Magnetic graphene-based nanotheranostic agent for dual-modality mapping guided photothermal therapy in regional lymph nodal metastasis of pancreatic cancer.

Biomaterials xxx (2014) 1e11

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Magnetic graphene-based nanotheranostic agent for dual-modality mapping guided photothermal therapy in regional lymph nodal metastasis of pancreatic cancer Sheng Wang a, c, 1, Qin Zhang d, e, 1, Xian F. Luo f, Ji Li a, Hang He a, Feng Yang a, Yang Di a, Chen Jin a, Xin G. Jiang g, Shun Shen b, g, h, **, De L. Fu a, * a

Pancreatic Disease Institute, Department of Pancreatic Surgery, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai 200040, China Key Laboratory of Medical Imaging Computing and Computer Assisted Intervention of Shanghai, Fudan University, Shanghai 200032, China Department of Surgery, Shanghai Medical College, Fudan University, Shanghai 200032, China d Department of Radiation Oncology, Shanghai Cancer Center, Fudan University, Shanghai 200032, China e Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China f Department of Radiology, Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200025, China g School of Pharmacy, Fudan University, Shanghai 201203, China h Key Laboratory of Smart Drug Delivery, Fudan University, Shanghai 201203, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 July 2014 Accepted 29 July 2014 Available online xxx

Although regional lymph nodes (RLN) dissection remains the only way to cure pancreatic cancer metastasis, it is unavoidably associated with sizable trauma, multiple complications, and low surgical resection rates. Thus, exploring a treatment approach for the ablation of drug-resistant pancreatic cancer is always of great concern. Moreover, reoperative and intraoperative mapping of RLN is also important during treatment, because only a few lymph nodes can be detected by the naked eye. In our study, graphene oxides modified with iron oxide nanoparticles (GO-IONP) as a nanotheranostic agent is firstly developed to diagnose and treat RLN metastasis of pancreatic cancer. The approach was designed based on clinical practice, the GO-IONP agent directly injected into the tumor was transported to RLN via lymphatic vessels. Compared to commercial carbon nanoparticles currently used in the clinic operation, the GO-IONP showed powerful ability of dual-modality mapping of regional lymphatic system by magnetic resonance imaging (MRI), as well as dark color of the agent providing valuable information that was instrumental for surgeon in making the preoperative plan before operation and intraoperatively distinguish RLN from surrounding tissue. Under the guidance of dual-modality mapping, we further demonstrated that metastatic lymph nodes including abdominal nodes could be effectively ablated by near-infrared (NIR) irradiation with an incision operation. The lower systematic toxicity of GO-IONP and satisfying safety of photothermal therapy (PTT) to neighbor tissues have also been clearly illustrated in our animal experiments. Using GO-IONP as a nanotheranostic agent presents an approach for mapping and photothermal ablation of RLN, the later may serve as an alternative to lymph node dissection by invasive surgery. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Magnetic graphenes Pancreatic cancer Regional lymph nodes Nanotheranostic

1. Introduction

* Corresponding author. Pancreatic Disease Institute, Department of Pancreatic Surgery, Huashan Hospital, 12 Central Urumqi Road, Shanghai Medical College, Fudan University, Shanghai 200040, China. Tel./fax: þ86 21 5288 8277. ** Corresponding author. School of Pharmacy, Fudan University, No. 131, Dong An Road, Shanghai 201203, China. E-mail addresses: [email protected] (S. Shen), [email protected] (D.L. Fu). 1 Both authors contributed equally to this work.

Pancreatic cancer is the fourth leading cause of cancer death in the United States in 2012 and has one of the worst prognosis among various types of cancers, with only 5% of patients surviving for 5 years after diagnosis and treatment [1,2]. Lymph node metastasis is the main metastatic pattern of pancreatic cancer and considered as the major cause of death [3]. Typically, pancreatic cancer first metastasizes to sentinel lymph nodes, and then quickly and latently spreads to other nearby lymph nodes (called regional lymph

http://dx.doi.org/10.1016/j.biomaterials.2014.07.064 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Wang S, et al., Magnetic graphene-based nanotheranostic agent for dual-modality mapping guided photothermal therapy in regional lymph nodal metastasis of pancreatic cancer, Biomaterials (2014), http://dx.doi.org/10.1016/ j.biomaterials.2014.07.064

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S. Wang et al. / Biomaterials xxx (2014) 1e11

nodes). RLN dissection remains the only treatment approach providing a chance for a cure [2,3]. However, less than 20% of patients are diagnosed with pancreatic cancer at an early resectable stage [2,4]. Even after radical resection for early stage pancreatic cancer, local recurrences and systemic metastases often occur within one year [2]. To improve the prognosis, many modalities such as surgery, radiotherapy, chemotherapy and a combination of all these modalities, have been used for the treatment of pancreatic cancer, unfortunately their outcome has been still very pessimistic [5,6]. Furthermore, these modalities can yield many side-effects, such as lymphatic leakage, body weight loss resulting from oral intake disturbance, systemic toxicity and a destructive “bystander” effect to neighboring cells [7e9]. Compare to radiotherapy, chemotherapy and surgical management, photothermal therapy (PTT) is less invasive and has attracted widespread attention [10]. PTT applies photo-absorbing agents to convert optical energy into heat, leading to the ‘burning’ of cancer cells [11e17]. Our research groups and the other have developed a large number of nanomaterials as PTT agents, such as various goldbased nanomaterials [10,18e21], carbon nanotubes [22] and graphene [23], all of which show strong optical absorbance in the NIR tissue optical transparency window. According to the previous study, sentinel lymph nodes model was always selected for the PTT of pancreatic cancer metastasis [10]. Actually, this doesn't meet clinical requirements. In the clinical practice, the only mapping and treatment of sentinel lymph nodes is not sufficient. More importantly, the potential metastasis of RLN should be traced and ablated. However, the treatment of metastatic lymph nodes by PTT has been rarely reported, mainly because the location of metastases in RLN are challenging due to their small size and fuzzy boundary with surrounding adipose and connective tissue [24]. In clinical practices, carbon nanoparticles, isosulfan blue and radioactive colloids (such as Tc-99 m sulfur colloid) are the most popular lymph tracing agents [24e27]. However, these lymph nodes mapping methods have some apparent disadvantages and limitations. For example, the visible dyes (carbon nanoparticles and isosulfan blue) have limit tissue penetration and are difficult to detect deeper lymph nodes [26]. Radioactive colloids have the harm of ionizing radiation and low spatial resolution [26,28]. To date, MRI has also been widely used for the preoperative location of lymph nodes, which provides perfectly three-dimensional soft tissue details [26,27,29e32]. Considering the cost effectiveness, it is difficult to use the complicated setup of MRI for real-time visualization during the surgical procedure, while dye mapping has the convenient and visual advantages. Unfortunately the versatile RLN tracing agents, with a combination advantage of MRI and dye mapping greatly needed for PTT are still scarce. Theranostics is a recently proposed concept that combines therapeutics and diagnostics, aiming to improve therapeutic efficiency with better planning and prognosis. Many versatile nanomaterials incorporating PTT and diagnostic imaging functions have been explored as a new generation platform in the field of nanomedicine [17,32,33]. Previously, Marites et al. and Zheng et al. have developed Gd-based or IONP-based dual-modality MRI and fluorescence imaging for sentinel lymph nodes mapping [26,28]. To our knowledge, the papers focused on dual-modality imaging for RLN were rarely reported, let alone both diagnosis and PTT. Herein, we developed magnetic GO as a nanotheranostic agent for mapping RLN and PTT of pancreatic cancer metastasis. Owing to its unique physicalechemical properties, GO has been extensively developed for applications in a large number of fields including biomedicine [34,35]. GO and its derivatives have shown great promise in the nanocarriers for drug [36e38] and gene delivery [39,40], PTT [14,23,41,42] as well as biomedical imagining [11,14,43]. By growing IONP on the surface of GO, the GO-IONP

composites have been applied as a nanotheranostic agent for photothermal destruction of cancers [11]. However, no attention has been focused to the feasibility of GO-IONP as a dual-modality lymphatic mapping and PTT agent for the treatment of metastatic lymph nodes in previous reports. In this work, GO-IONP was functionalized with polyethylene glycol (PEG). The obtained GOIONP-PEG with intrinsic black color serving as a visual dye, and superparamagnetism serving as a T2-MRI contrast agent, was firstly employed to solve a clinical problem of how to locate abdominal lymph nodes. Meanwhile, treatment efficacy of GO-IONP-PEG was also investigated through dual-modality mapping guided PTT. Meanwhile, we systematically assessed the safety of PTT to neighboring tissue and toxicity of GO-IONP-PEG for the major organs in nude mice. 2. Materials and methods 2.1. Materials N-(3-dimethylaminopropyl-N0 -ethylcarbodiimide) hydrochloride (EDC) was purchased from SigmaeAldrich. NH2-polyethylene glycol 5000 (NH2-PEG5000) was purchased from Seebio Biotech, Inc. (Shanghai, China). Iron (III) chloride hexahydrate, sodium acetate trihydrate, sodium polyacrylate, ethylene glycol (EG) and diethylene glycol (DEG) were purchased from Sinopharm Chemical Reagent Co., Ltd. CCK-8 and Calcein AM and propidium iodide (PI) were purchased from KeyGen BioTech (Nanjing, China). RPMI-1640 medium, fetal bovine serum (FBS), PenicillinStreptomycin solution and Trypsin-EDTA solution were purchased from Gibco (Tulsa, OK, USA). Carbon nanoparticles were purchased from Laimei Pharmaceutical Co., Ltd. (Chongqing, China). All the other chemicals were analytical grade, and purified water was produced by a Millipore water purification system. 2.2. GO-IONP coated with PEG GO and GO-IONP were synthesized according to the previous reports [44,45]. In order to increase hydrophilicity, the nanomaterials of GO-IONP were modified with NH2-PEG5000 by covalently linked method [40]. Briefly, 5 mg of GO-IONP and 25 mg of NH2-PEG5000 were dispersed with 5 mL of deionized water, and then sonicated in an ultrasonic bath for 5 min at room temperature. The mixture was added with EDC (0.5 mg/mL) following another sonication for 5 min, and then was stirred gently for 30 min at room temperature. Following the second time addition of EDC (1 mg/ mL), the mixture was sonicated for 5 min then stirred at room temperature for 6 h. Finally, the acquired GO-IONP-PEG was washed and purified using Millpore ultrafiltration tube (MWCO ¼ 100 kDa) for 5 times, and redispersed in deionized water. 2.3. Materials characterization The transmission electronic microscopy (TEM) images were taken by a Philips CM300 transmission electron microscope operating at an acceleration voltage of 200 kV. Atomic force microscopy (AFM) (XE-100, Park system) was used to observe the morphology of films. ICP-AES was performed to determine the concentration of iron contents in our GO-IONP nanocomposites by a P-4010 spectrometer (Hitachi, Japan). The magnetization characterization of the GO-IONP-PEG was measured using a vibrating sample magnetometer on a Model 6000 physical property measurement system (Quantum, USA) at 300 K. Raman spectra were recorded using a Renishaw spectrometer (model Invia Reflex) with 632.8 nm laser excitation. The Fourier-transform infrared (FTIR) spectra were recorder on a Magna-550 spectrometer (Nicolet, USA). The samples were dried and mixed with KBr to be compressed to a plate for measurement. UV/Vis absorption spectra were measured on a UV-3150 ultravioletevisible spectrophotometer (Shimadzu, Japan). T2-weighted images of GO-IONP-PEG nanocomposites with different iron concentration were obtained under a 3-T clinical MRI scanner. The stability of GO-IONP-PEG was also examined both in PBS and serum-containing cell medium at 37 C for 72 h. 2.4. In vitro temperature evaluation caused by NIR laser irradiation 150 mL aliquots with various concentration of GO-IONP-PEG were deposited into wells of a 48-well cell culture plate. Wells were illuminated using an 808 nm continuous-wave NIR laser (Changchun New Industries Optoelectronics Technology, Changchun, China) with 2 and 3 W/cm2 for 5 min. Pre- and postillumination temperatures were taken by thermocouple. 2.5. Cell experiment The BxPC-3 cells, human pancreatic cancer cells, originally obtained from the American Type Culture Collection, were routinely cultured in RPMI-1640 cell medium supplemented with 10% FBS, 100U/mL penicillin and 100 mg/mL streptomycin, at 37 C in 5% CO2 and 95% air atmosphere with >95% humidity. The cells (1 104 cells per well) were seeded in 96-well plates and incubated with various concentrations of GO-IONP-PEG for 24 h. Then, cell viabilities were determined by


S. Wang et al. / Biomaterials xxx (2014) 1e11 CCK-8 assay. To investigate the PTT efficiency of GO-IONP-PEG with NIR laser irradiation, BxPC-3 cells were incubated with and without GO-IONP-PEG with different GO concentration of 20 and 40 mg/mL for 2 h and irradiated by NIR laser (fluence: 2 W and 3 W/cm2) for 5 min. Subsequently, cell viabilities were determined by CCK-8 assay. For qualitative evaluation of the photothermal cytotoxicity of GO-IONP-PEG, BxPC-3 cells (1.5 105 cells per well) were seeded in 20 mm glass-bottom culture dish (NEST, China) and incubated with different GO concentration of 20 and 40 mg/ mL under 37 C for 2 h, then irradiated by NIR laser (2 W and 3 W/cm2) for 5 min. Afterwards, cells were co-stained by a mixture of Calcein AM and PI solution for 20 min. The samples were subjected to observe using a ZEISS LSM710 live cell confocal laser imaging System (Carl Zeiss, German). 2.6. Animal model Nude male mice, aging 6e8 weeks and weighing 18e21 g, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and housed in a specific pathogen-free animal facility. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University. The model of lymph node metastases was obtained by subcutaneous injection of 7.5 106 BxPC-3 cells suspended in 100 mL RMPI-1640 medium via right hind foot pad in nude mice [2,5]. Studies were done on these mice about 3 weeks after inoculation when the tumor diameter reached ~5.0 mm. 2.7. Dual-modality MRI-dye mapping of RLN In order to validate the feasibility of dual-modality mapping of RLN using GOIONP-PEG, 6 mice bearing lymph node metastases were randomized into two groups (n ¼ 3 per group). 50 mL of GO-IONP-PEG (8 mg/mL) or carbon nanoparticles as a control group, was separately injected into the right rear footpad of mice. After 24 h, MRI was performed with 3-T clinical MRI scanner equipped with small animal coil. The imaging parameters for T2-weighted were using a spin echo sequence (TR 3000 ms, TE 91 ms, FOV 100 mm). Then, these mice were sacrificed by anesthesia. The popliteal, inguinal, sciatic, iliac common and renal hilar lymph nodes were examined visually and photographed. The right popliteal, inguinal and sciatic lymph nodes were examined after surgical skin incision of corresponding region. The iliac common, renal hilar lymph nodes were exposed by a midline incision of the abdomen and retraction of the intestines. Scores of GO-stained black lymph node were defined as follows: 0 point, the color of the lymph node did not change at all; 1 point, lymph node turned gray; 2 point, the whole lymph node turned black [24]. The black lymph nodes injected GO-IONP-PEG were divided into about 1 mm3 tissue pieces and fixed in glutaraldehyde solution for TEM detection. 2.8. RLN photothermal ablation For in vivo PTT experiment, 20 mice bearing lymph node metastases were randomly divided into four groups (n ¼ 5 per group). One group was injected with NS into the right rear footpad without laser irradiation. The second group was injected with NS with laser irradiation at a power density of 2 W/cm2 for 5 min. The third group was injected with 50 mL of GO-IONP-PEG (8 mg/mL) without laser irradiation. The fourth group was injected with 50 mL of GO-IONP-PEG (8 mg/mL) with laser irradiation at a power density of 2 W/cm2 for 5 min. After 75% alcohol disinfection and asepsis layout, the popliteal and sciatic lymph nodes were exposed by a ca. 0.5 cm skin incision of corresponding region then irradiated by NIR laser through dual-modality mapping guided PTT. The iliac common lymph nodes were exposed by a ca.1.5 cm midline incision of the abdomen and retraction of the intestines, then irradiated by NIR laser through dye mapping. The whole operation was done in a sterile condition room. The temperature was recorded by an IR thermal camera (InfraTec, VarioCAM®hr research, German). At 15th day, the blood samples and main organs were collected and analyzed. The weight and histopathology of popliteal, sciatic and iliac common lymph nodes were examined for the assessment of therapeutic efficacy. To evaluate the safety of PTT using GO-IONP-PEG, Historical evaluation of, nerves, adipose tissues and blood vessels near the iliac common lymph nodes were implemented. 2.9. Statistical analysis Unpaired student's t test was used for between two-group comparison and oneway ANOVA with Fisher's LSD for multiple-group analysis. A probability (P) less than 0.05 was considered statistically significant. Results were expressed as mean ± standard deviation (SD) unless otherwise indicated.

3. Results and discussion 3.1. Synthesis and characterization of GO-IONP-PEG As shown in Fig. 1a, GO-IONP nanomaterials were synthesized by a solvothermal reaction [44,45]. The TEM images of GO-IONP displayed the numerous dark nanodots of IONP with diameters of 10 nm were uniformly grew on the surface of GO (Fig. 1b). D band

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(1352 cm1) and G band (1608 cm1) peaks could be seen due to the existence of GO in the Raman analysis of GO-IONP nanomaterials (Fig. S1, Supporting Information). Compared with GO, an extra peak in GO-IONP was also detected at lower Raman value (475 cm1) as a result of the formation of magnetite nanoparticles on the surface of GO. Fig. S2 showed the UV-vis spectra of aqueous dispersion of GO and GO-IONP. GO exhibited a strong band at ca. 230 nm because through the carboxylation process, which was similar with previous reports [46,47]. It was clearly seen that the adsorption peak at ca. 230 nm disappeared after solvothermal treatment, indicating the reduction of GO and magnetite nanoparticles were modified on the surface of GO. Moreover, the GOIONP has obvious NIR absorption. Thus, they could be employed for PTT. To prevent aggregation and decrease immunogenicity for in vivo applications, GO-IONP nanomaterials were further modified with PEG. Fig. S3a exhibited the FTIR spectra of GO-IONP-PEG. The absorption peak at ~571 cm1 was assigned to FeeOeFe vibration of magnetite nanoparticles, and 1102, 1563, 1692, 2873 cm1 were attributed to the CeOeC, eNeH, eC]O, eCH2 group, respectively. The absorption peak at 3442 cm1 could be assigned to the adsorbed water on the shell or the eOH group of the GO-IONP nanomaterials. Compared with FTIR spectra of GO-IONP (Fig. S3b), the Fig. S3a FTIR result confirmed the PEG has been successfully grafted on the surface of GO-IONP. No aggregation was observed when PEGylated GO-IONP nanomaterials were stored in PBS and cell medium containing 10% FBS at 37 C over a period of 72 h (Fig. 1c, (B) and (C)). However, GO-IONP nanomaterials without PEG decoration became precipitation in PBS during only 1 h (Fig. 1c, (A)). The morphology of GO-IONP-PEG was also characterized by AFM. The nanomaterial sheets were smaller than 200 nm and their thickness ranged from 3.7 to 10.2 nm (Fig. S4, Supporting Information). The GO-IONP-PEG showed well water solubility and physiological stability with hydrodynamic diameters of ca. 165.5 nm (Fig. S5, Supporting Information), which were an important prerequisite for the bioapplications. The weight ratio of Fe in GO-IONP nanocomposites was measured to 55.6% as determined by ICP-AES. The magnetic characterization (Fig. 1d) showed that our GO-IONP-PEG nanocomposites had superparamagnetic property and magnetization with saturation value of about 14.7 emu/g. Meanwhile, the magnetic GO-IONP-PEG in their homogeneous dispersion showed fast moment to the applied magnetic field and left the solution colorless (Fig. 1c, (D) and (E)) because of their strong magnetic responsivity. As shown in Fig. 1e, T2-weighted MRI images of GO-IONP-PEG gained on a 3.0-T MR scanner revealed a concentration-dependent darkening effect owing to high content of IONP components. The characterization results indicated that our as-prepared nanotheranostic agent of GO-IONP-PEG would be potentially useful for the mapping and PTT of metastatic lymph nodes. 3.2. In vitro temperature raised induced by NIR laser irradiation The GO-IONP-PEG may be effective thermal generators, which could absorb NIR by the GO ingredients. Absorbed NIR promotes molecular oscillation leading to efficient heating of the surrounding environment, which could be employed as thermal ablation agents for thermal destruction of tumor. In our study, the photothermal effect of GO-IONP-PEG with different concentration and illumination time were examined and compared ex vitro by irradiating NIR. The NIR-heating effect of GO-IONP-PEG was time and concentration-dependent. As seen from Fig. 2a, GO-IONP-PEG with higher concentration of 40 mg/mL performed markedly better than that of 10 and 20 mg/mL, the temperature was increased 34.4, 16.1 and 22.9 C, then reached to 60.4, 42.1 and 48.9 C for the concentrations of 40, 10 and 20 mg/mL, respectively. Moreover, the


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Fig. 1. (a) A schematic illustration of GO-IONP-PEG nanocomposites synthesis. (b) TEM image of GO-IONP-PEG, the arrows indicated the IONP and GO, respectively. (c) the stability of GO-IONP. A: Photo of the stability of GO-IONP in PBS. B and C: Photos of GO-IONP-PEG in PBS and serum-containing cell medium. D and E: Photos of GO-IONP-PEG solutions with and without an NdFeB magnetite. (d) Magnetization loops of GO-IONP-PEG. (e) T2-weighted MRI images of GO-IONP-PEG solution with different iron concentrations.

aqueous suspensions of GO-IONP-PEG also exhibited an increase in temperature with exposure time. In comparison, the normal saline (NS) as control group showed much less temperature change. The results made clear that optoelectronic excitation of GO-IONP-PEG by laser illumination occurred rapidly and the extra energies were efficiently transferred into molecular vibration modes to generate significant amounts of thermal energy. 3.3. Intracellular photothermal therapy To examine the feasibility of the GO-IONP-PEG in biomedicine, the nanomaterials were administered to BxPC-3 cells in 96-well plates, and each group was subdivided into groups of with and without NIR laser exposure. The quantitative evaluation of cell viability was performed by the Cell Counting Kit-8 (CCK-8) assay. The viability of untreated cells was assumed to be 100%. The cell viability remained approximately 84.33% when they were incubated with GO-IONP-PEG even at high GO concentration of 300 mg/ mL for 24 h (Fig. S6, Supporting Information), confirming the lack of inherent toxicity of our as-prepared nanomaterials. As we know, the nanotoxicity of GO is a debatable subject because of many variables affecting their chemical properties and structure. One characteristic that affects biotoxicity is the surface chemistry of GO. Although pristine GO has very hydrophobic surfaces, functionalization of the GO surface with PEG can reduce cellular

toxicity of the nanomaterials compared with its bare counterpart. We next applied GO-IONP-PEG for the photothermal ablation of cancer cells. The cytotoxic effects were shown in Fig. 2b. About 32.45% and 46.23% of cells were killed by GO-IONP-PEG with a power density of 2 and 3 W/cm2 for 5 min at an equivalent GO concentration of 20 mg/mL, respectively. And about 56.56% and 81.26% of cells were killed at an equivalent GO concentration of 40 mg/mL with the same power density and lighting time. In addition, direct irradiation of the cells showed almost no effect on cell viability, because the low light absorption by natural endogenous cytochromes of these cells caused minimal temperature elevation accounts for their high survival. The results demonstrated that our GO-IONP-PEG nanomaterials were cytotoxic against BxPC-3 cells in a power and concentration-dependent manner. Fluorescence images of calcein AM (green, live cells) and PI (red, dead cells) co-stained cells after photothermal ablation also confirmed the effectiveness of PTT using GO-IONP-PEG (Fig. 2c). Highest rate of killing BxPC-3 cell was observed at an equivalent GO concentration of 40 mg/mL exposed to laser illumination with a power density of 3 W/cm2 for 5 min. Once the power density was reduced to 2 W/cm2, some cells remained survival. Unheated cells (control) and cells incubated with lower concentration of GO-IONP-PEG with or without NIR laser treatment also showed limited damage, which could be due to insufficiently lethal temperature elevation. Therefore, using GO-IONP-



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Fig. 2. Photothermal ablation of cancer cells. (a) The photothermal effects of the PBS solutions of GO-IONP-PEG with the GO concentration of 10, 20 and 40 mg/mL were examined ex vitro by irradiating NIR (l ¼ 808 nm, 3 W/cm2) for 5 min. Temperature was measured by thermocouple. (b) Relative viabilities of GO-IONP-PEG with the GO concentration of 20 and 40 mg/mL treated BXPC-3 cells with or without NIR laser irradiation under 2 and 3 W/cm2 for 5 min. (c) Confocal fluorescence images of calcein AM (green, live cells) and propidium iodide (red, dead cells) co-stained BXPC-3 cells treated by GO-IONP-PEG at the GO concentration of 20 and 40 mg/mL with or without NIR laser irradiation under 2 and 3 W/cm2 for 5 min (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

PEG with appropriate laser irradiances, irradiation durations and nanoparticles concentration was required to eradicate tumors. 3.4. Dual-model MR imaging/dye mapping of regional lymph nodes The ability to the preoperative and intraoperative mapping of RLN is important in clinical management of patients with pancreatic cancer metastasis. Scholars around the world have tried to

elucidate the routes of lymph nodes metastasis in pancreatic cancer. Due to complex anatomical relationships of lymph nodes, the details about the patterns of pancreatic cancer spread still remain unclear. To our knowledge, mapping and treatment of metastatic RLN by nanotechnology has been rarely reported. In our work, GOIONP-PEG nanomaterials were attempted for the dual-modality mapping RLN. The mapping ability of GO-IONP-PEG was firstly examined. Fifty microlitre aliquots of GO-IONP-PEG or carbon


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nanoparticles as control group at the same concentration of 8 mg/ mL, were injected into the right rear footpad nude male mice bearing lymph nodes metastases, respectively. The MRI images of RLN before and post-injection of GO-IONP-PEG were shown in Fig. 3aed and eeh, and carbon nanoparticles in Fig. 3mep and qet. The GO-IONP-PEG could accumulate in RLN, enhancing susceptibility effects and reducing the nodal T2-weighted signal. It's worth mentioning that sciatic lymph nodes (the second station lymph nodes) and renal hilar lymph nodes (the fourth station lymph node) were all distinctly diagnosed. Noticeably, the inguinal lymph nodes

had no MRI enhancement effect, because they are not the main lymphatic drainage of mouse hind leg [24]. So, few GO-IONP-PEG nanomaterials could be delivered to the inguinal lymph nodes. Control group of RLN injected with carbon nanoparticles did not show any change in the T2-weighted signal (Fig. 3qet). The imaging results indicated that our GO-IONP-PEG nanomaterials had excellent lymphatic drainage characteristics and MRI skill, which were competent for the diagnosis of RLN metastasis. Subsequently, it's significant to distinguish the RLN hidden in fatty tissues in a blood-stained operative field. It is well known that

Fig. 3. Dual-modality mapping of RLN. (aed, eeh) The MRI images of RLN before and post-injection of GO-IONP-PEG. (mep, qet) The MRI images of RLN before and post-injection of carbon nanoparticles. (iel, uex) Dye mapping of RLN with GO-IONP-PEG or carbon nanoparticles, respectively.



carbon nanoparticles had been effectively used to map lymph node metastasis, so we conducted the lymphatic mapping study of GOIONP-PEG in the mice using carbon nanoparticles as a control. As seen from Fig. 3u,w,x, the popliteal, sciatic, iliac common and renal hilar lymph nodes were all labeled in black by the carbon nanoparticles (score: 2 point). Likewise, GO-IONP-PEG had also the good ability of visible mapping lymph nodes. RLN were all labeled in black (score: 2 point) by GO-IONP-PEG due to its intrinsic black color (in web version) (Fig. 3i,k,l). As well, the inguinal lymph nodes were not black staining in two groups (Fig. 3j,v) that could explain MRI images of inguinal lymph nodes using GO-IONP-PEG. Furthermore, by comparing the two imaging results of MRI and carbon nanoparticles dye, the complementary advantage using two tracer modalities has been clearly presented. It's well known that sciatic lymph node by the anterior anatomical approach is difficultly exposed because of the special anatomical site. However, according to the MRI result, it can be easily observed by the posterior anatomical approach. In addition, due to the signal interference with inferior vena cava, the iliac common lymph nodes are hardly diagnosed using MRI, while they can be located by dye trace (Fig. 3x). So the mapping results demonstrated our GO-IONP-PEG agent had the competence for the dual-modality mapping of RLN metastasis. TEM tissue slices (Fig. 4) further confirmed the existence of GO-IONP-PEG (IONP manifested as black mass with high electronic density) in metastatic lymph nodes that provided the platform for photothermal destruction of tumor because of the persistence of GO. 3.5. Dual-model lymph mapping guided PTT Encouraged by the effective photothermal outcome and dualmodality mapping of RLN metastasis by GO-IONP-PEG, we further investigated in vivo mapping-guided invasive photothermal treatment of metastatic lymph nodes. Mice bearing lymph node metastases were established by subcutaneous injection of BxPC-3 cells suspended in RMPI-1640 medium via right hind foot pad in nude mice (male, aging 6e8 weeks). Once the tumors of popliteal lymph nodes were measured about 5.0 mm in longest dimension, mice were randomized into four treatment groups (n ¼ 5 per group): NS treated (Group I), NS with laser treated (Group II), GO-IONP-PEG treated (Group III), GO-IONP-PEG with laser treated (Group IV). There was no statistical difference among group mean popliteal tumor volumes at the onset of treatment (P > 0.05). Twenty-four hours before irradiation, the tumor-bearing mice were injected with NS or GO-IONP-PEG solution. The metastatic lymph nodes

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were illuminated with an 808-nm NIR laser for 5 min (2 W/cm2; spot size, 5 mm) in Group II, IV. In a recent work, photothermal ablation of sentinel lymph nodes to inhibit tumor metastasis has been performed [10]. However, taking into account the lymph node metastasis characteristic of pancreatic cancer, this is not enough, and the potential metastasis of RLN should be treated. In clinical practice, the treatment of fourth station lymph node doesn't improve the healing, because it is considered distant metastasis. So, radical lymphadenectomy is performed to third station lymph nodes generally. In this study, we firstly carried out the PTT for the RLN including popliteal lymph nodes (the first station lymph nodes) (Fig. 5a), sciatic lymph nodes (the second station lymph nodes) (Fig. 5b) and iliac common lymph nodes (the third station lymph node) (Fig. 5c) under the guidance of dual-modality mapping of GO-IONP-PEG agent. Fig. 6a showed the infrared thermal images of popliteal lymph node, sciatic and iliac common lymph node surface in Group IV mouse. During the irradiation, the temperature rapidly increased 41.14, 29.6, 25.76 C within 5 min in the focal region respectively. In contrast, less temperature changed in Group II, and increased only 7.21, 8.24, 6.21 C within 5 min respectively. High temperature can lead to instantaneous coagulative necrosis and irreversible cell death. For the PTT of popliteal and sciatic lymph nodes, they were exposed by a skin incision. And for the PTT of splanchnic nodes (iliac common lymph nodes), they were exposed by a midline incision of the abdomen and retraction the intestines in a sterile operating room [24]. Significantly, we only spend 30 min to complete PTT of RLN with minimally invasive incision (Fig. 5d), which was contrast to the time-consuming, more bleeding of traditional radical lymphadenectomy. During the course of therapy, no mice died. At 15th days, mice were euthanized, popliteal, sciatic and iliac common lymph nodes were excised and weighted. The weights of popliteal lymph nodes for Group I, Group II, Group III and Group IV were 17.00 ± 1.4 mg, 18.22 ± 0.9 mg, 21.98 ± 1.33 mg and 6.50 ± 1.6 mg, respectively (Fig. 6b). Obviously, the antitumor efficiency of Group IV was particularly prominent and was superior to all the other groups (P < 0.001), showing an inhibition rate of 61.76%, 64.32% and 70.43% compared with Group I, II and III. The weights of sciatic and iliac common lymph nodes for Group I, Group II, Group III and Group IV were 4.82 ± 2.1 mg and 3.96 ± 1.1 mg, 4.11 ± 0.8 mg and 3.04 ± 0.5 mg, 4.52 ± 0.8 mg and 2.95 ± 0.6 mg and 1.97 ± 0.7 mg and 0.9 ± 0.2 mg, respectively (Fig. 6b), which also showed that Group IV was superior to all the other groups (P < 0.05). The treatment efficiency by PTT was further validated by MRI. As shown in Fig. 6d, the popliteal lymph node in Group IV was seriously

Fig. 4. TEM images of GO-IONP-PEG in metastatic popliteal lymph nodes. (a) GO-IONP-PEG was inside the cells of popliteal lymph nodes, (b) GO-IONP-PEG was inside the extracellular matrix.


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Fig. 5. The photographs of PTT of RLN.(a) Popliteal lymph node, (b) sciatic lymph node, (c) iliac common lymph node, (d) minimally invasive incision.

ablated exposed to laser irradiation, and its size after treatment got smaller compared to original one (Fig. 6c). Considering the smaller size, we didn't choose sciatic and iliac common lymph nodes for MRI assessment of treatment efficiency by PTT. Accordingly, histopathological examination with hematoxylin and eosin (H&E) staining showed the popliteal, sciatic and iliac common lymph nodes treated with GO-IONP-PEG with laser lighting were coagulative necrosis and apoptosis (Fig. 7b,c,d). However, a lot of tumor cells were observed in the control group (Fig. 7a), indicating that PTT with GO-IONP-PEG was an efficient tool for the therapy of metastatic lymph nodes. H&E staining also showed metastasis rates of the first station lymph nodes, second station lymph nodes and third station lymph nodes were 93.3%, 13.3% and 6.7%, respectively. So the only PTT of the sentinel lymph nodes in the previous studies is insufficient, the other second, third station metastatic lymph nodes will provide a reservoir of cancer cells leading to distant, lethal metastases, which demonstrates the necessity of photothermal ablation of RLN. 3.6. Therapeutic efficacy and toxicity of GO-IONP-PEG evaluated Last but not least, nanotoxicity has become the subject of concern in nanomedicine. In order to evaluate the safety of GOIONP-PEG with or without NIR lighting in vivo, all treated mice for Group I, Group II, Group III and Group IV were sacrificed at 15th days. Almost 0.5 mL of blood from each mouse was collected for hematology analysis, and the tissues for Group IV including brain, heart, liver, spleen, lung, kidney and tumor were harvested and further processed for H&E analysis. The results showed that the

parameters including white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), alanine aminotransferase (ALT), serum creatinine (SCR) in Group II, Group III and Group IV appeared to be normal in comparison with NS Group I (Table S1, Supporting Information). The images of H&E staining tissue sections of brain, heart, liver, spleen, lung, and kidney were performed in Fig. S7aef, showing no apparent organ damage or abnormality. Furthermore, a promising treatment using nanotheranostic agent for clinical applications should possess the capabilities of desired therapeutic effect but the minimum side injury, which is particularly important for intraperitoneal PTT because there are significant intraabdominal nerves, blood vessels and organs. To my knowledge, until now no relevant studies on the safety assessment of PTT have been reported. In this work, we further investigated the safety of the regional lymph photothermal dissection with dual-modality mapping by GO-IONP-PEG. The splanchnic lymph nodes were selected, because (1) they have more complex anatomical structure than the first station lymph nodes, (2) plentiful surrounding tissues, nerves and blood vessels are closer to the lymph nodes, so they required higher security during PTT. Histopathological analysis showed the iliac common lymph nodes by the treatment of GOIONP-PEG under laser irradiation with 2 W/cm2 and 5 min were effectively ablated (Fig. 6d). Meanwhile, the nerves, blood vessels and adipose tissues around the lymph nodes were amazingly intact after PTT, even the distance of normal tissues to the lymph nodes was only 100 microns (Fig. S7g,h). The safety results might be interpreted that the injected GO-IONP-PEG nanomaterials could easily be transported to RLN via lymphatic vessels with unique structure and hardly leak from one-way lymphatic system. Thus,



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Fig. 6. In vivo dual-modality guided PTT. (a) Infrared thermal images of RLN (popliteal lymph node, sciatic lymph node, iliac common lymph node with NS or GO-IONP-PEG under 808-nm laser irradiation at 2 W/cm2 for 5 min. (b) The weights of metastatic popliteal, sciatic, iliac common lymph nodes after NS treated, NS with NIR irradiation treated, GO-IONPPEG treated, GO-IONP-PEG with NIR irradiation treated. The antitumor efficiency of GO-IONP-PEG with NIR irradiation treated was superior to all the other groups (***P < 0.001, *P < 0.05). MRI images of metastatic popliteal lymph nodes (c) in the control group and (d) after PTT.

Fig. 7. Histological TUNEL analysis of metastatic lymph nodes. (a) Control group treated with GO-IONP-PEG without NIR irradiation, a lot of tumor cells were observed. Coagulative necrosis and apoptotic cells were observed in the (b) popliteal (c) sciatic and (d) iliac common lymph nodes treated with GO-IONP-PEG under NIR irradiation.


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when the NIR laser irradiated, the surrounding normal tissues without GO-IONP-PEG nanomaterials were impossible to generate high heat. These results further demonstrated that our nanocomposites could photothermal ablate the metastatic lymph nodes with low side injury characteristics under laser irradiation, and might open the future biomedical applications. 4. Conclusion A versatile lymphatic nanotheranostic agent was successfully constructed for dual-modality MRI-dye mapping of RLN, and well as effective photothermal ablation of lymph nodes with metastatic pancreatic cancer. In vitro studies showed GO-IONP coated with PEG had low toxicity and excellent ability for photothermal killing of cancer cell. In vivo studies, GO-IONP-PEG not only exhibited its powerful dual-modality lymphatic tracing capabilities and high efficiency of tumor ablation, but also showed lower systematic toxicity and excellent security of PTT to neighbor tissues. In addition, the strategy of RLN mapping using single local injection into the tumor was not only suitable for clinical practice, but also had higher specificity, simpler operation, higher security features, compared to lymph local injection and intravenous injection strategy. Therefore, GO-IONP-PEG may have a great potential for the RLN theranostic applications in future clinic practices. Acknowledgment The authors are grateful to Prof. Zhuang Liu, Dr. Liang Cheng, and Dr. Liangzhu Feng for their help in material supply, to Dr. Xiangrong Yu for his assistance with MRI. This work is supported by the National Natural Science Foundation of China (30901760, 81071884, 81301974, 81472221), Research Fund for the Doctoral Program of Higher Education of China (20110071110065), the New Outstanding Youth Program of Shanghai Municipal Health Bureau (XYQ2013090), and Zhuo-Xue Project of Fudan University. This work was supported by the 973 Project (2013CB911201). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.07.064. References [1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10e29. [2] Long J, Luo GP, Liu C, Cui XB, Satoh K, Xiao ZW, et al. Development of a unique mouse model for pancreatic cancer lymphatic metastasis. Int J Oncol 2012;41: 1662e8. [3] Fujii T. Extended lymphadenectomy in pancreatic cancer is crucial. World J Surg 2013;37:1778e81. [4] Shi S, Yao W, Xu J, Long J, Liu C, Yu X. Combinational therapy: new hope for pancreatic cancer? Cancer Lett 2012;317:127e35. [5] Yang F, Jin C, Yang D, Jiang Y, Li J, Di Y, et al. Magnetic functionalised carbon nanotubes as drug vehicles for cancer lymph node metastasis treatment. Eur J Cancer 2011;47:1873e82. [6] Wray CJ, Ahmad SA, Matthews JB, Lowy AM. Surgery for pancreatic cancer: recent controversies and current practice. Gastroenterology 2005;128: 1626e41. [7] Madani SY, Naderi N, Dissanayake O, Tan A, Seifalian AM. A new era of cancer treatment: carbon nanotubes as drug delivery tools. Int J Nanomed 2011;6: 2963e79. [8] Alexiou C, Schmid RJ, Jurgons R, Kremer M, Wanner G, Bergemann C. Targeting cancer cells: magnetic nanoparticles as drug carriers. Eur Biophys J 2006;35: 446e50. [9] Chen J, Wang L, Yao Q, Ling R, Li K, Wang H. Drug concentrations in axillary lymph nodes after lymphatic chemotherapy on patients with breast cancer. Breast Cancer Res 2004;6:474e7. [10] Okuno T, Kato S, Hatakeyama Y, Okajima J, Maruyama S, Sakamoto M, et al. Photothermal therapy of tumors in lymph nodes using gold nanorods and near-infrared laser light. J Control Release 2013;172:879e84.

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