Noninvasive in vivo spectroscopic nanorod-contrast photoacoustic mapping of sentinel lymph nodes
Introduction
Sentinel lymph node biopsy (SLNB) has become an axillary staging routine for breast cancer patients as an alternative to traditional axillary lymph node dissection (ALND) [1]. Sentinel lymph nodes (SLNs) are defined as the first draining lymph nodes. Metastatic cancer cells tend to orderly progress out of the original cancer site through the SLNs [1], [2]. In ALND, 10 or more lymph nodes are resected. By contrast, in SLNB, only the SLNs are biopsied to determine whether further lymph node dissection is necessary or not [3]. If the SLNs are cancer-free, the chances are low that the other lymph nodes are affected; thus, no more lymph node dissection is necessary. SLNB has become a standard procedure in axillary staging because it has less invasiveness and side effects than ALND. Furthermore, ALND does not improve the survival rate in comparison to SLNB while causing significant morbidity, such as lymphedema, limitations of arm and shoulder movement, and numbness in the upper arm.
In SLNB, blue dye (methylene blue or isosulfan blue) and radioactive isotope materials (99mTc) are administered near the cancer 5 min and 24 h before a surgery, respectively [2], [3], [4]. The injected tracers flow to the SLNs via the lymphatic system. The accumulated blue dye is visually detected through the surgery, while radioactive substances are detected by a Geiger counter [5]. Since blue dye molecules are small and can easily migrate to adjacent lymph nodes passing the SLNs, the blue dye-based method alone may have a high false positive rate [6], [7]. Because radioactive isotope materials are bigger (∼500 nm), the method using these substances is expected to have less migration to echelon lymph nodes. However, those bigger colloids are mostly retained in the interstitial space and significantly less accumulated in the lymph nodes [1]. Moreover, this method not only has poor spatial resolution, making SLN mapping difficult, but also uses harmful ionizing radioactive materials, which cause damage on connective tissues and also require a special facility to produce.
These complications initiated studies to find safe, noninvasive, and high-resolution SLN mapping methods, avoiding invasive surgeries and allowing minimally invasive staging by such as needle biopsy. Optical imaging is one of such methods because it is safe and non-ionizing. Further, it is more sensitive to imaging contrast than other conventional imaging methods, such as magnetic resonance imaging (MRI), X-ray computed tomography (X-ray CT), and positron emission tomography (PET). Therefore, near-infrared fluorescent type II quantum dots have been used as optical lymph node tracers and successfully applied to map SLNs in an animal model [8]. However, because of strong light scattering, this kind of purely optical imaging techniques based on diffuse light detections suffer from poor spatial resolution beyond the quasi-diffusive regime [9]. To overcome the poor spatial resolution, hybrid imaging modalities such as photoacoustic (PA) imaging, have been implemented by detecting ultrasound instead of light [10]. In PA imaging, a short pulsed laser illuminates biological tissue. Optical absorbers in the tissue absorb optical energy and expand because of thermo-elastic expansion, generating ultrasonic waves. As a result, PA imaging inherits high optical contrast and excellent ultrasonic spatial resolution. Because PA imaging uses multiply scattered photons, it has fairly deep imaging depth while maintaining satisfactory spatial resolution [10], [11]. The modality has been successfully employed to image various biological structures of small animals [10], [12] and also extended to clinical studies [13].
In this study, we have successfully mapped a SLN using spectroscopic photoacoustic imaging using gold nanorods in a rat model. Similar to other metal nanoparticles, gold nanorods have 5 orders of magnitude higher molar optical absorption than conventional absorbing dye molecules [14]. In addition, the surface plasmon resonance (SPR) peak wavelength of nanorods can be easily tuned because that depends on the shape, mainly on the aspect ratio of the axial diameter to the longitudinal length [14]. We showed that the gold nanorods can be used as new lymph node tracers. In addition, to identify the SLN out of blood vessels without taking a control image before the injection of tracers, we have proposed a spectroscopic PA mapping method.
Section snippets
Photoacoustic imaging system
The PA imaging system is schematically described in Fig. 1A. The system was designed to image deeply located biological structures [10]. For PA excitation, a Ti:sapphire laser (LT-2211A, LOTIS TII) pumped by a Q-switched Nd:YAG (LS-2137/2, LOTIS TII) laser was employed. The laser system has a 10 Hz pulse-repetition rate and about 10 ns pulse width. The laser beam with high optical energy was delivered to an object through two prisms and three optical parts. The three optical parts—a concave lens,
Results
Noninvasive in vivo PA SLN mapping with gold nanorods has been successfully accomplished in a rat model. An axillary region of a rat shown in Fig. 3A was scanned with the PA imaging system. Fig. 3B is an anatomical photograph taken after all image acquisitions, with the skin and surrounding fatty tissue removed, revealing a SLN. Insets are photographs of a top view and a bottom view of the dissected SLN, in which the nanorod-accumulated parts are in red color. Before the injection of the gold
Discussion
We demonstrated the feasibility of noninvasive in vivo PA SLN mapping by injecting gold nanorods into rats. In a lymphatic system, it is well known that smaller sized nanoparticles (<5 nm) migrate to SLNs faster than larger nanoparticles [5]. Methylene blue, which has a 0.7 nm in diameter and a 1.6 nm in length [6], takes 5 min to reach SLNs in humans. Radioactive isotope, 99mTc (∼1000 nm), takes up to 24 h. Spherical nanoparticles of 40 nm in diameter take about 4.6 min to reach SLNs of rats [18].
Acknowledgments
This research is sponsored in part by National Institutes of Health grants R01 EB000712 and R01 NS46214 (BRP). We gratefully acknowledge Nanopartz for their 10–808 nm Nanorods TEM image contribution. Thanks to Eunchul Cho for his assistance in measuring the absorbance of nanorods. L.V.W. has a financial interest in Endra, Inc., which, however, did not support this work.
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