cy7 molecules. Conjugation of the Cy7-peptide to GNS@PDA led to prominent fluorescence quenching (Figure 1c,d) due to the efficient nanometal surface energy transfer (NSET) effect resulting from the overlap between the fluorescent emission of Cy7 and the broad absorption of GNS@PDA and their spatial proximity.47 Notably, nanoprobes based on reversible Au-S bonds (here illustrated using GNS-Cy7 as control) run the risk of displacement from the gold surface by biothiols like glutathione (GSH),48,49 which might give ‘false positive’ signals (Figure S8). However, GNS@PDA-Cy7 based on an irreversible covalent bond can offer a more reliable alternative to Au-S bond with reduced signal distortion. FAP-activatable NIRF Imaging in Vitro. First, the catalytic kinetics of FAP towards GNS@PDA-Cy7 was investigated (Figure S9). The Michaelis-Menten constants (Km) and the catalytic rate constants (kcat) of FAP towards GNS@PDA-Cy7 were calculated to be 24.6 μM and 12.1 min-1, respectively. Therefore, the catalytic efficiency (kcat/Km) of FAP towards GNS@PDA-Cy7 was found to be 4.9 × 105 M-1 min-1, which indicates that the conjugated Cy7-KTSGPNQC can be effectively cleaved by FAP, although it is approximately 2-fold lower than the catalytic efficiency of FAP towards free Cy7-KTSGPNQC and other reported probes.38,50 7 ACS Paragon Plus Environment

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Afterwards, FAP-selective activation of GNS@PDA-Cy7 for NIRF imaging was systematically investigated in both noncellular and cellular level. Incubation of GNS@PDA-Cy7 with FAP enzyme led to a time- and FAP concentration-dependent increase in NIRF signal (Figure S10). In particular, incubation with 0.5 μg/mL FAP led to a 27-fold increase in fluorescence emission intensity of GNS@PDA-Cy7 within 6 h. In contrast, no NIRF signal was detected when GNS@PDA-Cy7/scr was used. Subsequently, we screened Mia-paca-2 cells (a human pancreatic cancer cell line with high expression of FAP) for in vitro and in vivo experiments, and HPNE cells (a human noncancerous pancreas cell line with no expression of FAP) as control (Figure S11). As expected, the supernatants of Mia-paca-2 cells incubated with GNS@PDA-Cy7 showed obvious fluorescence activation and strong NIRF signal (Figure 1e and Figure S12). In comparison, the supernatants of neither Mia-paca-2 cells incubated with GNS@PDA-Cy7/scr nor HPNE cells incubated with GNS@PDA-Cy7 displayed an obvious NIRF signal. Moreover, incubation of Mia-paca-2 cells with GNS@PDA-Cy7 resulted in a time-dependent activation of NIRF signal in the supernatants (Figure 1f). The time-dependent NIRF activation was also confirmed by flow cytometry analysis of Cy7 fluorescence signal in Mia-paca-2 cells

(Figure

1g,h).

Furthermore,

a

cell

number-dependent

activation

of

GNS@PDA-Cy7 was observed in Mia-paca-2 culture medium (Figure 1i). These data strongly support that GNS@PDA-Cy7 is an effective “turn on” nanoprobe for imaging FAP activity in cancer cells.


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Photothermal Properties. GNS@PDA is not only an extendable platform for building activatable NIRF nanoprobe, but also an excellent photothermal agent itself. To evaluate its photothermal properties, GNS@PDA with different concentrations were exposed to an NIR laser and the temperature changes of the solutions were monitored using an infrared thermal camera. Upon irradiation, GNS@PDA could effectively convert light energy to heat with a rapid increase of temperature in a concentration-dependent manner, but negligible temperature elevation was observed in water (Figure 2a). At the same GNS@PDA concentration (50 μg/mL), laser power-dependent temperature increases were also observed (Figure 2b,c). Notably, GNS@PDA achieved a higher temperature than its control GNS-PEG (55.5 °C vs. 52.7 °C), and the photothermal conversion efficiencies (η) of GNS@PDA and GNS-PEG were calculated to be 36.5% and 30.6%, respectively (Figure 2d,e and Figure S13). The enhanced photothermal conversion efficiency of GNS@PDA was derived from the auxiliary photothermal effect of PDA, as PDA itself serves as an outstanding photothermal agent.45 The photothermal conversion efficiency of GNS@PDA was also higher than that of some inorganic nanoparticles,51,52 but lower than that of semiconducting polymer nanoparticles and compact plasmonic blackbody.53,54 In addition, PDA layer can also improve the photothermal stability of GNS thanks to the restricted rearrangement of gold atoms,55 as the photothermal performance of GNS@PDA remained unchanged after three cycles of laser on/off, whereas the performance of GNS-PEG declined obviously (Figure 2f). Above all, GNS@PDA shows high photothermal conversion ability and excellent photothermal 9 ACS Paragon Plus Environment

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stability, which encourages further assessment of its photothermal cytotoxicity in vitro. Photothermal Killing in Vitro. Firstly, the biocompatibility of GNS@PDA was evaluated. While GNS-PEG exhibited dose-dependent cytotoxicity to HPNE cells, the cell viabilities of both HPNE cells and Mia-paca-2 cells were not influenced by GNS@PDA at concentration up to 100 μg/mL (Figure S14), suggesting the improved biocompatibility after PDA coating.56 Efficient plasmon-enhanced two-photon luminescence (TPL) from anisotropic GNS offered a convenient way to visualize its cellular uptake behavior, thus TPL images of Mia-paca-2 cells after treatment with GNS@PDA or GNS-PEG were acquired using a two-photon laser scanning confocal microscopy. As shown in Figure 3a, GNS@PDA exhibited much stronger signal inside cells than GNS-PEG, indicating the more efficient endocytosis of GNS@PDA compared with GNS-PEG. The low internalization efficacy of GNS-PEG was presumably due to the inhibitory role of PEGylation on cellular uptake.57,58 To assess the photothermal killing capacities of GNS@PDA and GNS-PEG, treated cells were double-stained with calcein AM (green fluorescence) and propidium iodide (PI, red fluorescence) to directly visualize live and dead cells, respectively. No cell killing was observed in the control, GNS@PDA only, GNS-PEG only or laser only groups (Figure 3b), as indicated by exclusively green fluorescence (live cells). In contrast, in the GNS@PDA/laser group, most of the cells were killed and displayed massively red fluorescence (dead cells). As a result of inferior photothermal performance and insufficient cellular internalization of GNS-PEG, the photothermal killing efficiency 10 ACS Paragon Plus Environment

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was largely compromised in the GNS-PEG/laser group. Quantitatively, the photothermal cytotoxicity was examined using the cell counting kit 8 (CCK-8) assay. Upon irradiation, cell viability decreased rapidly as the concentration of GNS@PDA increased, with a cell viability of less than 30% at a concentration of 100 μg/mL (Figure 3c). Consistent with the live/dead cell staining results, GNS@PDA exhibited stronger phototoxicity than GNS-PEG, which could be attributed to the PDA-enhanced cellular uptake and photothermal performance. We next investigated the penetration ability and phototoxicity of GNS@PDA in Mia-paca-2-derived stroma rich spheroids (Mia-SS). Mia-SS is a 3D tumor model to mimick the pathological penetration barrier of nanomaterials in solid tumor.59,60 TPL images of Z-stack scanning revealed that GNS@PDA exhibited deeper penetration and more accumulation than GNS-PEG owing to PDA-enhanced colloidal stability and cellular uptake, although both of them were detained mainly at the perimeter of Mia-SS (Figure 3d). Pretreated Mia-SS were then irradiated and photothermal cytotoxicity was visualized by PI staining. While the control Mia-SS showed few dead cells (Figure 3e), GNS@PDA/laser triggered extensive cell death within the 3D environment of Mia-SS owing to the lethal hyperthermia and its thermal conduction. In contrast, less cell death was observed in the GNS-PEG/laser group due to its less accumulation and inferior phototoxicity compared to GNS@PDA. Collectively, these data demonstrate that GNS@PDA is an effective TPL imaging agent as well as a potent PTT agent that holds great promise for in vivo tumor therapy.


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Anatomical and Functional Imaging in Vivo. Nanotheranostics that offer complementary anatomical and functional information of tumor are of great importance for precise therapy. Thus, we evaluated the multimodal imaging performance of this extendable nanoplatform in Mia-paca-2 tumor-bearing mice. Due to the strong X-ray attenuation capability of Au,61 GNS@PDA could serve as an excellent CT imaging contrast agent, as demonstrated by a linear increase in Hounsﬁeld units (HU) value as a function of concentration (Figure S15). Additionally, after intratumoral injection of GNS@PDA, the 3D reconstructed CT images of tumor-bearing mouse exhibited enhanced contrast at the tumor site (Figure 4a). GNS@PDA could also serve as an outstanding PAI agent owning to its strong NIR absorbance and efficient photothermal conversion. GNS@PDA was able to induce a strong PA signal, which strictly followed a concentration-dependent manner (Figure S16). To visualize the tumor morphology and the accumulation of nanomaterials in vivo, PA images of tumors were acquired at different time post intravenous injection of GNS@PDA or GNS-PEG (Figure 4b,c). Both of them exhibited enhanced PA signals at the tumor margin at 30 min post-injection and the PA signals reached the maximum values at 6 h post-injection due to the efficient tumor accumulation via enhanced permeability and retention (EPR) effect.62 Notably, the signals from GNS@PDA were not only stronger than those from GNS-PEG, but also located more uniformly inside the tumors, suggesting more efficient accumulation and penetration of GNS@PDA as a result of its improved colloidal stability and efficient internalization. The TPL images of tumor slices and quantification of Au content by 12 ACS Paragon Plus Environment

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inductively coupled plasma mass spectrometry (ICP-MS) further confirmed more tumor accumulation of GNS@PDA compared to GNS-PEG (Figure S17 and S18). MRI can provide clear soft tissue structure without ionizing radiation. To introduce MR imaging capability into GNS@PDA, Fe3+ was incorporated by utilizing the intrinsic chelating function of PDA,41 which led to the formation of GNS@PDA-Fe (Figure S19 and Table S1). T1-weighted MR images showed a linear correlation between the MR signals and Fe concentrations with a longitudinal relativity (r1) of 6.94 mM-1s-1. When GNS@PDA-Fe was used as a MR contrast-enhancing agent for tumor imaging, a clear tumor outline was delineated at 30 min post intravenous injection, indicating its efficient accumulation at the periphery of the tumor (Figure 4d). With the gradual accumulation and penetration of GNS@PDA-Fe, the tumor exhibited the strongest MR signal at 6 h post-injection, which was in agreement with PA imaging results. The diagnostic advantages of GNS@PDA could be further strengthened by complementing above anatomical imaging modalities with functional imaging modality. We then evaluated the FAP imaging ability of GNS@PDA-Cy7 in tumor-bearing mice, since western blot result supported that FAP expression was high in tumor and negligible in normal tissue (Figure S20). First, the specific NIRF activation of GNS@PDA-Cy7 was confirmed by intratumoral injection, as the tumor injected with GNS@PDA-Cy7 showed increased NIRF signals while the tumor injected with GNS@PDA-Cy7/scr did not (Figure S21). Afterwards, the FAP-activatable NIRF imaging was evaluated after its intravenous injection. In vivo 13 ACS Paragon Plus Environment

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whole-body imaging of mice revealed increased NIRF signals at the tumor sites and the signals reached the maximum values at 6 h post-injection (Figure 4e,f). Ex vivo results showed that the tumors from GNS@PDA-Cy7 treated mice had a much stronger NIRF signal than other organs (Figure 4g,h), which suggested the effective activation of GNS@PDA-Cy7 by FAP in tumor. Since FAP has been found to be over-expressed in over 90% of tumors and its high expression has been associated with tumor malignancy and poor prognosis,39,63,64 this “turn on” nanoprobe is valuable to reveal pathological information and detect tumor with high specificity and sensitivity. Photothermal Tumor Ablation. The adequate diagnostic information provided by multimodal imaging guided us to carry out precise PTT. At 6 h post intravenous injection, an infrared thermal camera was used to monitor the temperature variation and therapeutic response of tumor-bearing mice during laser irradiation (Figure 5a,b). Infrared thermal (IRT) imaging revealed that the tumor temperature increased rapidly and maintained at approximately 54.1 °C or 50.3 °C for mice injected with GNS@PDA or GNS-PEG, respectively. The better photothermal performance of GNS@PDA in vivo is derived from its greater tumor accumulation and higher photothermal conversion efficiency. As control, mice treated with saline showed only a slight increase in tumor temperature (38.1 °C). After a single irradiation, tumor volumes and body weights were recorded every other day until the end of experiment. Neither laser treatment nor GNS@PDA 14 ACS Paragon Plus Environment

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treatment showed any effect on tumor growth (Figure 5c,d,e). Encouragingly, GNS@PDA/laser treatment led to successful elimination of tumors in most mice without tumor regrowth, presumably due to its highly effective photothermal killing performance. The representative photos of tumor-bearing mice revealed that the tumor in GNS@PDA/laser group was homogenously ablated with a gradually healing scar at the initial tumor site (Figure S22). In contrast, tumor growth was initially inhibited in GNS-PEG/laser group and tumor regrowth was prominent at 6 days post-treatment. Moreover, hematoxylin and eosin (H&E) staining of tumor sections demonstrated much more extensive necrosis in GNS@PDA/laser group than that in GNS-PEG/laser group (Figure 5f). There were no obvious changes in body weight of the mice during the entire treatment period (Figure S23). To further assess the in vivo toxicity, the major organs and blood of mice were obtained for histopathological examination and serum biochemical analysis, respectively. The results demonstrated that there were neither noticeable histopathological changes in major organs (Figure S24), nor obvious changes in markers of liver or kidney function in any of the treatment groups (Figure S25). It was worth mentioning that the superior anti-tumor efficacy of GNS@PDA/laser treatment against large size tumors (~200 mm3) without noticeable side effects suggested its appealing clinical practice for tumor treatment. CONCLUSION We have successfully prepared a versatile nanoplatform based on GNS@PDA with intrinsic CT/PA/TPL/IRT and extended FAP-activatable NIRF as well as Fe3+-enabled MR imaging for precise cancer theranostics. The CT, PA and MR 15 ACS Paragon Plus Environment

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imaging provide detailed tumor positional information, the TPL imaging confirms efficient internalization and distribution of GNS@PDA, the NIRF “turn on” imaging reveals valuable pathological information of tumor, and the IRT imaging gives real-time therapeutic response during PTT. As a result of the precise guidance and the robust hyperthermia effect of GNS@PDA, homogenous photothermal ablation of tumors is fulfilled in a single treatment with good tolerance. These results strongly suggest that GNS@PDA offers an extendable and multifunctional nanoplatform with great clinical translation potential for cancer theranostics. Considering various replaceable imaging-related metal ions and tumor-associated protease specific substrates, this work encourages further exploration of GNS@PDA-based nanotheranostics for functional and anatomical imaging-guided PTT. EXPERIMENTAL SECTION Materials. Hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O), L-ascorbic acid (AA), silver nitrate (AgNO3), and trisodium citrate were purchased from Sigma-Aldrich. FAP cleavable peptide (KTSGPNQC) and scrambled peptide (KTGSPQNC) were customized from Top-peptide Co., Ltd. (Shanghai, China) with the purity more than 95%. Sulfo-Cy7 NHS ester and thiolated poly(ethylene glycol) (HS-PEG, MW 2000) were purchased from Xi’an Ruixi Biological Technology Co., Ltd. (Xi’an, China). Cell counting kit-8 (CCK-8), calcein acetoxymethyl ester (Calcein-AM), and propidium iodide (PI) were provided by Dojindo Laboratories. 35 mm confocal petri dishes (Cellvis, Mountain View, CA) were purchased from 16 ACS Paragon Plus Environment

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Hangzhou Xinyou Biotechnology Co., Ltd. (China). Other chemicals were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). All reagents were analytical grade and were used without further purification. Ultrapure water (resistivity higher than 18.2 MΩ·cm-1) was used in all preparations and all glassware was cleaned using aqua regia followed by repeated wash with ultrapure water.

Preparation of Cy7-labeled FAP Substrate. Water soluble sulfo-Cy7 NHS ester was

conjugated

to

the

N-terminal

lysine

of

the

FAP

substrate,

Ac-KTSGPNQC-CONH2. Sulfo-Cy7 NHS ester (2.5 mg, 3.2 μmol) was coupled with the peptide (5 mg, 5.7 μmol) in anhydrous DMF (500 μL) containing 1% TEA at room temperature in the dark for 2 h with vortexing. The final Cy7-KTSGPNQC product was purified by preparative reversed-phase HPLC and fractions containing the product were collected. The product was characterized by mass spectrometry and stored at -20

oC.

The Cy7-labeled scrambled peptide, Cy7-KTGSPQNC was

synthesized by the same method.

Peptide Cleavage by rhFAP. Recombinant human fibroblast activation protein α (rhFAP; R&D system, USA) was diluted to 0.2 μg/mL in assay buffer (50 mM Tris, 1 M NaCl, 1 mg/mL BSA, pH 7.5) and 50 μL of this solution was loaded into a 96-well plate. The reaction was started by adding 50 μL of 100 μM Cy7-KTSGPNQC or Cy7-KTGSPQNC (dissolved in assay buffer). After 3 h, the reaction products were analyzed by mass spectrometry.


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Preparation of GNS@PDA and GNS-PEG. Firstly, a 15 nm citrate-stabilized gold seed solution was synthesized and kept at 4 °C for long-term storage.65 Gold nanostar (GNS) was prepared by a seed-mediated surfactant-free method as previously reported.30 Briefly, 100 μL of seed solution was added to 10 mL of 0.25 mM HAuCl4 solution along with 10 μL of 1 M HCl under 700 rpm magnetic stirring. Simultaneously, 100 μL of 1 mM AgNO3 and 50 μL of 100 mM AA were injected quickly. Within 30 s, its color rapidly turned to greenish-black. Soon after, 100 μL of 10 mg/mL dopamine solution was added. Then, 20 μL of 1 M NaOH was added to accelerate the in situ self-polymerization of dopamine on the surface of GNS. After 10 min, the PDA-coated GNS (GNS@PDA) was collected by centrifugation (5000 rpm, 15 min) and washed twice by water. PEG stabilized GNS (GNS-PEG) was prepared as previously reported.44 Briefly, the GNS solution was added with 10 μL of 10 mM HS-PEG and allowed to stir for 2 h before collection.

Preparation of GNS@PDA-Cy7. The GNS@PDA-Cy7 was constructed by conjugating Cy7-KTSGPNQC to GNS@PDA via the Michael addition reaction between the thiol group of the cysteine and the quinone in PDA. In brief, GNS@PDA was re-suspended in 10 mM Tris buffer (pH 8.5) containing 100 μg/mL Cy7-KTSGPNQC. The combined solution was kept out of light under stirring at 4 °C overnight. The obtained product GNS@PDA-Cy7 was washed by repeated centrifugation (5000 rpm, 15 min). The amount of Cy7-KTSGPNQC conjugated onto the GNS@PDA was calculated by subtracting the amount of Cy7-KTSGPNQC remaining in the supernatant from the original amount added. The control probe 18 ACS Paragon Plus Environment

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GNS@PDA-Cy7/scr was synthesized by the same method. Another control probe Cy7-KTSGPNQC labeled GNS (GNS-Cy7) was prepared by forming Au-S bonds according to previous protocols.48

Characterization. Transmission electron microscopy (TEM) images were acquired using a Tecnai G2 20 S-TWIN TEM operating at an acceleration voltage of 200 kV. Fluorescence spectra were obtained on a fluorescence spectrophotometer (Hitachi F-4600, Japan). The molecular weight of the product was analyzed by matrix-assisted laser desorption ionization with time of flight (MALDI-TOF) mass spectrometry (Bruker, USA). The size distribution, polydispersity index and zeta potential of the samples were measured by dynamic light scattering using a Malvern Zetasizer Nano ZS90 (Malvern, UK). UV-vis absorbance spectra were recorded using a UV-Vis spectrophotometer (Lambda650, PerkinElmer, USA) in the range of 400-1000 nm.

Kinetic Assay. Various concentrations of Cy7-KTSGPNQC or GNS@PDA-Cy7 (0.5, 1, 2, 4, 8, 16, 32 μM) were incubated with rhFAP (0.2 μg) at 37 °C for 5 min in the assay buffer. After incubation, excess acetonitrile was added to terminate the reaction and the mixture was centrifuged at 5000 rpm for 15 min. The supernatant was filtered through the 0.22 μm PTFE filtration membrane before the released Cy7-KTSGP was analyzed using HPLC. The initial reaction velocity (pmol min-1) was calculated, plotted against the concentration of Cy7-KTSGPNQC or GNS@PDA-Cy7, and fitted to a Michaelis-Menten curve. The kinetic parameters were calculated from the Michaelis-Menten equation shown below: 19 ACS Paragon Plus Environment

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V = Vmax × [S]/(Km + [S])

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(1)

Where V is initial reaction velocity, Vmax is maximum enzymatic rate, [S] is substrate concentration, Km is Michaelis-Menten constant.

Activation of GNS@PDA-Cy7 by rhFAP. For the time-dependent enzymatic assays, either GNS@PDA-Cy7 or GNS@PDA-Cy7/scr (4 μM) was mixed with rhFAP (0.5 μg/mL) for 0, 1, 3 or 6 h. The fluorescence in the sample was visualized by a CRi Maestro imaging system and the emission spectra were recorded using a fluorescence spectrophotometer (excitation, 740 nm; emission, 760-860 nm). For dose-dependent studies, the GNS@PDA-Cy7 (4 μM) was mixed with different concentrations of rhFAP (0.1, 0.5 or 1 μg/mL) and the samples were analyzed as described above after incubation for 6 h.

Cell Culture and Animal Studies. The human pancreatic cancer cell lines Mia-paca-2, and the mouse fibroblast cell line 3T3, were maintained in DMEM medium (WISENT Inc., USA). The human noncancerous pancreas cell line HPNE was cultured in minimal essential medium (MEM, Hyclone). All cultures were supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2. BALB/c nude mice (female, 6-8 weeks age, 16-18 g body weight) were purchased from Vital River Laboratory Animal Technology Co., Ltd. All animal protocols were approved by the Institutional Animal Care and Use Committee of National Center for Nanoscience and Technology. To generate a xenograft model, Mia-paca-2 cells (2.5 × 106 cells) were suspended in a 100 μL PBS 20 ACS Paragon Plus Environment

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and Matrigel mixture (1:1, v/v; BD, USA) and subcutaneously transplanted into the right flank of each mouse.

Western Blot. Cells or tissues were lysed with RIPA buffer (Solarbio, China) containing 1 mM PMSF (Solarbio, China). Protein samples (50 μg) were electrophoresed on 10% SDS-PAGE gels and then transferred to PVDF membranes. These membranes were blocked and incubated with rabbit anti-human FAP polyclonal antibody (Abcam, ab53066) at 4 °C overnight. Subsequently, the membranes were washed and treated with a secondary antibody linked to horseradish peroxidase for 1 h at room temperature. The immunoreactivity was visualized by ECL reagents (Thermo Scientific, USA). GAPDH was used as an internal control.

Activation of GNS@PDA-Cy7 by Tumor Cells. To assess the time dependence of GNS@PDA-Cy7 activation, Mia-paca-2 cells were seeded into 12-well plates at a density of 5 × 104 cells per well and cultured for 2 days. The medium was discarded, and the cells were washed twice with PBS. The cells were then incubated with fresh DMEM (without FBS or phenol red) containing 100 μg/mL GNS@PDA-Cy7 (4 μM) for 0, 1, 3 or 6 h. After incubation, the supernatants were collected for fluorescence analysis and the cells were collected for flow cytometry analysis (BD Accuri C6, BD, USA).

To examine the cell number dependence of GNS@PDA-Cy7 activation, Mia-paca-2 cells were seeded into 48-well plates at a density of 0, 5 × 103, 1 × 104 or 2 × 104 cells per well and cultured for 2 days. The medium was discarded, and the 21 ACS Paragon Plus Environment

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cells were washed twice with PBS. The cells were then incubated with fresh DMEM (without FBS or phenol red) containing 4 μM GNS@PDA-Cy7 for 6 h. After incubation, the supernatants were analyzed by fluorescence spectrometer.

Cytotoxicity Studies. The cytotoxicity of GNS@PDA and GNS-PEG against HPNE cells and Mia-paca-2 cells was examined using the CCK-8 assay. Briefly, HPNE cells and Mia-paca-2 cells were seeded into 96-well plates at a density of 5000 cells per well and incubated for 24 h. Then the medium was replaced with fresh medium containing different concentrations of GNS@PDA or GNS-PEG. After further incubation for 24 h, cell viability was evaluated by a CCK-8 method according to the manufacturer’s protocol.

Measurement of Photothermal Performance. The photothermal effect of GNS@PDA was tested in 1.5 mL Eppendorf tubes at different concentrations. Each sample was irradiated with an 808 nm continuous laser (1.4 W/cm2) for 5 min. The photothermal effect of GNS@PDA (50 μg/mL) was also evaluated by irradiation with different power densities (0.7, 1.4 or 2.8 W/cm2). GNS-PEG irradiated at 1.4 W/cm2 was used as a control.

To further test the photothermal stability of GNS@PDA and GNS-PEG, they were irradiated at 1.4 W/cm2 for three cycles with a 5 min heating period and a 5 min cooling period. The temperature changes were monitored in real-time by an infrared thermal camera.


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Calculation of Photothermal Conversion Efficiency. GNS@PDA or GNS-PEG (50 μg/mL) was exposed to laser irradiation (1.4 W/cm2) for 6 min, and then the laser was turned off. The heating and cooling temperature of samples were recorded. Following Roper’s report,66 the photothermal conversion efficiency (η) was calculated according to the following equation:

(2) Where h is the heat transfer coefficient, S is the surface area of the container, Tmax is the maximum steady-state temperature of the sample solution, Tsurr is the ambient surrounding temperature, I is the laser power and A808 is the absorbance of the sample solution at 808 nm. Qdis represents the heat dissipation from the light absorbed by the water and container, which is measured independently. To calculate hS, another equation was introduced:

(3) Where m is the mass of sample, Cwater is the heat capacity of water (4.2 J g-1 k-1), and τs is the sample system time constant.

Cellular Uptake and Photothermal Killing in Vitro. Mia-paca-2 cells were seeded into confocal chambers for 24 h, and then incubated with 50 μg/mL of GNS@PDA or GNS-PEG for 6 h. Afterwards, the cells were washed twice with PBS and two-photon luminescence (TPL) images were obtained using a two-photon laser scanning confocal microscopy (Olympus FV 1000). 23 ACS Paragon Plus Environment

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To assess the photothermal killing effect on tumor cells, Mia-paca-2 cells were treated with 50 μg/mL of GNS@PDA or GNS-PEG for 24 h. The cells were then washed twice with PBS and irradiated with an NIR laser (808 nm, 1.4 W/cm2) for 5 min. After further incubation for 2 h, the cells were stained with both calcein AM (2 μM) and propidium iodide (PI, 4 μM). Finally, the live or dead cells were observed under the confocal microscopy (Zeiss LSM 710).

To quantitatively evaluate the photothermal cytotoxicity of GNS@PDA and GNS-PEG, Mia-paca-2 cells were seeded into 96-well plates at a density of 5000 cells per well and incubated for 24 h. Then the medium was replaced with fresh medium containing different concentrations of GNS@PDA or GNS-PEG. After further incubation for 24 h, the cells were washed twice with PBS, exposed to an NIR laser (808 nm, 1.4 W/cm2) for 5 min, and then incubated for another 2 h. Cell viability was assessed using a CCK-8 assay.

Penetration Studies and Photothermal Killing in Mia-SS. Mia-paca-2-derived stroma rich spheroids (Mia-SS) containing Mia-paca-2 and 3T3 fibroblasts were generated by an improved hanging drop method as described previously.41 Briefly, hanging drops of culture medium containing Mia-paca-2 (20,000 cells) and 3T3 (5,000 cells) were incubated for 3 days. Mia-SS were harvested and incubated with 50 μg/mL of GNS@PDA or GNS-PEG for 6 h. Then, Mia-SS were washed twice with PBS and two-photon luminescence (TPL) images were acquired using a two-photon laser scanning confocal microscopy through Z-stack scanning. 24 ACS Paragon Plus Environment

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To evaluate the photothermal killing ability in Mia-SS, pretreated Mia-SS were exposed to a NIR laser (808 nm, 1.4 W/cm2) for 5 min. After further incubation for 2 h, the Mia-SS were stained with PI (4 μM) and dead cells were observed under the confocal microscopy through Z-stack scanning.

CT Imaging for Diagnosis. The Hounsﬁeld units (HU) value of GNS@PDA in vitro was measured at different Au concentrations (0.63, 1.25, 2.5, 5, 10 mg/mL). For in vivo CT imaging, GNS@PDA (2 mg/mL, 20 μL) was injected directly into the tumor and CT images were acquired. Reconstructed 3D CT images of the mouse were done by the filtered back projection (FBP) method. The reconstruction kernel used a Feldkamp cone beam correction and SheppLogan filter.

PA Imaging for Diagnosis. The photoacoustic intensity of GNS@PDA in vitro was measured at different Au concentrations (0, 15.6, 31.2, 62.5, 125 μg/mL) on the InVision 128 MSOT system (iThera Medical, Germany). For in vivo photoacoustic imaging, a GNS@PDA or GNS-PEG suspension (Au: 4 mg/mL in PBS, 100 μL) was injected intravenously into tumor-bearing mice. The mice were anaesthetized and scanned at different time intervals by MSOT.

MR Imaging for Diagnosis. Fe3+ chelated GNS@PDA (GNS@PDA-Fe) was prepared by simply mixing 1 mL GNS@PDA (1 mg/mL) with 50 μL FeCl3 (0.2 M) at RT for 3 h. GNS@PDA-Fe was retrieved by centrifugation and washed twice to remove free ferric ions. The amount of chelated Fe3+ in GNS@PDA-Fe was measured by ICP-MS (NexION 300X, Perkin-Elmer, USA). 25 ACS Paragon Plus Environment

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The T1-weighted MR images of GNS@PDA-Fe in vitro were acquired at different Fe3+ concentrations (0, 31.2, 62.5, 125, 250 μM) using a 7.0 T small animal MRI instrument (BioSpec 70/20 USR, Bruker, Germany). For in vivo MR imaging, GNS@PDA-Fe (Au: 100 mg/kg, Fe: 0.5 mg/kg) was injected intravenously into tumor-bearing mice and MR images were acquired at different time post-injection.

NIRF Imaging for Diagnosis. Nude mice were inoculated with Mia-paca-2 cells on both left and right flanks. When the tumors reached ~200 mm3, mice were anesthetized and GNS@PDA-Cy7 (0.8 nmol, 50 μL PBS) was injected directly into the left tumor. The control probe GNS@PDA-Cy7/scr was injected into the right tumor. In vivo NIRF imaging was performed before and after intratumoral injection using a CRi Maestro in vivo imaging system with identical illumination settings (excitation, 740 nm; emission, 750-850 nm; exposure time, 2 s). The fluorescence emission was normalized to photons per centimeter squared per second (p/cm2/s). For ex vivo imaging, mice were euthanized at 6 h post-injection and major organs were obtained. Ex vivo images were acquired immediately with the same illumination settings as described above.

In order to investigate the FAP imaging ability of GNS@PDA-Cy7 after intravenous injection, GNS@PDA-Cy7 (13 nmol, 150 μL PBS) was injected intravenously into tumor-bearing mice. In vivo and ex vivo NIRF imaging was performed as described above.


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Therapeutic Efficacy of GNS@PDA in Vivo. When the tumors reached ~200 mm3, the mice were divided into five treatment groups: (a) Saline; (b) Saline + laser; (c) GNS@PDA (Au: 20 mg/kg); (d) GNS@PDA + laser; (e) GNS-PEG + laser. Mia-paca-2 tumor-bearing mice were anesthetized and the tumors were irradiated with an NIR laser (808 nm, 1.4 W/cm2) for 10 min at 6 h post injection of saline, GNS@PDA or GNS-PEG. Temperature increases were recorded using an infrared thermal camera. Tumor sizes and body weight were measured every other day after irradiation. Tumor volumes were determined by measuring their length and width with calipers according to the following formula:

(4)

Histological Analysis and Serum Biochemical Analysis. For histological analysis, tumors and major organs, including the heart, liver, spleen, lung and kidney, were collected at the end of the experiment, fixed in 10% neutral buffered formalin, processed routinely into paraffin, sectioned into 5 μm, and stained with H&E.

The serum samples were collected from the different groups and examined using a biochemical autoanalyzer. Liver function was evaluated by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, while kidney function was assessed by blood urea nitrogen (BUN) and creatinine (CRE) levels.

Statistical Analysis. Quantitative results are presented as means ± standard deviation. Statistical analysis was performed with SPSS 21.0 software. Student’s t-test 27 ACS Paragon Plus Environment

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was applied for comparison between two groups. Differences in tumor growth curves and tumor volumes among multiple groups were analyzed by one-way ANOVA. Student-Newman-Keuls test was used as post hoc test. P < 0.05 was considered to be statistically significant.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Tables S1; Figures S1-S25 (PDF) Financial interest statements The authors declare no competing financial interest. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] Author Contributions #Xuexiang

Han and Ying Xu contributed equally to this work.

ACKNOWLEDGMENT This work was supported by the grants from National Natural Science Foundation of China (Grant Nos. 31571021), National Key R&D Program of China (Grant Nos. 2018YFA0208900), Innovation Group of the National Natural Science Foundation of China (Grant Nos. 11621505), Frontier Research Program of the Chinese Academy of Sciences (Grant Nos. QYZDJ-SSW-SLH022), and the Key Laboratory of Biomedical Effects of Nanomaterials and Nanosafety, CAS. The authors are grateful to Dr. Dongliang Wang and Dr. Yuqing Wang at National Center for Nanoscience and Technology for CT and MRI experiments, respectively.

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Figures and Figure legends

Scheme 1. Scheme of extendable nanoplatform for functional and anatomical imaging-guided photothermal oncotherapy (PTT). (a) Synthesis of GNS@PDA from gold nanoparticles (GNP) and its function extensions. (b) Schematic illustration of GNS@PDA-based nanotheranostics for near-infrared fluorescence (NIRF), photoacoustic (PA), magnetic resonance (MR), computed tomography (CT), two-photon luminescence (TPL) and infrared thermal (IRT) imaging-guided PTT.


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Figure 1. Characterization and NIRF activation in vitro. (a) TEM image of GNS@PDA. Scale bar, 100 nm. Inset: enlarged micrograph. Scale bar, 20 nm. (b) UV-vis absorption spectra of Cy7-peptide, GNS@PDA and GNS@PDA-Cy7 solutions. (c) Bright and NIRF images of H2O, GNS@PDA, GNS@PDA-Cy7 and Cy7-peptide solutions. (d) NIRF emission spectra of H2O, Cy7-peptide, GNS@PDA and GNS@PDA-Cy7 solutions. (e) NIRF emission spectra of GNS@PDA-Cy7 incubated with HPNE or Mia-paca-2 cells for 6 h. (f) NIRF emission spectra of GNS@PDA-Cy7 incubated with Mia-paca-2 cells for different time. Inset: NIRF image of supernatants. (g) Flow cytometry results of Mia-paca-2 cells incubated with GNS@PDA-Cy7 for different time. (h) Mean fluorescence intensity (MFI) of Mia-paca-2 cells incubated with GNS@PDA-Cy7 for different time. The data are 42 ACS Paragon Plus Environment

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presented as the mean ± s.d. (n = 3). (i) NIRF emission spectra of GNS@PDA-Cy7 incubated with different numbers of Mia-paca-2 cells. Ex = 740 nm.


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Figure 2. Photothermal properties. (a) Temperature changes of GNS@PDA (0-100 μg/mL) under NIR laser irradiation (808 nm, 1.4 W/cm2). (b) Infrared thermal images during laser irradiation. (c) Temperature changes of GNS@PDA under various power density of laser irradiation (0.7, 1.4 or 2.8 W/cm2). (d) Heating and cooling curve of GNS@PDA. (e) Plot of cooling time versus negative natural logarithm of the temperature driving force. (f) Temperature changes of GNS@PDA and GNS-PEG during three on/off cycles of laser irradiation. The data are presented as the mean ± s.d. (n = 3).


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Figure 3. PTT in vitro. (a) Two-photon luminescence (TPL) images of Mia-paca-2 cells after GNS@PDA or GNS-PEG treatment. Scale bar, 20 μm. (b) Fluorescence images of calcein AM (green) and propidium iodide (PI, red) co-stained Mia-paca-2 cells after different treatments. Scale bar, 100 μm. (c) Photothermal cytotoxicity of GNS@PDA or GNS-PEG towards Mia-paca-2 cells after laser irradiation. The data are presented as the mean ± s.d. (n = 3). **P< 0.01. (d) TPL images of GNS@PDA or GNS-PEG penetration into Mia-paca-2-derived stroma rich spheroids (Mia-SS). Scale bar, 100 μm. (e) Fluorescence images of PI stained Mia-SS after laser irradiation and their 3D reconstruction. Scale bar, 100 μm.


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Figure 4. The anatomical (CT/PA/MR) and functional (NIRF) imaging in vivo. (a) 3D reconstructed CT images of tumor-bearing mice pre and post intratumoral injection of GNS@PDA. (b) In vivo PA images of tumor-bearing mice after intravenous injection of GNS@PDA or GNS-PEG. (c) Quantification of PA intensity at the tumor sites. The data are presented as the mean ± s.d. (n = 3). *P< 0.05; **P< 0.01. (d) In vivo MR images of tumor-bearing mice after intravenous injection of GNS@PDA-Fe. (e) In vivo NIRF images of tumor-bearing mice after intravenous injection of saline (left mouse) or GNS@PDA-Cy7 (right mouse). (f) Quantification 46 ACS Paragon Plus Environment

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of fluorescence intensity at the tumor sites after intravenous injection. The data are presented as the mean ± s.d. (n = 3). (g) NIRF images of excised tumors and major organs at 6 or 24 h post intravenous injection of GNS@PDA-Cy7. (h) Quantification of fluorescence intensity in excised tumors and major organs. The data are presented as the mean ± s.d. (n = 3). ***P< 0.001. The white dotted circles indicate tumors.


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Figure 5. PTT in vivo. (a) In vivo infrared thermal (IRT) images of tumor-bearing mice during laser irradiation (808 nm, 1.4 W/cm2) at 6 h post-injection of saline, GNS@PDA or GNS-PEG. The white dotted circles indicate tumors. (b) Temperature changes at tumor regions during laser irradiation. The data are presented as the mean ± s.d. (n = 4). (c) Tumor growth curves. The data are presented as the mean ± s.d. (n = 4). **P< 0.01; ***P< 0.001. (d) Photographs of the excised tumors in different groups. (e) Tumor weights of the excised tumors in different groups. The data are presented as the mean ± s.d. (n = 4). **P< 0.01; ***P< 0.001. (f) H&E staining images of tumor sections collected from mice in different groups. Scale bar, 500 μm. L, laser irradiation.


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Table of Contents (TOC) graphic

An extendable nanoplatform based on polydopamine-coated gold nanostar (GNS@PDA) is prepared for functional and anatomical imaging-guided photothermal oncotherapy. Such a versatile nanoplatform demonstrates extendable hexa-modal imaging and achieves homogeneous photothermal ablation of tumor in a single treatment, which holds great promise for precision diagnosis and effective treatment of tumor.


An Extendable Star-Like Nanoplatform for Functional and Anatomical

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