A Protein-Corona-Free T1–T2 Dual-Modal Contrast Agent for Accurate

Dec 8, 2015 - ABSTRACT: Precise nodal staging is particularly important to guide the treatments and determine the prognosis for cancer patients. Howev...
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A Protein-Corona-Free T1−T2 Dual-Modal Contrast Agent for Accurate Imaging of Lymphatic Tumor Metastasis Zijian Zhou,†,∥ Hanyu Liu,†,∥ Xiaoqin Chi,‡,∥ Jiahe Chen,§ Lirong Wang,† Chengjie Sun,† Zhong Chen,§ and Jinhao Gao*,† †

State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China ‡ Fujian Provincial Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma, Zhongshan Hospital, Xiamen University, Xiamen 361004, China § Department of Electronic Science and Fujian Key Laboratory of Plasma and Magnetic Resonance, Xiamen University, Xiamen 361005, China S Supporting Information *

ABSTRACT: Precise nodal staging is particularly important to guide the treatments and determine the prognosis for cancer patients. However, it is still challenging to noninvasively and precisely detect in-depth tumor metastasis in lymph nodes (LNs) because of the small size and high potential of obtaining pseudopositive results. Herein, we report the rational design of a T1−T2 dual-modal MRI contrast agent for accurate imaging of tumor metastasis in LNs using gadolinium-embedded iron oxide nanoplates (GdIOP). The GdIOP were modulated with suitable size in vivo through surface functionalization by zwitterionic dopamine sulfonate (ZDS) molecules. The efficient uptake of GdIOP@ZDS nanoparticles through drainage effect because of the presence of large amount of macrophages and dendritic cells generates both T1 and T2 contrasts in LNs. In contrast, the low uptake of protein-corona-free GdIOP@ZDS nanoparticles by melanoma B16 tumor cells promises pseudocontrast imaging of potential tumor metastasis in LNs. The combination of T1 and T2 imaging modalities allows self-confirmed detection of a metastatic tumor with about 1.2 mm in the minimal dimension in LNs, which is close to the detection limit of submilimeter level of MRI scans. This study provides an efficient and noninvasive strategy to detect tumor metastasis in LNs with greatly enhanced diagnostic accuracy. KEYWORDS: T1−T2 dual-modal, protein-corona-free, tumor metastasis, lymph node, accuracy

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potential of obtaining pseudopositive results.11 The identification of sentinel LNs is particularly important to reflect the status of tumor metastasis in distant LNs and determine the cancer treatment and decision-making, especially in breast cancer surgery. So far, lymphadenectomy and histologic strategy are still the gold standard to evaluate the tumor metastasis in LNs, which are invasive and were proven to be lack of accuracy.12 For safety concerns, patients have to suffer the removal of all nearby LNs after ablating a primary tumor. However, this treatment would bring a long-term lymphedema trouble to patients, such as localized fluid retention and tissue swelling, because of the inability to drain off the surrounding lymph fluid.13 Hence, there is a substantial need to develop a powerful probe capable of efficiently and accurately detecting metastatic tumors in LNs.

INTRODUCTION Tumor metastasis through bloodstream or lymphatic system is responsible for the vast majority of cancerous deaths, up to 90% from solid tumors.1,2 Although numerous efforts have been paid to against metastatic cancers, such as to boost the strength of immune responses to tumors and disrupt individual steps in the metastatic process, little progress has been made due to the complex mechanism related to tumor metastasis.3,4 Moreover, there is still a major gap between the accurate detection of metastatic tumors at preclinical level and the cure of cancers.5,6 It is worth noting that detection of circulating tumor cells in blood has been well demonstrated because of the ease of access,7,8 which may help doctors to decide whether additional treatments (e.g., chemo or radiotherapy) are needed after surgically removal of a primary tumor. Precise nodal staging is also particularly important to guide the treatments and determine the prognosis for cancer patients.9,10 However, it is still challenging to effectively detect in-depth tumor metastasis in lymph nodes (LNs) because of the small size and high © 2015 American Chemical Society

Received: September 8, 2015 Accepted: December 8, 2015 Published: December 8, 2015 28286

DOI: 10.1021/acsami.5b08422 ACS Appl. Mater. Interfaces 2015, 7, 28286−28293

Research Article

ACS Applied Materials & Interfaces Progress in nanotechnology has emerged numerous imaging agents working with single or multiple imaging modalities (e.g., CT and optics) that are able to identify and stage tumor metastasis in LNs.14−16 These advances can provide invaluable information for preclinical therapeutic decision-making when a primary tumor was found. Moreover, the combination of multiple imaging (e.g., CT and PET) and therapeutic strategies as antimetastatic nanomedicines has been developed. 17 However, these imaging strategies were limited by different penetration depths and spatial resolutions of different imaging machines, which hindered the further applications in practice. Among the variety of imaging techniques, magnetic resonance imaging (MRI) is one of the most powerful and noninvasive diagnostic tools which can provide excellent anatomical details for soft tissues.18 Contrast-enhanced MRI (either T1 or T2) detection of tumor metastasis in lymph nodes has been widely explored;19−22 however, the high potential of false positive signal usually leads to low sensitivity and poor accuracy in traditional single modal MRI study. Recently, we have developed an efficient strategy to engineer synergistically enhanced T1−T2 dual-modal contrast agents (DMCAs) for sophisticated MRI, which can provide enhanced diagnostic accuracy within one MRI machine.23,24 The combination of T1 and T2 imaging modalities promises “selfconfirmed” merits for diagnosing region of interests, which were employed to detect liver or liver cancers after accumulated in liver region due to the mononuclear phagocyte system (MPS).23,24 Considering that LNs consist of vast majority of macrophages and dendritic cells, the drainage of DMCAs in LNs may promise accurately imaging of LNs with combining T1 and T2 dual imaging modalities. Herein, we designed and evaluated gadolinium-embedded iron oxide nanoplates (GdIOP) as a novel DMCA for T1−T2 dual-modal imaging of tumor metastasis in LNs of mouse. The GdIOP benefit from the exposed metal-rich surfaces and the domain aggregation effect, which generate strong T1 and T2 contrasts under a wide range of magnetic fields.24 Moreover, we used zwitterionic dopamine sulfonate (ZDS) molecules as surface modifiers to render GdIOP with protein-corona-free character in biological environment.25−27 The “stealth” property of GdIOP@ZDS may lead to highly different uptake efficiency in the tumor cells and macrophages, that is, macrophages can recognize and eliminate GdIOP@ZDS as external materials by endocytosis but tumor cells do not. This phenomenon renders GdIOP possible to generate MRI contrasts between metastic tumor cells and normal LNs (Figure 1).

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Figure 1. Schematic illustration of T1−T2 dual-modal MRI of tumor metastasis in lymph nodes (LNs). The primary tumor at the mouse right leg may relocalize at the nearby popliteal LN through the process of tumor lymphatic metastasis. The protein-corona-free gadoliniumembedded iron oxide nanoplates (GdIOP) were injected in the sole of mouse right foot and was uptaken by the same LN, which then generates both T1 bright and T2 dark images in the LN. The potential metastatic tumor (red arrows) in LN was imaged by pseudocontrast phenomenon due to a relatively low uptake of contrast agents by metastatic tumors. flask, and the solution was heated to reflux for 2 h. The nanoparticles were then transferred to the lower layer of water phase. The products were collected by centrifugation and redispered in distilled water. Cellular Uptake Study of GdIOP@ZDS Nanoparticles. We evaluated the cellular uptake efficiency of GdIOP@ZDS nanoparticles with melanoma B16 and RAW 264.7 cell models. One mL of GdIOP@ZDS nanoparticles (50 μg/mL with respect to total metal ions) were added in a cell culture dish containing 1 × 106 cells. The groups (both melanoma B16 and RAW 264.7 cells) were then incubated with 3 and 6 h, respectively. After discarded the supernatant and washed twice with PBS, 1 mL of PBS containing Hoechst 33342 dyes was added and incubated for 5 min to stain the cell nucleus. The cell images were acquired by a Zeiss microscope (Axio Obserber A1). The cells in each dish were then counted and collected for ICP-MS study. Cytotoxicity Study. The cytotoxicity of GdIOP@ZDS nanoparticles was tested by MTT (3-(4,5-dimethylthiazol-2-y1)-2,5diphenyltetrazolium bromide) method using H melanoma B16 and RAW 264.7 cells as models. Cells were first seeded into a 96-well plate with a density of about 1 × 104 cells/well in DMEM at 37 °C, and were allowed to incubate for 24 h within 5% CO2 atomsphere. After the addtion of different concentrations (max. 110 μg/mL with respect to total metal ions) of GdIOP@ZDS nanoparticles, cells were incubated in the same conditions for another 24 h. 100 μL (0.5 mg/mL) of MTT was then added to each well and the plate was incubated for 4 h at 37 °C. After the addition of 100 μL DMSO per wells, the plate was kept at room temperature for 4 h. The OD490 value (Abs.) of each wells were measured by a MultiSkan FC microplate reader. Cell viability was calculated from OD490 value of experimental group by subtracting that of blank group. T1−T2 Dual-Modal MRI of LNs in Normal Mice. All the animal experiments were executed according to the protocol approved by Institutional Animal Care and Use Committee of Xiamen University. MRI studies were conducted at a 7 T MRI scanner (Varian 7 T micro MRI System). The mice were first anaesthetized and conducted with both T1 and T2 preinjection images at near the foot region of mice. The acquisition were performed by using fast spin echo multislice (fSEMS) sequence with the parameters as follows: TR/TE = 3000/ 120 ms (T2), TR/TE = 300/10 ms (T1), FOV = 60 × 60 mm2, 256 × 128 matrices, slice thickness =1 mm, number of slices =5, averages = 4. The GdIOP@ZDS nanoparticles were subcutaneously injected in the sole of mouse right foot with a dose of 50 μL (1 mg/mL with respect to total metal ions). The images (both T1 and T2) at 5 h postinjection (p.i.) were then acquired using the same parameters. The analysis of signal intensity in the popliteal LNs was performed by ImageJ software.29,30

EXPERIMENTAL SECTION

Synthesis of Gadolinium-Embedded Iron Oxide Nanoplates (GdIOP). The synthesis of GdIOP were reported elsewhere.24 Briefly, the complex of gadolinium oleate and iron oleate were prepared by mixing GdCl3 and FeCl3 with sodium oleate solution. The metal oleate complex (1 mmol) was dissolved in 1-octadecene (15 mL) in the presence of oleic acid (0.16 mL) as surfactant. The system was then degassed with N2 and heated to reflux for 2 h. After cooling to room temperature, excess isopropanol was added to precipitate the products. The black solid were collected by centrifugation and redispersed in hexane for further use. Preparation of GdIOP@ZDS Nanoparticles. The synthesis of zwitterrionic dopamine sulfonate (ZDS) was carried out according to reported procedures.28 ZDS-coated GdIOP nanoparticles were prepared through a ligand exchange reaction. ZDS (10 mg) were first dissolved in a mixed solution of 5 mL of ultrapure water and 10 mL of acetone. The GdIOP dispersed in hexane were added to the 28287

DOI: 10.1021/acsami.5b08422 ACS Appl. Mater. Interfaces 2015, 7, 28286−28293

Research Article

ACS Applied Materials & Interfaces

Figure 2. (a, b) TEM and HRTEM images of gadolinium-embedded iron oxide nanoplates (GdIOP), the interplanar distances of 0.30 and 0.21 nm are assigned to (200) and (400) planes of magnetite phase, respectively, fast Fourier transition (FFT) pattern indicates [100] zone axis along the perpendicular view of plates; (c) carton model of GdIOP where the green spheres refer to gadolinium species and the red color refers to Fe3O4{100} facet; (d) T1 and T2 relaxation enhancement phenomenon in the GdIOP nanoparticles, the exposed gadolinium and iron metals facilitate protons chemical exchange (T1) and the larger effective radius benefits protons diffusion (T2) process; (e, f) plots of 1/T2 and 1/T1 against concentrations of GdIOP@ZDS samples, the r2 and r1 values at 7 T are obtained from the slope of the related fitting lines; (g) T1−T2 dual-modal MR phantom study of GdIOP@ZDS samples performed at a 7 T MRI scanner, the metal concentrations represent to total metal ions (Fe and Gd) in GdIOP samples. T1−T2 Dual-Modal MRI of Tumor Metastasis in LNs. We first established a primary tumor model in 4-week-old (∼20 g) female ICR mice by subcutaneous injection of melanoma B16 cells (50 μL, 1 × 106 cells per mouse) in the hock of the right leg of mice (n = 6). The tumor-bearing mice were allowed to grow for 3 weeks and then selected to conduct the MRI study at a 7 T MRI scanner (Varian 7 T micro MRI System). Both T1 and T2 preinjection images at near the foot of mice were acquired with multiple slices. The acquisition parameters are as follows: TR/TE = 3000/120 ms (T2), TR/TE = 300/10 ms (T1), FOV = 60 × 60 mm2, 256 × 128 matrices, slice thickness = 1 mm, number of slices = 5, averages = 4. The GdIOP@ ZDS nanoparticles were subcutaneously injected in the sole of mouse right foot with a dose of 50 μL (1 mg/mL with respect to total metal ions). Both T1 and T2 postinjection (p.i.) images at the time points of 2, 4, and 6 h p.i. were then acquired using the same parameters. The signal intensity changes in the right popliteal LN (nearing the primary tumor) were finely analyzed using ImageJ software.

direction of the plane of GdIOP showed [100] zone axis of cubic phase, indicating that the obtained GdIOP are exposed with Fe3O4{100} facets. The energy dispersive X-ray spectroscopy (EDX) analysis indicated the presence of gadolinium species in GdIOP, where the percentages of Gd with respect to total metal ions (Fe and Gd) was 13.1% in GdIOP by inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis (Figure S1). Therefore, we proposed that the surface of GdIOP are distributed with both iron ions and gadolinium species (Figure 2c). Protons T1 relaxation enhancement is related to chemical exchange efficiency of protons in close vicinity of paramagnetic centers, following protons coordination and dissociation procedures.31,32 The iron-rich Fe3O4{100} facet and the exposed gadolinium species on GdIOP can provide sufficient paramgnetic centers for water chemical exchange, which may greatly increase T1 relaxation enhancement (Figure 2d). More importantly, the long-range order of magnetic spins in GdIOP was disturbed by embedded gadolinium species, which minimizes the influence of T2 decaying effect to the T1 contrast ability. On the other hand, protons T2 relaxation process is mainly related to protons diffusion around the local inhomogeneity field generated by magnetic particles.33 Therefore, because of the nonspherical platelet shape, GdIOP with larger effective radius may greatly enhance T2 contrast ability (Figure 2d). Moreover, the collection of magnetic spins in GdIOP due to the multidomain structure may also greatly enhance protons dephasing process around magnetic particles.24,34 These results indicated great potential for GdIOP to serve as a highly efficient DMCA. We then prepared and evaluated the MRI performance of GdIOP under a 7 T MRI scanner. The r1 and r2 relaxivities of

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RESULTS AND DISCUSSION Synthesis and Characterizations of GdIOP. The synthesis of GdIOP was strightforward following the procedures reported previousely.24 Briefly, iron oleate and gadolinium oleate complexes were mixed and heated to reflux in 1octadecene (ODE) solvent. The products were obtained by certrifugation after 2 h without further purification process. The transmission electron microscopy (TEM) image of GdIOP reveals square shaped uniform nanoplates with the side length of about 10 nm and the thickness of about 1.8 nm (Figure 2a). The high-resolution TEM (HRTEM) image showed interplanar distances of 0.21 and 0.30 nm, which can be assigned to (400) and (220) planes of magnetite phase (Figure 2b). The fast Fourier transition (FFT) pattern along perpendicular 28288

DOI: 10.1021/acsami.5b08422 ACS Appl. Mater. Interfaces 2015, 7, 28286−28293

Research Article

ACS Applied Materials & Interfaces

Figure 3. (a) Schematic illustration of GdIOP coated with zwitterionic dopamine sulfonate (ZDS) molecules, denoted as GdIOP@ZDS; (b) protein-corona-free characteristic of GdIOP@ZDS in the presence of serum proteins; (c) optical images of melanoma B16 (upper) and RAW264.7 (lower) cells after incubated with GdIOP@ZDS nanoparticles for 3 and 6 h. The control groups are incubated with PBS under the same conditions. The blue fluorescence indicates cell nucleus which were stained with Hoechst 33342 dyes, scale bar 50 μm for all images; (d) quantification of cellular uptake of melanoma B16 and RAW264.7 cells (1 × 106 each) after incubated with 1 mL of GdIOP@ZDS nanoparticles (50 μg/mL with respect to total metal ions) for 3 and 6 h, respectively, by ICP-MS measurements. The experimental groups were repeated three times, and each replicate was measured three times by ICP-MS to give the standard deviations in error bars.

host nanoparticles.26,28 The size of ZDS coated GdIOP were measured by dynamic light scattering (DLS) technique, which showed a steady hydrodynamic diameter of about 12.6 nm for GdIOP@ZDS before and after incubated with fetal bovine serum (FBS, 20%, v/v) and cell culture medium containing 10% FBS for 3 and 6 h (Figure S2). These results indicated a protein-corona-free characteristic for GdIOP@ZDS nanoparticles, which may show controllable behaviors with associated with cells or in vivo environment (Figure 3b). We then evaluated the cellular uptake efficiency of GdIOP@ZDS nanoparticles using different cell lines. We used macrophagy RAW 264.7 cells to mimic the systematic administration of nanoparticles by LNs, while melanoma B16 cells were used for comparison. After incubated with GdIOP@ZDS nanoparticles (50 μg/mL with respect to total metal ions) for 3 and 6 h, respectively, the supernatant of culture medium was washed away. Both RAW 264.7 and B16 cell groups showed little observerable agglomerations on cell surfaces in optical images (Figure 3c), indicating protein-corona-free characteristic in the culture medium. On the other hand, the Prussian blue staining experiments indicated efficient cellular uptake of GdIOP@ZDS nanoparticles by RAW 264.7 comparing to that by B16 cells (Figure S3). The ICP-MS measurements indicated that cellular uptake of GdIOP@ZDS by macrophagy RAW 264.7 cells is 9.9- and 6.1-fold higher than that by melanoma B16 cells at 3 and 6 h incubation time, respectively (Figure 3d). This phenomenon is probably due to the high uptake efficiency of nanoparticles by macrophages through phagocytosis procedure.37,38 Considering that LNs consist of a large number of macrophages and dendritic cells, the above results motivate us to investigate the ability of GdIOP@ZDS in imaging of LNs. It

GdIOP were obtained from the slops of reciprocals of relaxation time against concentrations with respect to total metal ions (Figure 2e, f). The GdIOP showed relatively high r1 and r2 values under 7 T magnetic field, which are 7.0 ± 0.3 and 160.9 ± 2.6 mM−1 s−1, respectively. The relatively high T2 relaxivity is dominated by the multiple domain structure within GdIOP, which exhibits enhanced T2 relaxation shorenting effect as perceived from that of magnetic nanoparticle aggregations.35 Moreover, the larger effective radius due to the nonspherical shape may also enhance the T2 relaxivity.24,34,36 The strong T1 relaxation enhancement can be ascribed to the exposed paramagnetic metals (Fe and Gd) on the surface, which greatly faciliate protons directly coordination and chemical exchange.34 The MR phantom results revealed excellent contrasts in both T1- and T2-weighted MRI, showing brighter and draker contrasts as increasing the concentrations of nanoparticles with respect to total metal ions, respectively (Figure 2g). It is known that magnetic nanoparticles with a single domain are prone to show prominent T2 contrast when the ratio of r2/r1 is higher than 10; however, the unique structure of GdIOP endows a strong T1 contrast even with the r2/r1 ratio of as high as 22.9. The simultaneous T1 and T2 contrasts may provide selfconfirmed diagnostic information with enhanced accuracy, which motivates us to further evaluate the feasibility of GdIOP in detecting small-sized LNs in small animal models. Cellular Uptake and Surface Coating Study. Prior to conduct in vivo studies, we used zwitterionic dopamine sulfonate (ZDS) molecules to render GdIOP water-soluble and with thin-layer coating on the surface (Figure 3a). More importantly, ZDS coating on nanoparticles was reported to be able to reduce nonspecific adsorption of serum proteins for 28289

DOI: 10.1021/acsami.5b08422 ACS Appl. Mater. Interfaces 2015, 7, 28286−28293

Research Article

ACS Applied Materials & Interfaces should be noted that GdIOP@ZDS nanoparticles are nontoxic in both RAW 264.7 and B16 cells after 24 h incubation even at a much higher dose (110 μg/mL) with respect to total metal ions (Figure S4). The uptake and transport of interstitial substances by LNs or lymphatic system are highly dependent on the sizes of the substances.39 It was reported that molecular agents (usually smaller than 5 nm) are prone to be transported quickly through the lymphatic vessels to the lower tier nodes,40,41 which may also diffuse out of the lymphatic vessels and contribute to the unexpected background signal during imaging.42 On the other hand, the large sized agents would be blocked and absorbed by nearby LNs.14,15 Therefore, the ideal size of imaging agents for tumor metastasis is the one large enough to enter and localize in LNs and small enough to skip the undesirable uptaken by metastatic tumor cells. The use of ZDS coated GdIOP may promise controllable particle size in complex biological environment, which strikes the right balance between the “inherent” size and “desirable” size of an agent for LNs imaging. T1−T2 Dual-Modal MRI of LNs. We first evaluated the ability of GdIOP@ZDS to imaging LNs of normal mice without a primary tumor by T1−T2 dual-modal MRI. The GdIOP@ZDS was subcutaneously injected in the sole of mouse right foot with a dose of 50 μL (1 mg/mL) of nanoparticles, which was prone to be administrated by peripheral LNs through the drainge effect. Owing to the suitable size in biological medium, the GdIOP@ZDS was efficiently internalized by macrophages and dendritic cells in LN. The T1 and T2 images of LN were acquired at a 7 T MRI scanner at pre- and 5 h postinjection (p.i.) of contrast agents (Figure 4a). We enlarged the region of interests (ROI) of the popliteal LN, which are normally in the size of about 2−3 mm (Figure 4a, insets). Compared to preinjection images, the T1 images showed brighter signal and the T2 images showed darker signal at 5 h p.i., indicating great potential for GdIOP@ZDS serving as a T1−T2 dual-modal MRI contrast agent in LN imaging. We then finely analyzed the images to quantify the signal changes ΔSNR (signal-to-noise ratio, ΔSNR = |SNRpost − SNRpre|/ SNRpre) in the popliteal LN.29 The results showed that both T1 and T2 imaging models revealed promising ΔSNR of about 63.5% and 71.6% (Figure 4b), respectively, which implies desirable diagnostic accuracy for LN imaging. The fate of injected GdIOP@ZDS nanoparticles may follow a way of elimination from body through feces and urine. It is noteworthy that the peripheral LN in the other sole of mouse left foot showed little to no contrast in both T1 and T2 images (Figure S5). We also conducted the similar procedures using a commercial gadolinium conjugated diethylenetriaminepentacetate (Gd-DTPA) complex as T1 contrast agent for comparison. The results showed only T1 contrast after 5 h p.i., while the T2 contrast is negligible (Figure S6). Considering the small size of LNs, it is a great challenge to show promising diagnostic accuracy using either T1 or T2 single imaging modality due to the potentially false positive or negative signals. The combination of T1 and T2 contrasts using dual-modal contrast agents benefits from the merits of an “AND” logic, which surpasses a single T1 imaging contrast with enhanced diagnostic accuracy especially for small lesions in LNs. To validate the uptake of GdIOP@ZDS in the popliteal LNs at the right side of mouse foot, we further conducted Prussian blue staining to detect the presence of iron in LNs after imaging. The popliteal LN at the left side of the same mouse was also dissected and used for comparison. From the digital

Figure 4. (a) T1−T2 dual-modal MRI study of mouse lymph node (LN) by subcutaneous injection of GdIOP@ZDS nanoparticles in the sole of right foot of a normal mouse with a dose of 50 μL (1 mg/mL with respect to total metal ions), insets show enlarged pictures of the popliteal LN (white arrow) near the right foot; (b) quantification of signal changes ΔSNR (signal-to-noise ratio) in LNs of both T1 and T2 images; (c, d) representative Prussian blue staining and optical images (insets) of LNs without uptake and with uptake of nanoparticles, respectively. The uptake of GdIOP@ZDS nanoparticles was indicated by black color of LNs in optical image and blue dots in Prussian blue staining slices, and vice versa.

images, the brownish black color of the right poplitheal LN indicates substantial uptake of GdIOP@ZDS particles (Figure 4c, inset). Conversely, the left poplitheal LN shows original color due to the little to no uptake of nanoparticles (Figure 4d, inset). The Prussian blue staining results also revealed blue dots for the right poplitheal LN, indicating the presence of iron probably resulted from the GdIOP particles (Figure 4c). These results are not observed in that of the left poplitheal LN (Figure 4d). The homogeneously distributed blue dots in the Prussian blue staining slice imply an efficient uptake of protein-coronafree GdIOP@ZDS nanoparticles, probably due to the presence of macrophages and dendritic cells in LNs. The suitable size of GdIOP@ZDS nanoparticles makes it a great candidate to be efficiently accumulated and retained in nearby LNs. More importantly, the protein-corona-free characteristic of GdIOP@ ZDS nanoparticles would generate a contrast between macrophages and lessions with low uptake of nanoparticles, which makes it possible to diagnose potential metastasis in LNs. T1−T2 Dual-Modal MRI of Tumor Metastasis in LNs. The ZDS coating renders GdIOP nanoparticles with proteincorona-free character in biological environment, which results in low uptake of GdIOP nanoparticles in tumor cells. The use of ZDS in this study is to maintain a desirable size after subcutaneously injection in living subjects, which is able to reduce the uptake by metastatic tumor cells but still retains high uptake by macrophages in LNs. Because of the low uptake of GdIOP@ZDS nanoparticles by melanoma B16 cells, we then established a melanoma tumor model at near the sole of mouse 28290

DOI: 10.1021/acsami.5b08422 ACS Appl. Mater. Interfaces 2015, 7, 28286−28293

Research Article

ACS Applied Materials & Interfaces

Figure 5. (a) T1−T2 dual-modal MRI study of mice lymph nodes (LNs) with a nearby primary tumor (yellow arrow) at the hock of mouse right leg, both T1 and T2 images were acquired at preinjection and 2, 4, and 6 h postinjection (p.i.) of GdIOP@ZDS nanoparticles with a dose of 50 μL (1 mg/mL with respect to total metal ions), the popliteal LN (white arrow) at the right leg was enlarged (insets, white circle) to analyze the hyperintense foci (HIF, red arrow); (b) quantification of the changes of contrast-to-noise ratio (ΔCNR) at the HIF at 2, 4, and 6 h p.i. of contrast agents; (c) Representative histology of the dissected popliteal LN stained with hematoxylin and eosin (H&E), the region of red dotted circle in the LN indicates the presence of melanoma B16 tumors; (d, e) H&E staining of normal LN and the primary tumor, respectively.

were confirmed by H&E staining, which showed similar histological morphology comparing to that of primary tumor (Figure 5c−e). MRI technique can provide promising anatomical details with the resolution usually at a submilimeter level. However, it is still a great challenge to reach such high accuracy using normal MRI scans, especially in living subjects because of the presence of motion artifact. The “AND” logic T1−T2 dual-modal imaging method outputs both bright and dark contrast images with self-confirmed merits, which promise significantly enhanced diagnostic accuracy. Moreover, this strategy also provides images with nearly the same temporal and spatial resolution, showing great feasibility in detecting a lession. It is noteworthy that the observed metastatic tumor in the LNs in our study is about 1.2 mm in the minimal dimension, which is close to the detection limit of MRI scans.

right foot and evaluated the possibility for GdIOP@ZDS nanoparticles pseudocontrast imaging of tumor metastasis in nearby LNs. The tumor-bearing mice (n = 6) were incubated for 4 weeks after subcutaneously injected with melanoma tumor cells, which allowed the exfoliation and relocation of tumor cells from the primary tumor region. To evaluate the possibility of GdIOP@ZDS nanoparticles in T1-T2 dual-modal imaging of potential tumor metastasis in LNs, we conducted MRI study by injection of GdIOP@ZDS nanoparticles with a dose of 50 μL (1 mg/mL with respect to total metal ions). Both T1 and T2 images at preinjection and 2, 4, 6 h p.i. time points were acquired. As shown in the Figure 5a, the popliteal LN at the right foot exhibited brighter contrast in T1 images and darker contrast in T2 images at 2 h p.i. comparing to that of preinjection images, respectively. These signal changes in the LN became more significant at 4 and 6 h p.i. time points, indicating consecutive administration of contrast agents into the LN. More importantly, we found a hyper-intense foci (HIF) in the upper right corner of the popliteal LN at both T1 and T2 models, showing draker contrast in T1 images and brighter signal in T2 images (Figure 5a, insets, red arrow). This phenomenon may be attributed to the low uptake of contrast agents in the HIF, indicating the presence of possible metastatic melanoma tumor cells. We used the changes of contrast-to-noise ratio (ΔCNR = | CNRpost − CNRpre|/CNRpre; where CNR = |SNRHIF − SNRLN|/SNRHIF) to quantify the ability of GdIOP@ZDS nanoparticles in imaging the potentially tumor metastasis in LNs.23,36 The results showed greatly enhanced ΔCNR after injected with GdIOP@ZDS nanoparticles for both T1 and T2 images with approximately 41.3% and 99.6%, respectively (Figure 5b). The presence of melanoma tumor cells in the HIF

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CONCLUSION In summary, we presented that GdIOP with highly efficient T1−T2 dual-modal MRI contrast abilities were able to detect tumor metastasis in LNs with enhanced diagnostic accuracy. We demonstrated that GdIOP@ZDS nanoparticles with protein-corona-free characteristic can be absorbed efficiently by the large amount of macrophages and dendritic cells in LNs, which resulted in T1−T2 dual-modal contrast images with feasible diagnostic accuracy. On the other hand, the low uptake efficiency of GdIOP@ZDS nanoparticles by melanoma B16 tumor cells generated pseudocontrast images in LNs using T1− T2 dual-modal MRI strategy. The combination of T1 and T2 imaging models can also reduce the potential false negative results by neither T1 or T2 single model, providing selfconfirmed information in diagnosis. The accurate and noninvasive detection of tumor metastasis in LNs is significantly 28291

DOI: 10.1021/acsami.5b08422 ACS Appl. Mater. Interfaces 2015, 7, 28286−28293

Research Article

ACS Applied Materials & Interfaces

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important for patients, especially when metastasis evaluation is urgently needed. This process can help doctors to decide which LNs should be removed specifically rather than removal of all nearby LNs. Our study provided a rational design of T1−T2 dual-modal imaging agents and outlined an efficient method to detect tumor metastasis in LNs, which may open up new avenues to develop more sophisticated strategy to diagnose clinically occult lessions.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b08422. Additional figures showing EDX profiles, dynamic light scattering measurements, Prussian blue staining of RAW 264.7 and melanoma B16 cells, cell viability study of melanoma B16 and RAW264.7, MR images of the mouse left popliteal lymph node, MRI study of mouse lymph node, quantification of signal changes in lymph nodes (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ∥

Z.Z., H.L., and X.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key Basic Research Program of China (2013CB933901, 2014CB744502, and 2014CB932004), National Natural Science Foundation of China (21222106, 21521004, 81370042, 81430041, and 81201805), Natural Science Foundation of Fujian (2013J06005 and 2013D014), and Fok Ying Tung Education Foundation (142012).

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REFERENCES

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DOI: 10.1021/acsami.5b08422 ACS Appl. Mater. Interfaces 2015, 7, 28286−28293