Highly Sensitive Fluorescence and Photoacoustic Detection of

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Highly Sensitive Fluorescence and Photoacoustic Detection of Metastatic Breast Cancer in Mice Using Dual-Modal Nanoprobes Xiangwei Lin,†,‡,⊥ Chengbo Liu,§,⊥ Zonghai Sheng,‡ Xiaojing Gong,§ Liang Song,§ Ruifang Zhang,∥ Hairong Zheng,*,‡ and Mingjian Sun*,†

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†

Measurement and Control Research Center, Department of Control Science and Engineering, Harbin Institute of Technology, Harbin 150001, China ‡ Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology and §Research Laboratory for Biomedical Optics and Molecular Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China ∥ Department of Ultrasound, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450000, Henan, China S Supporting Information *

ABSTRACT: The biomedical imaging of metastatic breast cancer, especially in lymphatic and lung metastasis, is highly significant in cancer staging as it helps assess disease prognosis and treatment. Using an albumin-indocyanine green dual-modal nanoprobe developed in our laboratory, in vivo fluorescence imaging and photoacoustic imaging of metastatic breast cancer tumors were performed separately. Fluorescence imaging at the near-infrared window features high imaging sensitivity but is generally limited by a low imaging depth. Thus, tumors can only be observed in situ whereas tumor cells in the lymph nodes and lung cannot be imaged in a precise manner. In contrast, photoacoustic imaging often helps overcome the limitations of imaging depth with high acoustic spatial resolution, which could provide complementary information for imaging cancer metastases. Ex vivo fluorescence and photoacoustic imaging were also performed to verify the tumor metastatic route. This study may not only provide insights into the design of dual-modal nanoprobes for breast cancer diagnosis but may also demonstrate the superiority of combined fluorescence imaging and photoacoustic imaging for guiding, monitoring, and evaluating lymphatic and lung metastatic stages of breast cancer with a high imaging specificity as well as sensitivity. KEYWORDS: breast cancer metastasis, indocyanine green, dual-modal nanoprobes, fluorescence imaging, photoacoustic imaging guided photothermal therapy of tumor metastasis.16 Meanwhile, Siegwart et al. reported size-controlled pH-activatable water-soluble probes for fluorescence detection of bone and liver micrometastasis of breast cancer.24 Although the progression of currently available fluorescent nanoprobes for the detection of metastatic breast cancer has been highly successful, the low penetration depth and poor resolution generally limit advanced applications.25−27 Therefore, the exploration of more feasible imaging tools and probes for the precise monitoring of the metastatic status of breast cancer remains a critical, albeit unmet, scientific goal. Compared with fluorescence (FL) imaging, photoacoustic (PA) imaging features the unique ability to visualize optical absorption and deep tissue penetration up to several centimeters,28,29 while still maintaining a high acoustic spatial

1. INTRODUCTION To date, breast cancer is one of the major causes of death, threatening the health of women around the globe.1−3 The death rate of metastatic breast cancer is particularly high due to high tumor recurrence and difficult prognosis assessment of early-stage cancer.4−6 The lymph and blood circulation systems are two main metastatic circulation routes, potentially carrying tumor cells to spread into other distant organs in the body.7−10 During cancer lymphatic metastasis, sentinel lymph nodes (SLNs) in proximity to xenograft tumors generally serve as the first site in which tumor cells colonize. By contrast, tumor cells may also enter blood circulation and the common metastasis sites, including lungs, bone, liver, and brain. In particular, the sentinel lymph nodes and the lung area are common primary targets for metastatic tumor formation.11−14 A series of imaging modalities and nanoprobes have been employed in the past for the imaging-guided screening of primary and metastatic tumors.15−23 For instance, Liu et al. reported carbon nanotube-based fluorescence (FL) imaging© XXXX American Chemical Society

Received: June 2, 2018 Accepted: July 16, 2018

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DOI: 10.1021/acsami.8b09142 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Schematic illustration of HSA-ICG dual-modal nanoprobes for imaging of lymphatic and lung metastases in mice.

resolution.30−35 Some specific PA probes, such as indocyanine green (ICG) and metallic nanoparticles, have been developed for the detection of breast cancer and SLN.36−41 For example, Wang et al. were the first to report PA detection of SLN with accumulation of methylene blue dye42,43 and gold nanobeacons.44 Meanwhile, Klode et al. reported the multispectral PA assessment of the metastatic status of SLN using the fluorophore indocyanine green.45 Although these contrastenhanced PA imaging methods generally help improve the detection sensitivity of SLN, applications of the PA imaging and PA nanoprobes for the simultaneous and highly sensitive detection of primary tumor and tumor metastasis still face enormous challenges. In this work, human serum albumin-assembled indocyanine green nanoprobes (HSA-ICG NPs) were developed as an FL and PA dual-modal contrast-enhanced probe for the simultaneous imaging of primary breast cancer tumors with SLN and lung metastasis (Figure 1). Both HSA46,47 and ICG48−50 acted via a programmed assembly strategy to form HSA-ICG NPs. The advantages of this detection method were as follows: (I) FL and PA dual-modality imaging could provide some comprehensive information on metastatic breast cancer detection. (II) The HSA-ICG NPs with inherent dual-modal contrast features exhibited a high cancer cell uptake efficacy and long-term retention in primary and metastatic breast cancer. (III) The prepared nanoprobes featured a high biocompatibility and non-antigenic properties. In light of this series of promising results, this unique FL and PA detection method may potentially be used in future clinical applications.

(TEM) imaging, ultraviolet−visible (UV−vis) absorption spectra, FL spectra, as well as dynamic light scattering (DLS) measurement.51 2.3. Cell Cultures. 4T1 breast cancer cells were purchased from Cyagen (China). The DMEM high-glucose medium with 10% FBS, 100 μg/mL streptomycin, 2 mM L-glutamine, and 100 U/mL penicillin were used for cell culture. 2.4. In Vitro Cellular Uptake and Cytotoxicity Assay. The cellular uptake experiments were consistent with previous processes,51 and a standard MTT assay could determine the relative cell viabilities, where 4T1 tumor cells were incubated with the corresponding NPs at different concentrations for 24 h. 2.5. Animals and Tumor Model. All animal experimental protocols were approved by the Animal Study Committee of the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. Female BALB/c mice (4−6 weeks old, weight 18−20 g) were obtained from the Medical Experimental Animal Center of Guangdong Province (Guangzhou, China). To develop the breast metastasis model, 4T1 murine breast cancer cells (1 × 106 cells/ mouse) were suspended in DMEM and then injected into the right hind foot sole of the mice. The tumor sizes were recorded to grow up to a volume of ∼100 mm3 before further imaging. At this time point, certain amount of breast tumor cells could spread into the nearest sentinel lymph node and lung area. The numbers of mice in the following FL and PA experiment is five to ensure the reliability of the imaging results. 2.6. In Vivo and ex Vivo FL Imaging. All in vivo and ex vivo FL imaging of the mice were performed using a commercial FL imaging system (CRi Maestro). 2.7. In Vivo and ex Vivo PA Imaging. A custom-made acousticresolution photoacoustic microscopy (AR-PAM) system52 was established for all PA imaging (Figure S1). This system was established by light illumination, signal acquisition, and postprocessing modules. The optical unit included a Q-switched Nd:YAG laser (Vibrant, 355 II HE) with optical parametric oscillator, emitting a 5 ns pulse width laser at a repetition rate of 10 Hz. For all PA imaging experiments, the wavelength was set to 780 nm to adapt the peak absorption of HSA-ICG NPs. The laser fluence in the cell study was set as 10 and 2 mJ/cm2 for in vivo and ex vivo animal imaging, respectively. This laser fluence was in accordance with all ANSI safety standards. The intensity of the laser at each pulse was recorded using a silicon photodiode (Thorlabs, SM05PD1A) to compensate for the laser energy fluctuation. A 10.0 MHz focused ultrasound transducer with 19.0 mm element diameter and 25.4 mm focal depth (Olympus, V315-SU) could acquire both PA and pulse-echo signals excited by an ultrasonic pulse-receiver (Olympus, 5073PR). Both the PA and pulseecho signals were amplified by 39 dB and then transferred and

2. MATERIALS AND METHODS 2.1. Materials. HSA, ICG, 4,6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phosphate buffer saline (PBS), fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), trypsin−ethylenediaminetetraacetic acid, and penicillin−streptomycin, as well as Alexa Fluor 488 Annexin V/propidium iodide cell apoptosis kit and Calcein-AM remain the same, as with our previous work.51 2.2. Preparation and Characterization of HSA-ICG NPs. HSA-ICG NPs were prepared via a programmed assembly strategy; see ref 51 in detail. The freshly prepared HSA-ICG NPs were subsequently characterized for transmission electron microscopy B


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Figure 2. Fabrication and characterization of HSA-ICG NPs. (a) Self-assembly process of HSA-ICG NPs. (b) TEM image of HSA-ICG NPs. (c) Size distribution of HSA-ICG NPs measured by DLS. Fluorescence emission spectra (d) and UV−vis absorption spectra (e) of free ICG and HSAICG NPs, respectively. digitalized by a data acquisition card (Gage, CS1422), inserted in a personal computer. The external trigger of the laser was used to synthesize the delay module, to acquire the PA signals first and then the pulse-echo signals after a delay time of 40 μs. Following the triggering of 8 pulses, a precision motorized three-dimensional scanning stage (Zolix, PSA2000) was moved to the next position. At each scanning step (100 μm), the acquired signals were averaged for a total of 8 times and the PA images were reconstructed by an enveloped detection method, after the compensation of laser energy variation. The corresponding ultrasound and photoacoustic (US/PA) images were co-registered and displayed using Image J. 2.8. H&E Staining. The cryogenic slides were fixed with 10% formalin (30 min) and then were further treated with alcohol solutions with different concentrations (100, 95, and 70%) after washing. Hematoxylin staining was continued for about 3 min and washed for 1 min, whereas Eosin staining was performed for about 1 min. The slides were washed, treated with xylene, and mounted with Canada balsam. Nikon Eclipse 90i microscopy was used to obtain the H&E staining images. 2.9. Examination of SLN and Lung Metastasis. To examine the nearest SLN and lung metastatic status, the mice were sacrificed 4 h after HSA-ICG NPs were injected via tail vein injection. Major organs (primary tumor, nearest SLN, and lung) were collected and soaked in Fekete’s solution at room temperature for 2 days. For histology examination, the nearest SLN and lung in different mice groups were extracted, fixed in 10% formalin, treated routinely with paraffin, sectioned at 6 μm thickness, and finally stained with

hematoxylin and eosin. The representative H&E staining images of lymphatic and lung metastasis in breast cancer are shown in Figure S2.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of HSA-ICG NPs. HSA-ICG NPs were programmably assembled by HSA and ICG (Figure 2a), and Figure 2b is the corresponding TEM image of HSA-ICG NPs, which exhibited well-defined spherical structures and homogeneously dispersed individual particles with diameters ranging from 25.0 to 35.0 nm. Its average hydrodynamic diameter was measured to be 75 ± 2.4 nm via DLS measurements, as shown in Figure 2c. The fluorescence emission spectra of both free ICG and HSA-ICG NPs (10 μg/mL) after loading of ICG in HSA NPs are shown in Figure 2d, whereas Figure 2e shows UV−vis absorption spectra. Compared with free ICG, it could be determined that the absorption spectra did not change significantly after loading of ICG; however, a suitable optical absorption performance could still be observed. Furthermore, the fluorescence emission spectra indicated that the fluorescent intensity decreased due to a high fluorescence quenching effect caused by local aggregation of ICG.53 3.2. PA Performances of HSA-ICG NPs. PA spectra is shown in Figure 3a, which was also measured by the AR-PAM system. Noteworthily, two absorption peaks for these two kinds of NPs were located in the same wavelength (780 nm). C


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Figure 3. PA properties of the HSA-ICG NPs. (a) PA spectra of the indicated nanoprobes. (b) PA intensity of indicated nanoprobes diluted in ultrapure water versus number of laser pulses. (c) PA signal at 780 nm in the time domain of different mass concentrations of indicated nanoprobes. (d) PA intensity of the same mass concentration in (c) as a function of mass concentration. Error bars represent standard deviations of PA intensity measurements.

increasing cell number. The minimum detected cell numbers in PA detection were ∼1000 cells and ∼100 cells in FL imaging. This result showed that HSA-ICG NPs may potentially serve as FL and PA dual-modal probes for sensitive dual-modality FL and PA imaging of metastatic breast cancer with improved cellular uptake, accumulation, and enhanced cancer cell targeting. 3.4. In Vivo FL Imaging of Primary, Lymphatic, and Lung Tumor Metastases. An animal model of the breast tumor metastasis was established (as shown in Figure 1), and the nearest SLN and lung area were collected for histology assessment of the tumor metastases (Figure S2). In vivo FL imaging was performed to investigate the biodistribution of HSA-ICG NPs in tumor-bearing mice, revealing its ability to track the metastatic route of breast cancer during whole-body imaging. The non-tumor-bearing mice injected with HSA-ICG NPs were designed as the control group. After tail vein injection of the indicated nanoprobes (100 μL, 0.5 mg/kg), Figure 5 depicts a typical FL intensity distribution within the mouse as a function of time. From the normalized FL imaging obtained from the back of the mice (Figure 5a), welldetectable FL intensities were found only in the tumor in situ. Furthermore, no fluorescent signal in the control group could be detected, whereas the signal intensity gradually increased in the other two groups. More importantly, a much more significant accumulation over time could be determined in the foot of the HSA-ICG NP-treated mouse. This latter finding exhibited that the HSA-ICG NPs featured an enhanced permeability and retention (EPR) effect with efficient tumortargeting ability. Upon turning over the mouse (Figure 5b), it was found that the FL intensities started to gradually increase from the primary tumor toward internal metastatic regions, such as the nearest SLN and lung area (yellow circle, purple circle, and

Therefore, this wavelength was set as the laser excitation in the following experiments. Figure 3b shows the photostability test of these nanoprobes, where the self-assembled HSA-ICG NPs exhibited an improved photostability compared to that of the free ICG in the continuous laser shot. Potentially, this particular feature may be beneficial in the long-term monitoring of a pathophysiological process. The PA detection sensitivity of nanoprobes at different concentrations was measured (Figure 3c shows the PA signal in the time domain) and analyzed (Figure 3d depicts the quantitative analysis), where the minimum detection concentration was determined additionally. The corresponding results showed that the PA intensity gradually increased with concentration, demonstrating an approximate linear relationship in a mass concentration range of 20−140 μg/mL. 3.3. In Vitro Cellular Uptake and Quantitative Assay. The cellular uptake studies of free ICG and HSA-ICG NPs were evaluated by confocal laser scanning microscopy, as shown in Figure 4a,b. The murine 4T1 breast cancer cells were separately incubated with these nanoparticle species for 3 h. FL intensity of free ICG could not be observed, whereas a significantly stronger FL intensity was detected in the 4T1 cells incubated with HSA-ICG NPs. This result indicated that certain amount of HSA-ICG NPs could enter the tumor cells and localize in the cytoplasm, with an enhanced cellular uptake efficiency due to albumin receptor-mediated endocytosis pathway in 4T1 cells.51,54 In vitro PA and FL experiments were also performed in an effort to evaluate the detection sensitivity with the cell inclusion of HSA-ICG NPs. The original PA signals and FL intensities are shown in Figure 4c,e, and the corresponding quantitative analysis is shown in Figure 4d,f, respectively. The control group exhibited negligible intensities prior to incubation with HSA-ICG NPs, whereas both the PA and FL intensities increased linearly with D


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Figure 4. In vitro cellular uptake and detection results. Fluorescence confocal microscopy of 4T1 cells incubated with free ICG (a) and HSA-ICG NPs (b). The three images from left to right in each row separately show the ICG, DAPI, and their overlay FL (scale bar: 60 μm), respectively. PA signal generated in different cell numbers (c) and corresponding statistical analysis (d). FL intensity detected in equal cell numbers (e) and corresponding statistical analysis (f). Error bars represent standard deviations of intensity measurements.

whereas the clearance rate in the free ICG-treated mice was rather rapid (∼8 h). This finding indicated that both the tumor and the tumor-targeting nanoprobes are prerequisites in

green square in the third line of Figure 5b, respectively). As expected, in the control group, no significant FL signal accumulation could be observed in major metastatic organs, E


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Figure 5. In vivo and ex vivo FL imaging with quantitative analysis. (a) FL intensity distribution of the mouse back as the indicated control group and separate tail vein injections of free ICG and HSA-ICG NPs into tumor-bearing mice. (b) FL intensity distribution in the same sequence after the same mouse was turned over. The tumor region, nearest SLN, and the lung were separately labeled as follows: yellow, bright blue, and green dashed line. (c) The ex vivo FL imaging of the excised major organs, nearest SLN, and primary tumor. (d) The corresponding quantitative analysis of FL intensities, which are shown as mean ± standard deviations (n = 5). (*) p < 0.05, (**) p < 0.01.

tracking breast tumor metastasis. In the tumor-bearing mice injected with HSA-ICG NPs, significant and enhanced FL intensities could be determined during the same observation period in the primary tumor region, nearest SLN, and lung area. More importantly, the accumulation of the nanoprobes in the tumor region was far more significant compared to that in the three other tumor-free feet. However, it is worth noting that some accumulation inconsistencies were found due to the different metabolic characteristics in mice and the low penetration depth of FL imaging. Therefore, in an effort to restore consistency, major metastatic organs were extracted for ex vivo FL assessments. As illustrated in Figure 5c, to further validate lymphatic and lung metastasis in breast cancer, ex vivo FL imaging of the excised major organs (heart, liver, spleen, lung, and kidney), nearest SLN, and tumor was performed 24 h after tail vein injection of related NPs. Quantitative analysis results of the FL signals of these organs are further shown in Figure 5d. The ex vivo imaging results showed that HSA-ICG NPs could accumulate in the tumor metastatic routine. Comparing the injection of HSA-ICG NPs with free ICG, the FL intensities were significantly higher in the metastatic regions, even after

24 h. However, detectable FL signals could not be found in the control group. This result indicated that HSA-ICG NPs exhibited specific tumor-targeting ability with enhanced EPR effect, potentially useful for effective FL imaging in the longterm monitoring of breast cancer metastases. 3.5. In Vivo PA Imaging of Primary, Lymphatic, and Lung Tumor Metastases. In vivo PA imaging was carried out to investigate breast tumor metastases and to provide complementary information on metastatic depth. As a dualmodal nanoprobe, HSA-ICG NPs also showed an excellent PA performance. Therefore, primary tumors, metastatic SLN, and the lung area could be imaged using the custom-designed ARPAM system. The same mice used for FL imaging could also be used for PA imaging. Both the control and experimental groups were assigned in the same order as the groups used in FL imaging. Figure 6 shows the cross-sectional co-registered US/PA images of the metastatic tumor area as a function of time after tail vein injection of the indicated nanoprobes (100 μL, 0.5 mg/kg). Ultrasound imaging could provide position information and rough contour estimates of the tumors, whereas PA images demonstrated the depth-resolved biodisF


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Figure 6. In vivo PA imaging and corresponding quantitative analysis. US/PA images of primary tumor region (a) and nearest SLN (b) obtained pre-, 10 min, 2, 4, 8, and 24 h post tail vein injection of HSA-ICG NPs into the non-tumor-bearing mice, free ICG and HSA-ICG NPs into the tumor-bearing mice, respectively. Quantitative analysis of PA intensities in the region of interest of the primary tumor (c) and nearest SLN (d), which are shown as mean ± standard deviations (n = 5). G


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Figure 7. Ex vivo PA imaging and corresponding quantitative analysis. Maximum amplitude projection of the excised tumor, nearest SLN, and lungs 24 h post tail vein injection of the indicated nanoprobes (a) and corresponding quantitative analysis (b), which are shown as mean ± standard deviations (n = 5). (*) p < 0.05, (**) p < 0.01.

Figure 8. In vivo toxicity study of HSA-ICG NPs. Representative H&E-stained images of major organs, including heart, liver, spleen, lung, and kidney collected from mice sacrificed 7 days after injection of indicated nanoprobes.

the results of FL imaging. Such complementary information could be crucial for both assessing the cancer metastasis stage and image-guided tumor therapy. Note that PA intensities before the injection of any nanoprobes were primarily from the blood absorption at 780 nm, and the PA images revealed the contrast enhancement in the metastatic area due to the accumulation of indicated nanoprobes. However, the animal lung area cannot be imaged due to the severe PA signal attenuation in the irregular alveolar air. Therefore, upon in vivo PA imaging of the primary tumor and nearest SLN, ex vivo PA imaging of above organs was further explored, with particular attention paid to the lung area. Figure 7a depicts the PA signal distribution in the primary tumor, nearest SLN, and lung area, exhibiting a tendency and features similar to those in FL imaging (Figure 5c). A quantitative analysis of the PA signal distribution in these tumor areas was also performed, as illustrated in Figure 7b. In doing so, we found that more significant PA signal intensities were present in the HSA-ICG NPs (∼2-folds compared with free ICG and ∼6-folds compared with the control group), providing further evidence for the efficient tumor-targeting ability of the developed HSAICG NPs to track the metastatic route of breast cancer. 3.6. Biotoxicity Evaluation. The in vitro biotoxicity of HSA-ICG NPs was evaluated, and no significant decrease of cell viability could be observed 24 and 48 h post injection of HSA-ICG NPs at concentrations varying from 0.6 to 40 μg/ mL (Figure S3). Figure 8 shows the in vivo H&E staining of

tribution of indicated nanoprobes in the metastatic tumor areas. Figure 6a shows the PA signal distribution in the primary tumor at the cross-sectional orientation of the indicated nanoprobes. It could be determined that the in-depth PA signal increase in the group treated with HSA-ICG NPs was much more intense. However, the other groups exhibited weaker PA signal intensities with reduced nanoprobe accumulation over time. The corresponding quantitative analysis at the specific regions of interest is illustrated in Figure 6c, and the results further verified the above findings. Results of US/PA imaging of lymphatic breast cancer metastases are shown in Figure 6b. In the control group, the HSA-ICG NPs were found to concentrate at the nearest SLN around 10 min after tail vein injection. Most likely, this finding was due to a rapid clearance process and nonspecific targeting. The PA signal intensities started to enhance gradually over the course of 2 h and were then found to decrease in the free ICG-treated group. In contrast, the overall PA signal intensities in the HSA-ICG NPs treated group were higher and the signal accumulation area was larger compared to those in the group injected with free ICG. The corresponding quantitative statistic findings further confirmed this result, as shown in Figure 6d. Using PA imaging, local and depth information of the tumor and nearest SLN could be obtained, demonstrating the specific tumor-targeting ability of HSA-ICG NPs to breast cancer tumors with enhanced PA intensities, which agreed well with H


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(Grant No. 2016YFA0202702), Development Plan of Chinese Academy of Sciences and Wego Group (Grant No. 2017011), S h e n z h e n B a s i c R e s e a r ch P r o g r a m ( G r a n t N o . JCYJ20160429115309834), and Key Laboratory for Magnetic Resonance and Multimodality Imaging of Guangdong Province (Grant No. 2014B030301013)

major organs (heart, liver, spleen, kidney, and lung) harvested from a representative mouse on day 7 after injection with HSA-ICG NPs and PBS as control group. Compared with the control group, no obvious tissue damages could be observed in the HSA-ICG NP-treated group. In addition, ex vivo imaging results for the analysis of in vivo clearance of HSA-ICG NPs after imaging were also provided, as shown in Figure S4.

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4. CONCLUSIONS In summary, tracing the metastatic status of tumor cells represents an important goal for the diagnosis and therapy of breast cancer. For the first time, our experimental results demonstrated that a high accumulation and long-term retention of HSA-ICG NPs in the metastatic breast cancer could be determined. In virtue of this self-assembled specific tumor-targeted probe, i.e., prolonged circulation time and excellent dual-modality FL/PA properties, in vivo and ex vivo FL and PA imaging could provide complementary planar distribution and in-depth penetration information of the nanoprobes for tracking the lymphatic and lung metastasis in breast cancer. Moreover, the dual-modality FL imaging and PA imaging may cross-validate the results. In our study, FL imaging exhibited an improved planar sensitivity compared with PA imaging, whereas PA imaging provided a high acoustic spatial resolution for depth-resolved tumor information that generally cannot be obtained using planar FL imaging. Taken in concert, this study suggests that the HSA-ICG NPs may offer great potential for the clinical translation of dual-modal imaging of lymphatic and lung metastatic breast cancer.

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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.8b09142. Schematic illustration of acoustic resolution photoacoustic microscopy; results of H&E staining images of primary tumor, lymphatic, and lung metastasis in breast cancer, cytotoxicity of HSA-ICG NPs against 4T1 cells; photograph and fluorescence images of collected urine and droppings of mice 24 h post injection of HSA-ICG NPs (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H.Z.). *E-mail: [email protected] (M.S.). ORCID

Mingjian Sun: 0000-0001-8719-524X Author Contributions ⊥

X.L. and C.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (Grant Nos. 61371045 and 11574046), Natural Science Foundation of Shandong Province (Grant Nos. ZR2017MF041 and ZR2018MF026), Science and Technology Development Plan Project of Shandong Province (Grant Nos. 2016GGX103032 and 2018GGX103047), National Key R&D Project from Minister of Science and Technology of China I


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