Dual-Modality Noninvasive Mapping of Sentinel Lymph Node by

Article pubs.acs.org/molecularpharmaceutics

Dual-Modality Noninvasive Mapping of Sentinel Lymph Node by Photoacoustic and Near-Infrared Fluorescent Imaging Using DyeLoaded Mesoporous Silica Nanoparticles Zhiguo Liu,† Pengfei Rong,‡ Lun Yu,† Xintong Zhang,† Cejun Yang,‡ Fei Guo,‡ Yanzhong Zhao,‡ Kechao Zhou,§ Wei Wang,*,‡ and Wenbin Zeng*,† †

School of Pharmaceutical Sciences, Central South University, Changsha, 410013, P. R. China Department of Radiology, the Third Xiangya Hospital, Central South University, Changsha, 410013, P. R. China § State Key Lab Powder Met, Central South University, Changsha, 410083, P. R. China ‡

S Supporting Information *

ABSTRACT: The imaging of sentinel lymph nodes (SLNs), the first defense against primary tumor metastasis, has been considered as an important strategy for noninvasive tracking tumor metastasis in clinics. In this study, we developed an imaging contrast system based on fluorescent dye-loaded mesoporous silica nanoparticles (MSNPs) that integrate nearinfrared (NIR) fluorescent and photoacoustic (PA) imaging modalities for efficient SLN mapping. By balancing the ratio of dye and nanoparticles for simultaneous optimization of dualmodality imaging (NIR and PA), the dye encapsulated MSNP platform was set up to generate both a moderate NIR emission and PA signals simultaneously. Moreover, the underlying mechanisms of the relevance between optical and PA properties were discovered. Subsequently, dual-modality imaging was achieved to visualize tumor draining SLNs up to 2 weeks in a 4T1 tumor metastatic model. Obvious differences in uptake rate and contrast between metastatic and normal SLNs were observed both in vivo and ex vivo. Based on all these imaging data, it was demonstrated that the dye-loaded MSNPs allow detection of regional lymph nodes in vivo with time-domain NIR fluorescent and PA imaging methods efficiently. KEYWORDS: mesoporous silica nanoparticles (MSNPs), sentinel lymph nodes (SLNs), multimodality imaging, photoacoustic (PA) imaging, near-infrared fluorescence (NIR) imaging

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INTRODUCTION

For example, MRI is capable of providing precise threedimensional tomography, but it is limited by low target sensitivity. On the contrary, PET and optical methods both can image the target with good sensitivity, yet they suffer from low spatial resolution or tissue penetration. With those regards, multimodality imaging, to harness the strengths of different imaging methods by definition, has become both attractive and promising for both small animal and human studies.22,23 As a result, the newly emerged hybrid imaging platform comes as a strategy applied with characteristics superior to those of any of its constituents considered alone.24,25 Toward these goals, multimodality methods for SLN imaging have been widely exploited. In several recently reported studies, PET/MRI/NIR trimodal nanoprobes have been designed to image the SLNs.17,19,26 In the case of the radioguided method,

The lymph nodal status of cancer is considered to be an important prognostic indicator for patients who were diagnosed with many forms of solid cancer.1−4 Tumor-induced lymph angiogenesis in the peritumoral area and in tumor-draining lymph nodes (LN) has been found to promote metastatic cancer spread in several experimental models and clinical studies.5,6 Moreover, the presence of sentinel lymph node (SLN) metastases represents a critical staging parameter to determine the prognosis and the course of therapy for the majority of cancer patients.7,8 Current techniques for the lymphatic mapping of the SLN involve radionuclide imaging, optical imaging, MRI imaging, and photoacoustic tomography imaging. With the development of nanotechnology, several categories of nanoprobes have been investigated for identification of SLNs, including quantum dots (QDs),9−13iron oxide, 14 gold nanoparticles (NPs), 15,16 rare-earth-based NPs,17−19 carbon-based materials (carbon nanotubes and graphenes),20 and perfluorocarbon-based NPs.21 However, each imaging modality has its own strengths and limitations. © XXXX American Chemical Society

Received: October 15, 2014 Revised: June 19, 2015 Accepted: July 1, 2015

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DOI: 10.1021/mp500698b Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

mL of deionized water,10 mL of ethanol, and 0.30 mL of NH3· H2O (28%−30%) were stirred for 10 min at room temperature. Then, various volumes of APS-dye solution (C = 10 mM, DMF) and 80% of TEOS (0.4 mL) were mixed with vigorous stirring for 4 h, and the additional TEOS (0.1 mL) was added at a rate of 5 μL/min. (Note: a stepwise addition of TEOS is necessary to keep the total amount of TEOS and hydrolyzed TEOS in the system at any time below the critical nucleation concentration to preserve nonfluorescent particles forming.) After 12 h of stirring, the resulting particles were collected by centrifugation (14,000 rpm), and then washed three times with ethanol and deionized water. After sonication for 10 min, the final particles (Cy754-MSNPs) were obtained. The resulting particles were analyzed by transmission electron microscopy (TEM, H-7600, Hitachi, Tokyo, Japan). Hydrodynamic size and zeta potential of the particles were determined via dynamic light scattering (DLS) (Zetasizer NANO ZS, Malvern, U.K.). The measurements were carried out in deionized water. The UV−vis and fluorescence spectra of particles were recorded on a UV−vis spectrophotometer (UV-2450, Shimadzu, Japan) and fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan), respectively. Determination of Dye Molecules per MSNP.30 The loading efficiency of the dye was determined by UV−vis spectroscopy. The presence of unencapsulated dye was determined by centrifuging. The concentration of Cy754-APS in the supernatant was determined spectrophotometrically. The TEM characterization showed that the Cy754 doped MSNPs were uniform in size (∼35 nm). The average volume of MSNP was obtained from the TEM, see eq 1.

the detection is possible in deep lymph nodes. However, some certain shortcomings (i.e., the relatively high costs of the radioactive probes) inhibit the application of these methods. Moreover, the high radioactivity at the primary injection site often interferes with the intraoperative detection of nearby lymph nodes.27,28 Therefore, low-cost, less invasive, and more accurate multimodality imaging for SLN localization should be further developed. Optical/photoacoustic dual-modality imaging, combining fine ultrasonic resolution with optical absorption contrast, is an emerging technique for in vivo noninvasive imaging.29 Compared with nuclear imaging methods, both fluorescence and PA imaging modalities are safer for patients and medical staff because of utilizing nonionizing radiation. In recent comparison research with PA and fluorescence contrast, Achilefu’s group successfully performed the SLN mapping in rats by employing NIR dye loaded perfluorocarbon (PFC) nanoparticles, and demonstrated that the enhanced contrast allows detection of regional lymph nodes in vivo with time-domain optical and photoacoustic imaging methods.21 Although the research consolidated the relevance between PA and fluorescence imaging, it would be of necessity to further disclose the underlying mechanisms of how the enhanced PA contrast yields. To further explore the potential of these nanoparticles for SLN imaging in vivo, not only the studies of the tumor metastasis model but also the underlying mechanisms of relevance between optical and PA properties at the molecular level should be investigated. In this research, we developed NIR dye (Cy754, with the emission wavelength of 795 nm) doped mesoporous silica nanoparticles (Cy754-MSNPs) that possess the core/shell structure. The NIR dye Cy754 was covalently doped into the MSNPs to form a homogeneous core. In order to improve the photostability of MSNPs, an additional thin silica shell was chosen to install the outer layer. Then, we investigated how the dye in the particle behaves in different conditions and further, for the first time, discovered the mechanism of the signals observed in both optical and PA images produced from one dye. Furthermore, the Cy754MSNPs were applied for PA and fluorescence dual-modality imaging to visualize T-SLNs in a 4T1 tumor metastasis model. Due to their high stability, signals from tumor draining SLNs are detectable up to 2 weeks. These results demonstrated the feasibility of lymph node mapping of both modalities. Above all, MSNPs loaded with NIR dye provide a strategic approach to bridge the disparate contrast reporting mechanisms of fluorescence and PA imaging methods.

V = 4/3π(d /2)3

(1)

where V is the average volume and d is the diameter determined by TEM. The density of the MSNPs (corresponding to that of amorphous silica, ca. 2.2 g/cm3) allowed the estimation of the number of MSNPs in the suspension obtained at the end of the synthesis. As we know the total number of Cy754 dye molecules in the suspension, the number of Cy754 dye molecules per MSNP is obtained from eq2.

Np = NCy754 /NNPs

(2)

where Np is the number of Cy754 dye molecules per MSNP, N Cy754 is the total number of Cy754 dye molecules encapsulated in MSNPs, and NNPs is the total number of MSNPs. In Vitro NIR Fluorescence Imaging. The NIR fluorescence images of the dye doped MSNP were obtained using an IVIS imaging system (Lumina II, Caliper, USA) consisting of an excitation filter (750 nm band-pass filter) and a cold charge-coupled device (CCD) camera (Orca ERG; Hamamatsu Photonics, Hamamatsu City, Japan) with an emission filter (810 nm band-pass filter). The nanoparticles were transferred into a 0.2 mL eppendorf tube. NIR fluorescence images of these samples were acquired by the same exposure time (0.5 s). Photothermal Imaging of Cy754-MSNPs. The photothermal images of Cy754-MSNPs in solution were obtained using FLIR E40 equipment running on FLIR tools systems, in conjunction with an 808 nm laser (Xi’an Sampling Laser Techinc Institute). The whole system is set up by our group, including photothermal imaging equipment, laser illuminator (808 nm), laser diode, lens, fiber, and specimen table. Different samples of Cy754-MSNPs in deionized water (5, 10, 25, 50, 80,

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EXPERIMENTAL SECTION Materials. Tetraethoxysilane (TEOS, 99%), (3aminopropyl)triethoxysilane (APS, 99%), methanol (99.9%), ethanol (99.9%), dimethylformamide (DMF), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 99%), ammonium hydroxide (NH3·H2O, 28−30%), N,N-diisopropylethylamine (DIEA, 99.9%), and 2,2,2-tribromoethanol−tert-amyl alcohol were purchased from SigmaAldrich (Berlin, Germany). Milli-Q-water with a resistance greater than 18 MX was used for all experiments (Millipore, USA). All chemicals and reagents were of analytical grade. Synthesis and Characterization of Dye Doped MSNs. Cy754 doped MSNs were synthesized according to known literature procedures.30 Briefly, (3-aminopropyl)triethoxysilane dye (APS-dye) was conjugated by stirring Cy754 NHS ester in an APS DMF solution (nAPS/ndye = 2.4) for 4 h. Separately, 0.38 B


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Molecular Pharmaceutics Scheme 1. Synthetic Routes of Cy754-MSNPs

Sigma-Aldrich). A Cy754-MSNP solution (4 mg/mL, 50 μL) was injected intradermally into the sole of the foot. Thirty minutes later, NIR fluorescent imaging was observed on an IVIS imaging system at different time points (0, 1, 3, 24 h, 2, 3, 5, and 14 d). NIR images (0.5 s exposure) of mouse lymph nodes were acquired using a band-pass excitation filter (730− 765 nm) as an excitation light source and a band-pass emission filter (810−875 nm) for imaging. At the end of 14 d, the mice were sacrificed, and the major organs were collected and subjected to ex vivo imaging with an IVIS imaging system. In Vitro and in Vivo Photoacoustic Imaging. We investigated five different concentrations of Cy754-MSNPs (0.25, 0.5, 1, 2, and 4 mg/mL). The PA signal from blank MSNPs was used as comparison. Particle samples were injected into polyvinyl chloride tubes (S-54-HL, 720994, Tygon microbore tubing; 1.5 mm inner and outer diameters, respectively) located in a small water tank below the transducer of the PAM system to capture PA images. The use of Cy754MSNPs as a PA imaging contrast agent was also investigated in tumor-bearing nude mice. A solution (100 μL, 4 mg/mL) of particles were administered by intradermal injection in the footpads of rats. For animal studies, a 754 nm laser was used. PA images were acquired 1 h for postinjection. The merged image was acquired by overlaying differential images of the SLNs on the images of blood vessels. When final imaging was performed, animals were euthanized 1 h after. Ex vivo PA imaging was implemented by dissected lymph nodes and imaging.

and 120 μM) were placed in a series of eppendorf tubes irradiated by an 808 laser (0.5 W/cm2) for 5 min, respectively. The temperature signals were analyzed with FLIR tools systems. Photostability Test. Cy754, ICG, and Cy754-MSNPs dissolved in deionized water were placed in glass vials, with almost the same UV absorbance by adjusting concentration. Samples were exposed to light under a W−halogen lamp (200 W). The distance between lamp and samples is 30 cm. The samples were exposed for 7 h.The fluorescence intensity was measured every 1 h using the fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) and an IVIS imaging system (Lumina II, Caliper, USA), respectively. In Vitro Cytotoxicity Assay. Cell cytotoxicity was measured by MTT assay. The present study was undertaken to investigate the cytotoxicity assay of Cy754-MSNPs in vitro using a murine dendritic cell line (DC2.4 cells). DC2.4 cells (2 × 104/well, reside in the lymph nodes) were placed in a 96 well plate in RPMI medium supplemented with 10% heatinactivated fetal bovine serum, 5 × 10−5 M 2-mercaptoethanol, 50 IU/mL penicillin, and 50 mg/mL streptomycin. After incubating the cells with blank MSNPs or Cy754-MSNPs for 24 h, MTT (10 mL/well of a 5 mg/mL MTT stock solution in PBS) was added directly to each well and the plates were incubated at 37 °C for 4 h. For a colorimetric MTT assay, dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to solubilize formazan, and absorbance was measured at 562 nm. 4T1 Tumor Lymph Node Metastasis Model. The tumor model was established in 4−6 week old female BALB/c mice by subcutaneous injection of 5 × 106 4T1 cells transfected with firefly luciferase to the hock of the right leg of mice.31 Tumor metastatic progression of sentinel lymph node was evaluated after injection of luciferase substrate for 15 min in an IVIS imaging system. Animal procedures were performed according to the regulations provided by Animal Laboratory Center of the Third Xiangya Hospital. In Vivo Fluorescence SLN Imaging. For NIR fluorescent imaging, the BALB/c mice were anesthetized with 300 μL of a 2.5% avertin solution (2,2,2-tribromoethanol−tert-amyl alcohol,

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RESULTS AND DISCUSSION Synthesis and Characterization of Dye-Loaded MSNPs. Silica matrix is optically transparent that allows light to pass through the matrix efficiently, and provide a stable environment which is resistant to both chemical attack and mechanical stress. Silica surfaces can also be easily modified by conventional organosilane chemistry.32 Usually, there are two strategies to prepare the dye doped MSNPs. One way is to encapsulate dyes into the MSNPs noncovalently. The other is to conjugate dyes on the surface of the particles covalently C


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Figure 1. (A) TEM images of Cy754-MSNPs. The left was at low magnification, and the right was at high magnification. The photographs of water, blank-MSNPs, and Cy754-MSNPs (B, above), and the diameter distribution curves for Cy754-MSNPs (B, below) from DLS measurement. (C) UV−vis spectra of blank MSNPs, Cy754, and Cy754-MSNPs. (D) Fluorescence emission spectra of blank MSNPs, Cy754, and Cy754-MSNPs. (E) Zeta potentials of Cy754-MSNPs and blank MSNPs. (F) Dependence of PA signals on the nanoparticle concentration. The error bars indicate the standard deviation for each measurement (n = 4).

using dye-APS. However, the dye loaded noncovalently might leak from the MSNPs easily, especially for the dye with good water solubility. In exposed way fluorescent molecules are easily photobleaching or detached from the particle, which may comprise in vivo application and imaging quantification. Alternatively, we doped Cy754 dye molecule into MSNPs with core/shell structure to prevent photobleaching and falloff of the dye molecules in vivo.33 Here, the Stöber method was used as a principal method for the preparation of MSNPs.34,35 The fluorophore (Cy754) was synthesized according to a previous report.36 Cy754-NHS ester was prepared in house (Scheme S1 in the Supporting Information). Compared with ICG, an FDA-approved NIR dye, it has several attractive features, such as good water solubility, high fluorescence quantum efficiency, and less self-aggregation.36 As shown in Scheme 1, (3-aminopropyl)triethoxysilane-Cy754 (APSCy754) was first prepared, and was then added into the reaction of the particle fabrication under diluted TEOS with aqueous ammonia as a catalyst. Consequently, APS-Cy754 was embedded into particles by the binding of APS to silicon oxide. In order to prevent photobleaching and falloff of the dye molecules’ outer layer, additional TEOS was used to form a thin shell outside the dye doped core. After purification of the dye doped particles by centrifugation, the Cy754-MSNPs with core/shell structure were obtained. To further verify the superiority of the method in the process of NP formulation using APS-Cy754, we chose ICG for comparison and prepared

ICG-MSNPs by the same process as mentioned above. Figure S1 in the Supporting Information shows the photographs of Cy754-MSNPs, and ICG-MSNPs in eppendorf tubes after centrifugation. Due to the conjugation of Cy754 into the MSNPs, Cy754-MSNPs display dark green color at the bottom of the eppendorf tube (right). As the compared group, ICGMSNPs display almost no color at the bottom of tube, and most of the free ICG still stayed in the supernatant solutions (left). Since ICG have sulfonate groups and are negatively charged,37 it is difficult for the molecules to be capsulated by a negatively charged silica layer under neutral pH conditions due to electrostatic repulsion. These results indicated that the dye covalently doped into MSNPs was superior to that encapsulated noncovalently, especially for the dye with good water solubility and negatively charged. Before proceeding to in vivo application for imaging, the major features of the nanoprobes were first characterized in vitro, including particle size and zeta potentials, NIR fluorescence, and PA properties. First, we measured the average sizes and distributions of these particles using transmission electron microscopy. As shown in Figure 1A, the diameter of particles is about 35 nm. In addition, the size of the Cy754MSNPs was also measured by dynamic light scattering (DLS), whose result indicated that the mean hydrodynamic diameter of the particle was approximately 46 nm (Figure 1B). The size measured by TEM was found to be slightly smaller than those measured by DLS. We deduce that the possible explanation for D


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Figure 2. (A) In vitro NIR fluorescence image (pseudocolor). (B) The fluorescence intensity (red line) and PA signals (blue line) of the Cy754MSNPs in water according to the concentration of Cy754. (C) Photothermal images of various concentrations of dye (0, 5, 10, 25, 50, 80, and 120 μM) of Cy754-MSNPs solution exposed to the 808 nm laser (0.5 W/cm2) for 5 min. (D) The relationship of samples’ temperature and PA signals according to the concentration of Cy754 after laser illumination. (E) Fluorescence stability of Cy754 and Cy754-MSNPs was determined by using the NIR fluorescence images and fluorescence intensity (F) in water at room temperature at different time points. The data are shown as mean ± standard deviation (SD). The error bars indicate the standard deviation for each measurement (n = 4).

this phenomenon may be that the TEM images were obtained after the air-drying process, in which the particles shrink and the size gets smaller than under water. It was reported that particles both smaller than 5 nm and larger than 1000 nm were not ideal for lymph node imaging.16 In vivo NIR fluorescencebased lymph node mapping requires NPs with a diameter in the 20−80 nm range, as already reported.38 The size of Cy754MSNPs (∼35 nm) we developed was just in the range mentioned above, and the particles may be very suitable for lymph node imaging. Zeta potentials of Cy754-MSNPs and blank MSNPs were recorded at pH 7.0 as shown in Figure 1E. The zeta potential of blank MSNPs is −25.22 ± 3.22 mV, due to the existence of −OH groups on the surface of MSNPs. After conjugation with APS-Cy754, the negatively charged dyes on the surface of particle led to a slight decrease in zeta potential (−30.05 ± 4.35 mV). Further investigation of the fluorescent properties for Cy754-MSNPs is warranted. As shown in Figure 1C, blank MSNPs do not show characteristic absorption peaks of Cy754 in the range of 300−800 nm. After conjugation with Cy754, the absorption of Cy754-MSNPs is similar to that of Cy754, indicating no changes to the Cy754’s chromophore upon conjugation. The emission spectra of the Cy754 and Cy754MSNPs agreed well in water at 25 °C, and no spectral shifts

were observed in the emission spectrum of the Cy754-MSNPs (Figure 1D). These results suggest that Cy754 was successfully loaded, and Cy754-MSNPs did not influence the optical spectrum of Cy754. Strong fluorescence signal from Cy754MSNPs were also identified with IVIS imaging system in Figure 1D (inset), validating the potential ability of the probes for optical imaging. The behavior of the NIR dye inside the nanoparticle was very different from that in solution. The distance between the dyes in the nanoparticle was getting shorter than that in solution, which causes the quenching of fluorescence for the dye doped nanoparticles compared to that of equivalent concentrations of free dye. The quenched energy might lead to the PA signals appearing. Taking MSNPs with 120 μM dye loaded MSNPs, for example, PA signals for dye-loaded MSNPs were measured in solution using a noncontact PA microscopy system. It was observed that no PA signal (comparable to the background signal) was detected from blank MSNPs. As shown in Figure 1F, the PA intensity was dependent on the concentration of the nanoprobes ranging from 0.25 to 4 mg/mL. Also, as we expected, fluorescence quenching of the MSNPs was observed obviously compared to that of equivalent concentrations of Cy754. It was found that the Cy754-MSNPs did produce a E


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Molecular Pharmaceutics Table 1. Quantitative Features Dealing with the Synthesis of Cy754-MSNPsa NP dye concn (μM) 5 10 25 50 80 120 a

TEOSa converted (%) 96 97 94 96 95 97

av vol (cm3) −16

5.8 × 10

density (g/cm3) 2.2

Cy754-APS molecules used

Cy754 molecules per NP

intensity (au)

× × × × × ×

14 28 57 133 256 385

180 220 230 130 70 30

3.8 7.5 1.5 3.0 4.8 7.2

1016 1016 1017 1017 1017 1017

The amount of TEOS used was 0.2804 g.

higher PA signal than that of free dyes in vitro (Figure S2 in the Supporting Information). Optimization for Fluorescence and PA Signal. Next, for optimal fluorescence and PA signal, dye doped MSNPs were prepared at the previous condition except the concentration of dye (from 5 to 120 μM). The change in fluorescence intensity of the Cy754-MSNPs with the different amount of Cy754 molecule was investigated. NIR fluorescence imaging was performed on a series of Cy754 doped MSNPs using an IVIS imaging system (Figure 2A). The fluorescence intensity was highest at the concentration of 25 μM, and as the concentration was increased up to 120 μM, the fluorescence signal decreased (Figure 2B, red line). On the contrary, the PA signal was increased significantly (Figure 2B, blue line). In order to acknowledge and understand the principle of the dyes in nanoparticles, the number of dyes encapsulated in one nanoparticle was calculated. As shown in Table 1, fluorescence of Cy754-MSNPs drops continuously when number of encapsulating dye molecules goes from 57 to 385. We attribute the decrease of fluorescence to the homo-Förster resonance energy transfer (HFRET) effect.39 The HFRET interaction happens between close pairs of dye molecules (excited one and unexcited one) with small Stokes shift of the original dyes. The Cy754 with a small Stokes shift (∼40 nm) makes efficient HFRET occur between dye molecules inside a nanoparticle. With the increase of dye molecules per nanoparticle, the distance between the dyes gets shorter and HFRET interaction becomes stronger, which led to the decrease of fluorescence of Cy754 doped MSNPs. Further, these experiments indicated that the MSNPs loaded with 80 μM dyes (about 256 dye molecules encapsulated in one particle) afford moderate signals for both fluorescence and PA imaging. Therefore, we chose the MSNPs loaded with 80 μM dyes as the contrast agent for the following research. Mechanism of the Relevance of Optical and PA Properties. Different from the fluorescent probes requiring high fluorescence quantum efficiency, the normal probe for PA imaging should be of minimal fluorescence emission. The PA imaging probes might enhance efficiency of the conversion of absorbed light energy to heat and subsequently to generate an acoustic signal. Usually, the PA/NIR-combined modality requires such a probe with a moderate fluorescence quantum efficiency to produce both a fluorescence signal and a PA signal simultaneously. As mentioned above, the MSNPs loaded with 80 μM dyes possess moderate signals for both fluorescence and PA imaging. In order to better understand the process of PA signal generation, the underlying mechanisms should be further investigated. We hypothesized that the HFRET incorporation of NIR dyes in MSNPs causing a photothermal effect would improve PA contrast. We explored a series of dye doped MSNPs by photothermal imaging system. The photothermal

imaging was investigated by monitoring the MSNP solutions of various concentrations of dye (5, 10, 25, 50, 80, and 120 μM) irradiated by a NIR laser (808 nm, 0.5 W/cm2). As the concentration of dyes increased, the color of the photothermal images continuously changed from purple (corresponding to low temperature) to bright red (corresponding to high temperature) (Figure 2C), whereas the images of blank MSNPs showed little change. These results indicated that the dye doped MSNP samples could absorb and convert NIR light to a substantial amount of heat energy in a concentration dependent manner. As shown in Figure 2D, the temperature was elevated from room temperature (25.6 °C) to 37.2 °C for different samples of Cy754-MSNP solution, and slightly increased for blank MSNPs. As we expect, with the increase of number of the dye molecular (Cy 754) doped per MSNP, the signals for photothermal (Figure 2C,D) and PA images (Figure 2B, the blue line) were gradually improved. The changes for both studies were very consistent with each other. Easily understood, with the increased number of dye molecules per nanoparticle, the distance between the dyes was getting shorter, causing stronger HFRET interaction. Finally, the increased HFRET effect led to more absorbed NIR light converted to photothermal effect, which improved the PA contrast signals. Photostability Test. Conventional organic fluorophores usually suffer from their poor photostability, which limits their imaging time. A long tracking time is desirable for bioanalysis and disease diagnosis. The photostability of Cy754-MSNPs was compared with that of free Cy754. When the fluorescence intensities of Cy754 and Cy754-MSNPs were examined for 2 h at 25 °C, the intensity of the free dyes was decreased nearly 70% (Figures 2E and 2F). Moreover, it was found that the process of photobleaching was faster for ICG than that of Cy754 (Figure S3 in the Supporting Information). This result ensured that the Cy754 we synthesized was a more suitable candidate than ICG for preparing nanoparticles for long time imaging. In contrast, the fluorescence intensity of the Cy754MSNPs showed no significant decrease (less than 8%) over time compared with the Cy754 (nearly 70%) at 25 °C. These results suggested that the photostability of Cy754 was improved when loaded on the mesoporous silica nanoparticles. Cytotoxicity of Cy754-MSNPs. The most important requirement for a probe to be used in in vivo applications is a low cytotoxicity. A cytotoxicity analysis was therefore performed on a dendritic cell line by measuring the cell viability with a colorimetric MTT assay. This cell line was chosen because the dendritic cell is a typical cell in residing in the lymph node and may be suitable as a model cell line for testing viability levels. For the cytotoxicity assay, we examined the dendritic cell viability after incubation with different concentrations of the Cy754-MSNPs for 24 h using the F


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results indicate that both blank MSNPs and dye loaded MSNPs exhibit no obvious cytotoxicity to DC2.4 cells in the concentration range from 1.5 to 6.0 mg/mL. These results suggest that the dye loaded nanoparticles have good biocompatibility for in vivo applications. Tumor Metastasis Model and in Vivo Fluorescence SLN Imaging. Regional lymph node metastasis represents the first step of tumor dissemination for a variety of common human cancers.3,4,40 Herein, we developed a mouse model using 4T1 murine breast cancer cells and performed long-term and optical imaging with Cy754-MNSPs for SLN mapping and indirect tumor metastasis tracking. Before hock inoculation in the left leg of Balb/C mice, 4T1 cells were stably transfected with firefly luciferase. After 2 weeks, an obvious localized bioluminescent signal was observed by in vivo (Figure 4A) imaging using an IVIS imaging system, confirming that tumor cells metastasized to SLNs (left side). No signal was detected in SLNs (right side). Moreover, the investigation of NIR fluorescent imaging was carried out (Figure 4B). As expected, the result was consistent with that of bioluminescent imaging. To mimic the administration route of the SLN imaging agents, the Cy754-MNSPs were subcutaneously injected in the sole of the foot and then in vivo optical imaging was acquired at different time points by IVIS imaging system. The fluorescence signal of metastasized SLNs was rapidly increased and reached a maximum at 1 h (Figure 4C). A positive signal was also

MTT assay method (Figure 3). We can see that nearly 100% of cell viabilities can be observed in a wide range of concentrations

Figure 3. Cytotoxicity of the blank MSNPs and Cy754-MSNPs determined for mouse DC2.4 cells after 24 h of incubation for each group. The data are showed as mean ± standard deviation (SD). The error bars indicate the standard deviation for each measurement (n = 6).

after incubation for 24 h (from 1.5 to 6.0 mg/mL). It is shown that, even incubated with high concentration (12.5 mg/mL) for 24 h, more than 82.7% of the DC2.4 cells still survived. The

Figure 4. Bioluminescence (A) and NIR fluorescence (B) images of 4T1 tumor model (right) and control group (left) were tracked after injection with substrate of luciferase or Cy754-MSNPs, respectively. Right sentinel lymph node: tumor metastasis (T-SLN). Left sentinel lymph node: normal lymph node (N-SLN) as control. (C) NIR fluorescence of tumor metastatic lymph nodes after injection of Cy754-MSNPs at different time points (1 h, 3 h, 1 day, 2 days, 3 days, 5 days, and 14 days) for the 4T1 tumor model group (top) and control group (bottom). (D) Ex vivo NIR fluorescence images of isolated organs from the Cy754-MSNPs group (left) and control group (right). Organs are displayed one after another from left to right: SLNs, hearts, livers, spleens, lungs, kidneys. (E) Quantitative analysis of the fluorescence signal of T-SLN and N-SLN at different time points. The data are showed as mean ± standard deviation (SD). The error bars indicate the standard deviation for each measurement (n = 4). G


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Figure 5. Noninvasive PA imaging. Sequential PA maximum intensity projection (MIP) frames captured before (A), and 1 h postinjection (B) of the Cy754-MSNPs. (C) Bright-field, fluorescent, and PA images of ex vivo lymph nodes 1 h after intradermal administration of Cy754-MSNPs. (D) Time course of region of interest quantification of PA signal (n = 4). (E) Comparison of PA image contrasts quantified from ex vivo results, and fluorescence intensity from ex vivo results. The data are showed as mean ± standard deviation (SD). Error bars represent standard deviation. *p < 0.05. BV, blood vessels; LN, lymph node.

observed over the liver region at 1 h, indicating that the injected Cy754-MNSPs entered into blood circulation and were subsequently captured by the liver. After 6 h, the signal of metastasized SLNs was still very strong and gradually decreased over time, which might be due to macrophage migration and slow degradation of Cy754-MNSPs. Metastasized SLNs had a more rapid uptake rate compared with normal SLNs. More importantly, metastasized SLNs showed significantly stronger signals than normal SLNs in the control group at all the time points (except 0 h) examined (*p < 0.05), confirmed by the quantification analysis of signals showed in Figure 4E. The strong fluorescent signal observed in metastasized SLNs could be explained by several reasons. One is increased number of macrophages induced by tumor mediated inflammation-like reaction.41,42 In addition, lymphangiogenesis induced by tumor also enhances the delivery and uptake of Cy754-MNSPs in metastasized SLNs.43,44 Finally, the uptake ability of macrophages to foreign materials may also be improved by the activation of tumor cells. Two weeks after Cy754-MNSP administration, an obvious signal of metastasized SLNs was still detectable, showing the long-term imaging ability of the Cy754MNSPs. Ex vivo imaging confirmed the distribution of Cy754MNSPs in metastasized SLNs (Figure 4D), which is consistent with in vivo results. Moreover, metastasized SLNs have strong fluorescence signal, while almost no signal is detected in metastasized SLNs at 6 h. These imaging data verified our speculation that the increased number of activated macrophages and lymphangiogenesis are responsible for increased accumulation of Cy754-MNSPs in metastasized SLNs. However, the enlarged volume of metastasized SLNs itself may not be helpful for the uptake of the Cy754-MNSPs. These

results indicate that the metastasized SLN uptake of Cy754MNSPs is related to tumor metastasis to some extent, implying that SLN mapping by Cy754-MNSPs might be useful for longterm tumor metastasis tracking. Photoacoustic Imaging. PA imaging was carried out using the same mouse model described above after subcutaneous injection of contrast agent solutions in the front paws. The information on the PAI system has been provided in PAI System Characterization in the Supporting Information. A 750 nm laser with moderate laser energy at ∼6 mJ/cm2 was used for PA probe detection. The vasculature below the skin surface was displayed with transverse (axial) maximum intensity projection (MIP). As shown in Figure 5A, lymph nodes were undetectable preinjection, lacking any intrinsic optical absorbers, in contradistinction to the adjacent blood vessels containing highly absorbing red blood cells. Following preinjection image acquisition, Cy754-MSNPs (100 μL, 4 mg/mL) were injected intradermally into the foot. At 1 h postinjection, sentinel lymph nodes were easily visualized (Figure 5B), indicating the accumulation of Cy754-MSNPs in the right lymph nodes. Moreover, the contrast of the blood vessel was also slightly increased at 1 h after injection, indicating that the injected MSN-probes entered into blood circulation. To quantify the PA signals, identical regions of interest (ROI) were selected in each MIP image indicated by the red dashed circle shown in the image of Figure 5B. At two time points examined (0 and 1 h), the signals from metastasized SLNs were confirmed by the quantification analysis of PA imaging presented in Figure 5A,B. The average PA image contrasts of SLNs (n = 3) at 0 and 1 h injection are 3.7 ± 1.3 and 22.3 ± 3.4, respectively. The signals at 1 h showed significantly stronger than that at 0 h (*p < 0.05) H



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(Figure 5D). From the in vivo PA analysis, we confirmed that the remarkable difference between the two groups clearly demonstrated the good targeting ability of Cy754-MSNPs for SLN mapping. Finally, the animals were euthanized and lymph nodes were removed. Fluorescence and PA imaging were performed ex vivo to confirm the local signal to the lymph node and to assess the signal levels observed in vivo relative to direct detection. Obvious PA and fluorescence signals were observed for the left lymph node (T-SLN), whereas the signals were too weak to detect in one dissected lymph node from the right (NSLN) (Figure 5C). The ex vivo results observed for both PA and fluorescence image agreed well with the in vivo result. As shown in Figure 5E, T-SLNs showed significantly stronger signals than N-SLNs (*p < 0.05), confirmed by the quantification analysis of the signals, respectively. These results demonstrated the feasibility of Cy754-MSNPs for PA and fluorescence mapping of lymph nodes. Based on all these imaging data, a possible clinically related imaging strategy should be as follows: It is feasible that PA be used for localization with fine ultrasonic resolution at early time points. Then NIR imaging can be used to provide imaging guidance during surgery.

AUTHOR INFORMATION

Corresponding Authors

*Tel/fax: 0086-731-82650459. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We are grateful to National Natural Science Foundation of China (30900377, 81271634) and Hunan Provincial Natural Science Foundation of China (12JJ1012).

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CONCLUSION In this study, we have successfully developed a dual-modal imaging probe based on mesoporous silica nanoparticles and accomplished noninvasive, in vivo imaging of tumor draining SLNs using dual imaging modalities with optical and PA imaging. The combination of NIR fluorescence dye and mesoporous silica nanoparticles (MSNPs) dramatically improved the photostability and retention time of the NIR fluorophore under physiological conditions in the SLN. Enhancement of the PA signal by Cy754-loaded MSNPs resulted from fluorescence quenching. It is expected that the nanoparticles loaded with dye Cy754 display a greater acoustic signal due to their physical and chemical properties. In a 4T1 tumor metastasis mode, a faster uptake rate and higher uptake of the multifunctional MSN-probes were observed in metastasized SLNs compared with normal SLNs, confirming the feasibility of these MSNs as contrast agents to map SLNs and identify tumor metastasis. Based on all these data observed, it was found that the imaging results from different modalities are consistent and complementary. Further studies will focus on optimization of particle size, surface modification of imaging tags, and delivery route for potential clinical translation. To the best of our knowledge, this is the first report showing the feasibility of SLN mapping based on dye doped MSNPs for NIR fluorescent and PA dual-modality imaging in vivo. The dye doped MSNPs are expected to be applicable as excellent contrast agents for SLN imaging in human clinical practice.

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Article

ASSOCIATED CONTENT

S Supporting Information *

Synthesis of Cy754-NHS ester (Scheme S1), extinction coefficient and quantum yields for Cy754 and Cy754-MSNPs, the photographs of Cy754-MSNPs and ICG-MSNPs in eppendorf tubes before and after centrifugation (Figure S1), Cy754-MSNPs for both PA and NIR imaging compared to that of equivalent concentrations of Cy754 (Figure S2), fluorescence stability of Cy754 and ICG (Figure S3), and the characterization of PAI system. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/mp500698b. I


Article

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Dual-Modality Noninvasive Mapping of Sentinel Lymph Node by

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