Enhanced CT imaging of human laryngeal squamous carcinoma and indirect CT lymphography imaging using PEGylated PAMAM G5·NH2-entrapped gold nanoparticles as contrast agent

Colloids and Surfaces A: Physicochem. Eng. Aspects 497 (2016) 194–204

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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa

Enhanced CT imaging of human laryngeal squamous carcinoma and indirect CT lymphography imaging using PEGylated PAMAM G5 ·NH2 -entrapped gold nanoparticles as contrast agent Fang Shi a,b , Chen Peng c , Yue Yang a,b , Yan Sha a , Xiangyang Shi d,e,f , Haitao Wu a,b,∗ a

Department of Otolaryngology—Head and Neck Surgery, Eye Ear Nose & Throat Hospital, Fudan University, Shanghai, China Shanghai Key Clinical Disciplines of Otorhinolaryngology, China c Department of Radiology, School of Medicine, Shanghai Tenth People’s Hospital Affiliated with Tongji University, Shanghai, China d State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai, China e College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, China f CQM—Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390 Funchal, Portugal b

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• Hep-2 xenograft tumor model and the SLN in rabbits after administration of the PEGylated Au DENPs could be efficient imaged. • The formed PEG-Au DENPs were noncytotoxic in the given concentration range. • The PEG-Au DENPs could be uptaken predominantly in the lyososomes of the cells.

a r t i c l e

i n f o

Article history: Received 14 November 2015 Received in revised form 23 January 2016 Accepted 5 February 2016 Available online 5 March 2016 Keywords: Dendrimers Gold nanoparticles In vivo CT imaging Tumors Indirect CT lymphography

a b s t r a c t We report the utilization of dendrimer-entrapped gold nanoparticles (Au DENPs) modified by polyethylene glycol (PEG) with good biocompatibility for enhanced computed tomography (CT) imaging of human laryngeal squamous carcinoma and indirect CT lymphography imaging in New Zealand rabbits. In this work, PEG-modified amine-terminated poly(amidoamine) dendrimers of generation 5 (G5·NH2 ) were used as templates to synthesize Au DENPs, followed by acetylation of the remaining dendrimer terminal amines to generate PEGylated Au DENPs. The formed PEGylated Au DENPs was used for both enhanced CT imaging of human laryngeal squamous carcinoma cells (Hep-2 cells) and the xenograft tumor mode, and indirect CT lymphography imaging in New Zealand rabbits. In vitro cytotoxicity assay, flow cytometry analysis, and cell morphology observation revealed that the formed PEGylated Au DENPs were non-cytotoxic at a Au concentration up to 400 ␮M for 24 h and indicated their good biocompatibility. Transmission electron microscopy data confirmed that the PEGylated

∗ Corresponding author at: Department of Otolaryngology—Head and Neck Surgery, Eye Ear Nose & Throat Hospital, Fudan University, 83 Fenyang Road, Shanghai 200031, China. Fax: +86 21 64377151. E-mail address: [email protected] (H. Wu). http://dx.doi.org/10.1016/j.colsurfa.2016.02.005 0927-7757/© 2016 Elsevier B.V. All rights reserved.

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Au DENPs was uptaken dominantly by the lysosomes of the cells. The PEGylated Au DENPs enabled not only enhance CT imaging of a xenograft tumor model in nude mice after intravenous injection of the particles, but also effective indirect CT lymphography imaging in rabbits. These findings suggest that the designed PEGylated Au DENPs can be used as a potentially effective contrast agent for CT imaging of various biological systems and different kinds of carcinoma, especially lymphatic mapping and human laryngeal squamous carcinoma. © 2016 Elsevier B.V. All rights reserved.

1. Introduction As formally introduced in 1999 and rapidly developed in the latest decade, molecular imaging (MI) represents an advanced technology in medical imaging area. It has the ability to detect and quantitatively measure the function of biological and cellular processes in vitro and in vivo. As one of the most important MI technologies, computed tomography (CT) is a reliable and widely used clinical imaging technique with better spatial and density resolution than other imaging modalities. The key component in CT imaging of biological systems is to design suitable contrast agents with better biocompatibility, longer imaging time, and more desirable imaging sensitivity and specificity [1–3]. The commercially available CT imaging agents used in clinics are usually iodinated small molecules (e.g., Omnipaque) that have severe drawbacks such as renal toxicity at a high concentration, short imaging time, and non-specificity [4–6]. To overcome these drawbacks, various nanoparticle (NP)-based contrast agents for CT imaging have been designed and have received considerable attention in recent years, just as it is in the case of, gold nanoparticles (AuNPs) due to its higher X-ray absorption coefficients than that of Omnipaque [7,8]. These advantages become particularly apparent when CT is used to diagnose diseases in the head and neck region, such as laryngeal squamous carcinoma. Head and neck cancer (excluding cancers of the nasopharynx) accounts for an estimated 549,000 global cancer diagnoses each year [9], making it the 7th most common cancer worldwide (6th among men). Squamous cell carcinoma makes up over of 90% of these cancers [10]. Despite significant advances in clinical treatment and in the understanding of its biology, as the predominant type of head and neck cancer today, laryngeal squamous carcinoma is a devastating malignancy that severely affects patients’ quality of life, with compromise of ability to talk and swallow. In this kind of cancer, lymph node staging and localization of pathological lymph nodes are necessary for doctors to choose adjuvant or surgical therapy and are major factors for the prognosis of head and neck cancer patients. The high incidence, mortality rate and disability rate associated with laryngeal squamous carcinoma has prompted numerous exhaustive efforts to develop various technologies for the diagnosis of this cancer and the staging of lymph node. While diagnosing metastasis of an enlarged lymph node with computed tomography (CT) or magnetic resonance imaging (MRI) is relatively easy, clinically negative lymph nodes (cN0) may harbor occult metastases and it is difficult to distinguish occult metastatic lymph nodes from non-metastatic lymph nodes when they are still normal-sized. Determining the status of the sentinel lymph node (SLN) is helpful [11] and indirect CT lymphography (CT-LG) has succeeded in identifying SLN by mapping and visualizing SLNs in the breast, oesophagus, lung, skin, and head and neck, using the non-ionic, iodinated, water-soluble contrast agent (e.g., Omnipaque, Iopamidol) [12–17]. More recently, the utilization of AuNPs has been believed to have an increasing potential as molecular probes for X-ray CT imaging, for they offer several advantages over conventional iodine-based agents. First, gold element have a higher X-ray attenuation intensity than iodine because of its higher atomic number and electron

density, endowing it in principle with a greater ability to enhance contrast for more sensitive CT imaging. Second, AuNPs, such as dendrimer-entrapped gold nanoparticles (Au DENPs) [18–20], dendrimer-stabilized gold nanoparticles (Au DSNPs) [21,22] and polymer-coated gold nanoparticles [23] are reported to be nontoxic at a certain concentration range. Third, it is easy to modify the surface of AuNPs with functional moieties such as targeting agents, imaging dyes, or specific biomarkers for a range of MI applications. Finally, proper surface treatment of AuNPs can help them to prolong their blood circulation time via effectively avoiding removal by the reticuloendothelial system (RES) [24]. Producing AuNPs with prolonged blood circulation time is crucial in tumor imaging via both a “passive” mechanism through an EPR effect and an “active” mechanism through a receptor-mediated endocytosis pathway [25,26]. Dendrimers are a class of highly branched, monodispersed, synthetic macromolecules with well defined composition and architecture [27]. Unique structural properties allow them to be used as templates or stabilizers to synthesize dendrimer-entrapped AuNPs (Au DENPs), which have the additional benefit of being stable not only in water, phosphate buffered saline (PBS), and cell culture medium but also at different temperature and pH conditions [28,29]. The art of dendrimer chemistry allows one to synthesize Au DENPs or Au DSNPs with terminal amines transformed to acetyl functional groups, significantly avoiding nonspecific cell membrane binding and toxicity [30–34]. In our previous reports [29] and [35], we show that Au DENPs prepared using amine-terminated poly(amidoamine) dendrimers of generation 5 (G5·NH2 ) modified by PEG monomethyl ether (G5·NH2 -mPEG) can be acetylated for in vivo blood pool CT imaging of mice after intravenous injection and for in vivo CT imaging of a xenografted tumor model in mice planted with a human lung adenocarcinoma cell line (SPC-A1 cells). In normal rabbits, oval or round enhanced cervical lymph nodes in each side of the neck were visualized by indirect CT lymphography using iopamidol [36]. However, it is uncertain whether others tumors and lymph nodes could be visualized by PEGylated Au DENPs. In the present study, we systematically studied the biocompatibility, and X-ray attenuation characteristics of the PEGylated Au DENPs. Cytotoxicity assay and cell morphology were used to assess the cytocompatibility of the particles. The particle distribution within the cells was observed with transmission electron microscopy (TEM), after which the CT imaging performance was evaluated in vivo using the Hep-2 xenografted tumor model. Indirect CT lymphography imaging was performed with the particles injected into the tongue submucosa in New Zealand rabbits. Finally, pharmacokinetic studies were performed by inductively coupled plasma-atomic emission spectrometry (ICP-AES) to estimate the half-decay time of the PEGylated Au DENPs. 2. Experimental section 2.1. Materials Ethylenediamine core G5·NH2 dendrimers with a polydispersity index less than 1.08 were from Dendritech (Midland, MI).

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Fig. 1. UV–vis spectra (a) and TEM image (b) of the PEGylated [(Au◦ )300 -G5·NHAc-mPEG] DENPs.

Fig. 2. Representative microscopic images of Hep-2 cells: (a) negative control cells without treatment with the [(Au◦ )300 -G5·NHAc-mPEG] DENPs (200×), the cells incubated with [(Au◦ )300 -G5·NHAc-mPEG] DENPs at the concentration of [Au] (b) 100 ␮m and (c) 300 ␮m for 24 h (200×), respectively.

Fig. 3. CCK-8 assay of the viability of Hep-2 cells treated with [(Au◦ )300 -G5·NHAc-mPEG] DENPs at different concentrations of [Au] for 24 h (n = 4).

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Fig. 4. Flow cytometry analysis of Hep-2 cells treated without (a) or with [(Au◦ )300 -G5·NHAc-mPEG] DENPs at the concentration of [Au] 100 ␮m (b) and 300 ␮m (c), respectively for 24 h (n = 3).

Fig. 5. TEM images of Hep-2 cell: (a) negative control cells without treatment, (b) cells incubated with [(Au◦ )300 -G5·NHAc-mPEG] DENPs at the concentration of [Au] 300 ␮m for 24 h. (c) a higher-magnification image of the black dotted line area in (b) to clearly show the [(Au◦ )300 -G5·NHAc-mPEG] DENPs in the cytoplasm and the cell nucleus. In all images, the white arrow shows the cell nucleus. The black arrow shows the domain of AuNPs.

PEG monomethyl ether with one end of maleimide (mPEG-MAL) was from Shanghai Yanyi Biotechnology Corporation (Shanghai, China). All other chemicals were obtained from Aldrich and used as received. Hep-2 cell line (a human laryngeal squamous carcinoma cell line) was purchased from Shanghai Cell Bank, the Chinese Academy of Sciences (Shanghai, China). Penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Sigma (St.

Louis, MO). Trypsin-EDTA, Dulbecco’s PBS, RPMI 1640 medium, and bovine serum albumin were obtained from GIBCO-BRL (Gaithersburg, MD). The water used in all the experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with a resistivity higher than 18 m cm. Regenerated cellulose dialysis membranes (molecular weight cut-off, MWCO = 10,000) were acquired from Fisher.

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Fig. 6. Representative transectional enhanced micro-CT images (a) and CT values (b) of the xenografts Hep-2 tumor in nude mice before and after injected with [(Au◦ )300 G5·NHAc-mPEG] DENPs for 6 and 24 h. The star ‘∗’ in (a) indicates the location of the tumor.

2.2. Synthesis of [(Au◦ )300 -G5·NHAc-mPEG] DENPs Twenty molar equivalents of mPEG-MAL (61.76 mg, 0.031 mmol) dissolved in dimethylsulfoxide (DMSO, 5 mL) were added dropwise to a solution of G5·NH2 dendrimers (40.16 mg, 1.54 × 10 − 3 mmol) in DMSO (10 mL) with vigorous magnetic stirring. The stirring process was continued for 3 days to complete the reaction, and the reaction mixture turned to a faint yellow color. Then the reaction mixture was extensively dialyzed against PBS buffer (3 times, 4 L) and water (3 times, 4 L) for 3 days to remove the excess of reactants and by-products, followed by lyophilization to obtain the G5·NH2 -mPEG product. The procedure to synthesize [(Au◦ )300 -G5·NHAc-mPEG] DENPs was adopted from previously reported methods [28], [29], [37]. The [(Au◦ )300 -G5·NHAc-mPEG] DENPs were prepared using sodium borohydride reduction chemistry with the molar ratio of gold salt to G5·NH2 -mPEG at 300:1. Briefly, solution was added into an aqueous solution of G5·NH2 -mPEG (20 mg, 10 mL) under vigorous stirring. After 30 min, an icy cold NaBH4 solution (5 mL, water/methanol (v/v = 2:1)) with 5 times molar excess to the Au salt was added to the gold salt/dendrimer mixture under stirring, and the reaction mixture turned to a deep-red color within a few seconds. The stirring process was continued for 2 h to complete the reaction. The final Au DENP products are denoted as [(Au◦ )300 -G5·NH2 -mPEG] DENPs. Then, the [(Au◦ )300 -G5·NH2 -mPEG] DENPs were further acetylated to neutralize the dendrimer terminal amine groups [29], [34], [38]. Briefly, triethylamine (54.7 ␮L) was added to an aqueous solution of [(Au◦ )300 -G5·NH2 -mPEG] DENPs (25 mL, 21.12 mg) under magnetic stirring. After 30 min, acetic anhydride (31.0 ␮L, 324 mm, 4 times molar excess of the total primary amines of G5 PAMAM) was added into the DENP/triethylamine mixture solution while stirring, and the mixture was allowed to react for 24 h. The aqueous solution of the reaction mixture was extensively dialyzed against PBS (3

times, 4 L) and water (3 times, 4 L) for 3 days to remove the excess of reactants and by-products, followed by lyophilization to obtain the [(Au◦ )300 -G5·NHAc-mPEG] DENPs. 2.3. Characterization techniques UV–vis spectra were collected using a Lambda 25 UV/Vis spectrometer (Perkin Elmer, USA). Samples were dissolved in water (pH 6.0) at room temperature (25 ◦ C) before the experiments. TEM was performed using a JEOL 2010F analytical electron microscope (JEOL, Japan) operating at 200 kV. An aqueous solution of Au DENPs (1 mg/mL) was dropped onto a carbon-coated copper grid and air dried before measurements. The size-distribution histogram of particles was measured using ImageJ software (http://rsb.info.nih.gov/ ij/download.html). 300 NPs from different TEM images were randomly selected to analyze the size. 2.4. Cell culture Hep-2 cells were continuously cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37 ◦ C and 5% CO2 in a humidified incubator. 2.5. Cytotoxicity of [(Au◦ )300 -G5·NHAc-mPEG] DENPs Hep-2 cells (4 × 105 ) were plated in each well of 6-well cell culture plates. When the cells were grown to about 80% confluence, [(Au◦ )300 -G5·NHAc-mPEG] DENPs ([Au] = 100 and 300 ␮M) were incubated with the cells for 24 h. The cell morphology was observed under an optical microscope (200 × magnification, Nikon, Tokyo, Japan). The viability of cells treated with the [(Au◦ )300 -G5·NHAc-mPEG] DENPs was then measured with the Cell Counting Kit-8 (CCK-8;

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Dojindo Laboratories, Kumamoto, Japan). A total of 4 × 103 cells in 100 ␮L of the medium were seeded in a 96-well plate and incubated for 24 h to obtain sufficient cell growth. Then the medium was replaced by RPMI 1640 medium containing different concentrations of Au DENPs ([Au] = 0–400 ␮M) and cells were incubated for 24 h at 37 ◦ C. After treatment, the medium was aspirated and cells were washed with PBS buffer for three times. And then 100 ␮L fresh RPMI 1640 medium was added to each well. Thereafter, 10 ␮L of CCK-8 solution was added to each well of the plate and the plate was incubated at 37 ◦ C for 4 h. Absorbance was measured at 450 nm as an indicator of cell viability using a microplate reader (Bio-tek). The toxicity of the [(Au◦ )300 -G5·NHAc-mPEG] DENPs was further examined by flow cytometric detection of cell cycles and apoptosis. Hep-2 cells were seeded in 6-well cell culture plates at a density of 4 × 105 cells per well in quadruplication and allowed to grow to confluence for 24 h. Then, after replacing the medium with fresh medium containing different concentrations ([Au] = 100 and 300 ␮M), the cells were incubated for 24 h at 37 ◦ C in CO2 incubator, after which the culture medium was discarded and the cells were harvested by trypsinization and centrifugation, washed with PBS buffer, and fixed in citrate buffer for 2 h. The cells were then centrifuged to remove citrate buffer and resuspended with PBS buffer with a cell concentration of 1 × 106 cells/mL. The cell suspensions were incubated with trypsogen for 3 min and then incubated with RNase for 3 min. Subsequently, the cells were stained with propidium iodide (PI) for 15 min and then the PI-stained cells were measured by flow cytometry (FACSCalibur, Becton Dickinson, USA) in red (FL2) channel at 488 nm. The cell cycle profiles, including G0G1, G2-M, and S phases were analyzed by using Cellquest software (FACSCalibur, Becton Dickinson, USA). 2.6. Cellular uptake of

[(Au◦ )300 -G5·NHAc-mPEG]

DENPs

For further TEM imaging of the distribution of [(Au◦ )300 G5·NHAc-mPEG] DENPs within the cells, Hep-2 cells were plated in 6-well cell culture plates with a density of 4 × 105 cells per well in RPMI 1640 medium with 10% FBS in a humidified incubator(37 ◦ C, 5% CO2 ) for 24 h to grow to about 80% confluence. Then, the culture medium containing 300 ␮M [Au] was added to each well and cells were incubated for 24 h at 37 ◦ C. The culture medium was discarded and the cells were washed with PBS buffer, trypsinized, centrifuged, washed for three times with PBS buffer again, and finally fixed with 2.5% glutaraldehyde in 0.2 m phosphate buffer (pH 7.2) for 12 h at 4 ◦ C and post-fixed with 1% OsO4 in 0.2 m phosphate buffer (pH 7.2) for 2 h at 4 ◦ C. After additional washing in PBS buffer, the cells were dehydrated and embedded with Epon 812 (Shell Chemical, UK), followed by polymerization. Then, the embedded cells were sectioned with a thickness of 75 nm using a Reichart Ultramicrotome. The sections were then mounted onto 200-mesh copper grids and counterstained with uranyl acetate and lead citrate for 5 min, respectively before TEM measurements. TEM was performed using an H600 transmission electron microscope (Hitachi, Japan) with an operating voltage of 60 kV. 2.7. In vivo enhanced micro-CT imaging of hep-2 xenograft tumor model Animal experiments were carried out according to protocols approved by the institutional committee for animal care and also in accordance with the policy of the National Ministry of Health. Male 4- to 6-week-old BALB/c nude mice (n = 3, Shanghai SLAC Laboratory Animal Center, Shanghai, China) were subcutaneously injected in the right side of their back with 1 × 106 cells/mouse. When the tumor nodules had reached a volume of 1.0 ± 0.15 cm3 after approximately 3 weeks post-injection, the tumor was ver-

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ified by gross specimen and HE staining. The mice were placed in a scanning holder, and then scanned using a micro-CT imaging system (eXplore Locus, GE Healthcare, London, Ontario, Canada) with 80 kV, 450 ␮A, and a slice thickness of 45 ␮m. CT scanning was performed before and after intravenous (100 ␮L, [Au] = 0.1 m) injection of [(Au◦ )300 -G5·NHAc-mPEG] DENPs at time points of 6 and 24 h post-injection, respectively. Images were reconstructed on a micro-CT imaging workstation using the following parameters: voxel, 45 ␮m × 45 ␮m × 45 ␮m; display field of view, 10–25 mm. CT values were acquired on the same workstation using the software supplied by the manufacturer. To further verify that the PEGylated Au DENPs has good biocompatibility at the given concentration range for enhanced CT imaging, the mice’s different organs including heart, lung, liver, spleen, kidneys and tumor at 24 h post injections were tested by HE staining and observed using a light microscope. 2.8. In vivo biodistribution of [(Au◦ )300 -G5·NHAc-mPEG] DENPs After micro-CT scanning, the above tumor-bearing mice were sacrificed. Different organs including the heart, lung, stomach, spleen, liver, intestines, kidney, testicle, blood, tumor, and brain were extracted and weighed. The organs were cut into 1–2 mm2 pieces and incubated in aqua regia solution for 4 h. Finally, the Au content was measured by a Leeman Prodigy ICP-AES system (Hudson, NH03051, USA). 2.9. Localization of the sentinel lymph node (SLN) in normal rabbits via indirect CT lymphography (CT-LG) imaging Dunne et al. studied New Zealand white rabbit lymph node topography and found that dye injected into the tongue drained first into the rostral mandibular lymph nodes, and secondarily into the caudal mandibular lymph nodes. This is similar to humans [39], so we chosed the rabbits for the CT-LG imaging.The details of indirect CT lymphography imaging were adopted from previously reported methods [17], [36] and [40]. Six 12-month-old New Zealand White rabbits (Shanghai Slac Laboratory Animal Center, Shanghai, China) weighing each which 2.5–3.0 kg did not receive any treatment were fixed to a specially designed CT Table in the supine position with the neck extended. And then they were scanned using a Transverse CT scanning system (Siemens SOMATOM Sensation 10 Forchhein, Bavaria, Germany), which was operated at 120 kV and 150 mAs with a 7- to 12-cm field of view and a 512 × 512 matrix. Each rabbit underwent a contiguous 1.5 mm thick CT scan. Indirect CT lymphography images were obtained from the tongue to the sternum. Before the particles was injected, CT images were recorded to eliminate the possibility of confounding calcification in the lymph nodes. After [(Au◦ )300 G5·NHAc-mPEG] DENPs (500 ␮L, [Au] = 0.1 m) was injected gently into the right ventrolateral submucosa of the tongue, contiguous CT images were obtained at 6 and 24 h, respectively. After CT scanning, the above rabbits with the particles injected were sacrificed. An incision was made at the marked site. The target lymph nodes were identified carefully and precisely by referring to the detailed anatomy on the CT lymphography images to ensure that these nodes corresponded in position and size. 3. Results and discussion 3.1. Synthesis and characterization of [(Au◦ )300 -G5·NHAc-mPEG] DENPs Under the same experimental protocol described in our previous reports [28], [29] and [37], we were able to obtain PEGylated Au DENPs with Au salt/G5 dendrimer molar ratio of 300:1. UV–vis

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Fig. 7. Optical microscopic image of HE-stained the mice’s different organs (including heart, lung, liver, spleen, kindney and tumor) at 24 h post intravenous injections and the untreated control organs. The magnification for tumor section was set at 200×.

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Fig. 8. The biodistribution of [(Au◦ )300 -G5·NHAc-mPEG] DENPs in different organs and xenograft Hep-2 tumor. The data were obtained by ICP-AES at 6 h and 24 h post-injection in Hep-2 xenografted BALB/c nude mice models.

Fig. 9. Representative transectional indirect CT lymphography images (a) and CT values (b) of the sentinel lymph node in the normal New Zealand White rabbits before and after injected with [(Au◦ )300 -G5·NHAc-mPEG] DENPs for 6 and 24 h. The star “↑” in (a) indicates the location of the sentinel lymph node.

spectroscopy and TEM were utilized to characterize the synthesized [(Au◦ )300 -G5·NHAc-mPEG] DENPs (Fig. 1). The PEGylated [(Au◦ )300 -G5·NHAc-mPEG] DENPs displays a surface plasmon resonance (SPR) peak of AuNPs at around 510 nm (Fig. 1a), in agreement with literature [30]. Fig. 1b shows the TEM images of [(Au◦ )300 G5·NHAc-mPEG] DENPs. It is clear that the size of AuNPs have a relatively uniform size distribution with a mean diameter of 3.15 nm, which suggesting that the formation and growth of AuNPs are effectively restricted within the G5·NH2 -mPEG templates. The [(Au◦ )300 -G5·NHAc-mPEG] DENPs in a powder form can be dissolved in water, PBS buffer, and cell culture medium with good colloidal stability for at least 6 months at room temperature. In general, the acetylated Au DENPs were stored at −20 ◦ C in a dried form.

3.2. Cytotoxicity of [(Au◦ )300 -G5·NHAc-mPEG] DENPs Before applying PEGylated [(Au◦ )300 -G5·NHAc-mPEG] DENPs for in vivo CT imaging of the Hep-2 xenograft tumor model, extensive cytotoxicity studies were performed. [(Au◦ )300 -G5·NHAcmPEG] DENPs were tested the cytotoxicity of Hep-2 cells by observation of cell morphology, CCK-8 assay of cell viability, and flow cytometric analysis of cell cycles and apoptosis. The morphology of Hep-2 cells before and after incubation with the [(Au◦ )300 -G5·NHAc-mPEG] DENPs was checked by optical microscopy (Fig. 2). The results showed that even if the concentration of [Au] was up to 300 ␮M and incubated with the cells for 24 h, the cells did not display any morphological changes when compared with the untreated control cells. These results indicated that [(Au◦ )300 -G5·NHAc-mPEG] DENPs was non-cytotoxic, and did not

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have any effect on the morphology of the Hep-2 cells at a relatively higher concentration for a longer time than that of acetylated Au DENPs used in our previous report [41]. To further examine the cytotoxicity of the [(Au◦ )300 -G5·NHAcmPEG] DENPs on the Hep-2 cells, the concentration-dependent effect of the [(Au◦ )300 -G5·NHAc-mPEG] DENPs on the cell viability was analyzed by CCK-8 viability assay (Fig. 3). It was clear that cells treated with different concentrations of [(Au◦ )300 -G5·NHAc-mPEG] DENPs (the concentration of [Au] is 200, 300, and 400 ␮M, respectively) had approximately similar cell viability compared with the untreated negative control cells (p > 0.05, n = 4). The non-toxicity of the [(Au◦ )300 -G5·NHAc-mPEG] DENPs at a concentration of [Au] up to 400 ␮M suggested that PEGylation modification was a powerful strategy to significantly improve the biocompatibility of Au DENPs for biomedical imaging applications, especially for the circumstances requiring the particle concentration to be sufficiently high. Cell cycle is an important parameter of cell biology. Cell cycle damage is one of the important features for the cytotoxicity [42]. Cell phase distribution is generally analyzed by the determination of DNA contents, and the fraction of DNA content in sub-G1 phase is an indicator of cell apoptosis [43] and [44]. To investigate the influence of the PEGylated [(Au◦ )300 -G5·NHAc-mPEG] DENPs on the cell apoptosis, the treated cells were analyzed by flow cytometry (Fig. 4). The sub-G1 fraction of Hep-2 cells incubated with [(Au◦ )300 -G5·NHAc-mPEG] DENPs at the concentration of [Au] 100 ␮M and 300 ␮M were 1.09 ± 0.15 and 1.31 ± 0.05%, respectively in the quadruplication experiment. We showed that there was no statistically significant difference from that of the untreated negative control cells (1.09 ± 0.15%, p > 0.05), which indicated that [(Au◦ )300 -G5·NHAc-mPEG] DENPs had no effect on the cell cycle of Hep-2 cells (Table 1). These results further confirm that the [(Au◦ )300 -G5·NHAc-mPEG] DENPs were non-cytotoxic at the given concentration range and should be amenable for CT imaging applications.

3.3. In vitro cellular uptake of [(Au◦ )300 -G5·NHAc-mPEG] DENPs For further identification of the distribution of the [(Au◦ )300 G5·NHAc-mPEG] DENPs in the subcellular compartments, TEM imaging of cells was performed (Fig. 5). It was clear that after incubated with [(Au◦ )300 -G5·NHAc-mPEG] DENPs ([Au] 300 ␮M) for 24 h, numerous high electron-staining particles could be found in the cytoplasm of the cells (Fig. 5b). The higher-magnification images further demonstrate that the uptake of [(Au◦ )300 -G5·NHAcmPEG] DENPs was dominantly located in the lysosomes (Fig. 5c). In sharp contrast, there were no electron-staining particles in the cytoplasm of the untreated control Hep-2 cells (Fig. 5a). The TEM results confirmed that the [(Au◦ )300 -G5·NHAc-mPEG] DENPs were internalized by the cells instead of sticking to the surface of the cells. The internalization of the PEGylated [(Au◦ )300 -G5·NHAc-mPEG] DENPs likely occurs through two distinct mechanisms: phagocytosis and diffusion via cell walls as has been showed by previous literature data [45] and [46]. Furthermore, TEM imaging data also showed that the incubation of [(Au◦ )300 -G5·NHAc-mPEG] DENPs with a concentration of [Au] as high as 300 ␮M did not appre-

ciably affect the cell morphology, further corroborating the CCK-8 viability assay data.

3.4. Enhanced micro-CT imaging of hep-2 xenograft tumor model in vivo The great biocompatibility of [(Au◦ )300 -G5·NHAc-mPEG] DENPs drove us to pursue their applicability for in vivo CT imaging of xenotransplanted tumor model in BALB/C nude mice. To investigate the applicability of using [(Au◦ )300 -G5·NHAc-mPEG] DENPs for in vivo enhanced CT imaging of Hep-2 cells, the xenotransplanted Hep-2 tumor model was established in BALB/C nude mice. After intravenous injection of a PBS buffer solution containing [(Au◦ )300 G5·NHAc-mPEG] DENPs (168.76 mg in 4 mL PBS buffer, [Au] = 0.1 m) into a mice through tail vein, CT scan was performed and the images were collected. The enhanced micro-CT imaging results were quite inspiring. Fig. 6a shows the tumor CT images before and after intravenous injection of [(Au◦ )300 -G5·NHAc-mPEG] DENPs. It was clear that the tumor site showed an obvious enhancement with a significantly higher CT value after administration of [(Au◦ )300 -G5·NHAc-mPEG] DENPs when compared with that before injection. Moreover, we found that [(Au◦ )300 -G5·NHAc-mPEG] DENPs could diffuse into the entire tumor area with time, and the tumor region and the margin of the tumor region gradually turn to be clearer and sharper. The CT values of the tumor area at different time points increased gradually (Fig. 6b). At the time point of 6 h after injection of the [(Au◦ )300 -G5·NHAc-mPEG] DENPs, the CT value of tumor area was significantly higher than that before injection (p < 0.05;Fig. 6b). Even at the time point of 24 h post-injection, the CT values of the tumor area were all still significantly higher than that of the tumor area before injection(p < 0.05; Fig. 6b). These results clearly indicate that [(Au◦ )300 -G5·NHAc-mPEG] DENPs could not only enhance the laryngeal tumor in vivo but also be in part transported to the tumor site via EPR effect and then ingested by tumor cells [47], [48], [49] and [50], in agreement with our previous study [41]. These results confirmed that the particles can be slowly delivered to the tumor site via intravenous injection, allowing for effective enhanced CT imaging of tumors, which was very important for early-stage diagnosis of unknown tumors. Furthermore, the xenografted Hep-2 tumors were confirmed by HE staining (Fig. 7f), which showed the features of Hep-2 cells. After applying [(Au◦ )300 -G5·NHAc-mPEG] DENPs for in vivo enhanced CT imaging of the Hep-2 xenograft tumor model, the biocompatibility was tested by HE staining. HE staining was used to check the morphology of the mice’s different organs (including heart, lung, liver, spleen, kindney) and tumor at 24 h post injections. It was clear that all the organs and tumor did not display any appreciable morphological changes after treatment with the [(Au◦ )300 -G5·NHAc-mPEG] DENPs at a high concentration ([Au] = 0.1 m) when compared with the untreated control organs (Fig. 7). These results indicated that the PEGylated Au DENPs had a good biocompatibility and did not affect the morphology of the mice’s different organs and Hep-2 xenograft tumor in a certain concentration window.

Table 1 Apoptosis and cell cycle analysis of Hep-2 cells after incubation with the [(Au◦ )300 -G5·NHAc-mPEG] DENPs at the concentration of [Au] 100 ␮m and 300 ␮m for 24 h (mean ± S.D., n = 3). Group

Apoptosis (%)

Control 100 ␮m 300 ␮m

1.09 ± 0.15 0.81 ± 0.16 1.31 ± 0.05

Cell cycle (%) G0–G1 80.24 ± 2.46 80.07 ± 1.05 81.15 ± 1.86

G2-M 9.47 ± 1.41 9.07 ± 0.57 7.46 ± 0.73

S 10.29 ± 1.05 10.85 ± 0.82 11.39 ± 1.20

G2/G1 1.8 1.78 1.78

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Understanding the biodistribution of the [(Au◦ )300 -G5·NHAcmPEG] DENP is crucial for their uses as nanoprobes for in vivo CT imaging. The biodistribution of the [(Au◦ )300 -G5·NHAc-mPEG] DENPs in different organs including heart, lung, stomach, spleen, liver, intestines, kidney, testicle, blood, tumor, and brain at 6 h and 24 h post injections was analyzed by ICP-AES (Fig. 8). It was clear that for both 6 h and 24 h time points after the intravenous injections, spleen had the most significant Au uptake. The less uptake of Au in both liver and kidney, which were all known to have the reticuloendothelial system (RES) organs [51], suggested that the [(Au◦ )300 -G5·NHAc-mPEG] DENPs are able to escape the reticuloenthelial system (RES) located in the liver and pass through the renal filter, thereby allowing for an efficient transport of the particles to the tumor tissue via the passive EPR effect. Overall, our biodistribution studies showed that the [(Au◦ )300 -G5·NHAc-mPEG] DENPs could be uptaken by tumor tissues, allowing for effective CT imaging of tumor. And it also mean that the most particles metabolized by hepatic metabolism and renal metabolism. 3.5. Indirect CT-LG imaging findings Prior to injection of the contrast agent [(Au◦ )300 -G5·NHAcmPEG] DENPs, no noticeable lymph node calcification was visualized on CT, and cervical lymph nodes could not be clearly identified (Fig. 9a). After the particles injection, indirect CT-LG revealed a rapid enhancement of the ipsilateral afferent SLN (Fig. 9a). Only one enhanced SLN, that were all in the same position, lateral to the larynx-trachea region, was visualized in the ipsilateral side of the neck on CT-LG images. The average maximum diameters of the enhanced SLNs that displayed an oval or round shape on CT-LG images were 0.533 ± 0.05 cm. It was clear that there was no statistically significant difference between that of the actual average maximum diameters of the SLNs by measureing the anatomy (0.541 ± 0.08 cm, p > 0.05). The gradually increasing enhancement was obtained 10 min after [(Au◦ )300 -G5·NHAc-mPEG] DENPs injection and at the time point of 6 h post injection, peak enhancement achieved. The CT value of SLNs was significantly higher than before injection (p < 0.05;Fig. 9b), with the enhancement time lasting till 24 h post-injection. There was no enhancement of contralateral nodes. The actual position of the particle-stained SLNs at dissection was consistent with the position indicated using CT-LG. Therefore, indirect CT-LG combined with [(Au◦ )300 -G5·NHAc-mPEG] DENPs injection could be utilized for SLN identification with longer imaging time [17] and [40]. And CT-LG offered selective and effective enhancement of the SLN without causing serious local or general adverse reactions, except minor and temporary swelling at the injection site [52]. This is a preliminary study investigating the utility of indirect CT-LG combined with [(Au◦ )300 -G5·NHAc-mPEG] DENPs injection in normal head and neck SLNs. Future research could focus on other types of head and neck SLNs(e.g.,the metastatic SLNs) as well as human applications. 4. Conclusion In summary, a new use of PEG-modified dendrimer-based Au DENPs for enhanced CT imaging of cancer cell in vivo and in indirect CT-LG was reported in this study. Micro-CT imaging studies showed that Hep-2 xenograft tumor model and the SLN in rabbits after administration of the particles two different injection routes could be efficient imaged. Besides, combined morphology observation, CCK-8 viability assay, and flow cytometric analysis of cells revealed that the formed PEG-Au DENPs were non-cytotoxic in the given concentration range. TEM imaging studies further confirmed that the PEG-modified Au DENPs could be uptaken predominantly in the lyososomes of the cells. Moreover, given the unique structural

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characteristics of dendrimer chemistry that could be further functionalized with various targeting ligands, we anticipate that the PEG-modified Au DENPs could be used as nanoprobes for targeted CT imaging not only of human laryngeal squamous carcinoma, but also of different types of cancer, especially for early-stage diagnosis of cancer.

Acknowledgments This research is financially supported by grants No.PW2012D-4 from the Health Bureau of Shanghai New Pudong District of China, No. 13ZZ008 from the Shanghai Municipal Education Commission of China, and No. 09411967500 from the Shanghai Municipality Science and Technology Commission of China. Xiangyang Shi. gratefully acknowledges the Fundac¸ão para a Ciência e a Tecnologia (FCT) and Santander bank for the Chair in Nanotechnology.

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