To demonstrate an application for using GNRs as a nanoprobe for high resolution imaging of SLN, we used two different optical imaging modalities, OCT and PT-OCT. shows the three-dimensional projection view of a dissected SLN at 48 hours after GNRs injection obtained with the OCT system. The inset in is a top view photograph of a dissected SLN, in which the areas of high GNRs uptake can be observed with a dark color. shows a cross-sectional cut of the volumetric SLN dataset at a depth of 240 µm from the surface obtained with the OCT system, and it delineates the typical morphological features of the SLN. It can be observed, that the OCT image cannot detect the nanoparticle accumulation because of the low scattering contrast that is present on the GNRs. However, in the PT-OCT image (), the phothothermal properties of the GNRs significantly enhance the imaging contrast. The PT-OCT image delineates the uptake of GNRs throughout the SLN, and by combining both the OCT and PT-OCT images we can distinguish the distribution of GNRs in several SLN structures. The intravenously injected GNRs migrate out of the venules and mostly accumulate in the superficial sinus, which is a channel that filters foreign organisms. Also, the GNRs accumulate at the cortex surrounding follicles, where mainly B cells and intrabecular sinus are filtered.
Figure 3 (a) Three-dimensional OCT projection image of a dissected SLN at 48 hours after GNRs injection. (b) 3D OCT view of SLN morphology with a cross-sectional cut at a depth of 240 µm below the top surface. (c) The same as in (b) but cross-sectional (more ...)
Using the PT-OCT system, it is possible to monitor the distribution of GNRs within the SLN structures with high resolution as a function of time and at defined depths. depicts several PT-OCT cross-sectional images of a lymph node at a depth of 120, 240, 360, 480, 600, 720 and 840 µm below the surface, and at two time points (12 hrs and 96 hrs). At 12hr post-injection (), GNRs were present at qualitatively consistent levels. At 96 hrs () the GNRs uptake by the SLN was significantly increased at all the depths.
Figure 4 Cross-sectional PT-OCT images was obtained at different depths (120, 240, 360, 480, 600, 720 and 840 µm) below the surface by slicing the 3D data cube [shown in (a)] acquired from the SLN at the time point of (b) 12 and (c) 96 hours after GNRs (more ...)
Time dependent lymph node uptake of nanoparticles has been investigated by other researchers and typically focused on time points in the range of 6–120 hrs post-injection.18, 19
Also, the ability of surface modification of nanoparticles to enhance lymphatic drainage has been demonstrated.19
To further evaluate the kinetics of PEG modified GNRs uptake, PT-OCT images were acquired at 0, 0.25, 4, 8, 12, 24, 48, 96, 140, 320, 456 and 672 hrs after the 0.8 nM GNRs injection. This enabled us to visualize the time-dependent GNRs uptake within the SLN. presents projection images of the GNRs uptake within SLN at the specified time points. To quantify the GNRs uptake kinetics in SLN, the average concentration of GNRs within the SLN was calculated, by summing the PT-OCT signal strength (i.e. optical pathlength changes) across the whole SLN and then dividing it by the SLN volume. This value was compared with a calibration curve () that was obtained using tissue phantoms made with known concentration of GNRs. presents the average concentration of GNRs within the SLN as a function of time. It is observed that the concentration reaches a peak of ~ 17 pM at 96 hours, and after that the concentration slowly declines. Compared to the initial GNRs injection (0.8 nM), the maximal lymph node uptake of about 2 % can be achieved with GNRs at 96 hr.
The thermal effect upon the pulsed laser irradiation is the physical base for PT-OCT to measure the GNRs accumulation within the SLN. However, the increase of temperature in tissue is small. Based on a mathematical model developed by Adler et. al.
the estimated increase of temperature for a ~17 pM is ~ 0.1 °C, which is way below the threshold that would cause laser-induced tissue injury.
The use of GNRs enables us to understand the functionality of the SLN in many ways using PT-OCT. First, the GNRs are a source of high contrast that allows us to obtain high resolution images of different SLN structures. Second, the use of PT-OCT may be useful in delineating the migration pattern of the GNRs within different SLN structures as a function of time. And finally, the PT-OCT images allow the quantification of the circulation times of the GNRs. The miniaturized catheter has been developed for OCT to successfully image the interstitial tissue morphology in vivo
Therefore, we expect that such miniaturized catheter can be integrated, in future, with the current PT-OCT probe so that in vivo
imaging of GNRs uptake within the SLN would be feasible.
An ideal SLN contrast agent should be small enough to rapidly drain to the lymphatic tissue but large enough to stay within the lymphatic system during the procedure. The migration speed of certain contrast agents has been reported to be dependent on the shape, size (smaller sized nanoparticles migrate faster than larger sized nanoparticles) and surface composition.6, 22
The GNRs that were used for this study had an average length of 45 nm, and the surface was functionalized with PEG-SH to produce long blood circulation lifetimes and biocompatibility. PEG has well established stealth effect that can protect GNRs from extraneous matter by serum proteins and also it can reduce the clearance by reticuloendothelial system.23
Also, PEG-modified GNRs shows lower cytotoxicity and improved in vivo
circulation following intravenous injection in murine models.24
We have used GNRs as a nanoprobe for enhancing 3D high resolution images of in situ SLN samples from a mice model. Compared to conventional scattering OCT, the PT-OCT system can monitor the distribution of GNRs within specific SLN structures through time. The uptake of GNRs within a SLN is slow (reaching a peak at ~96 hours) indicating that the GNRs have long circulation times. The resulting SLN images show that GNRs are suitable as a contrast agent. Although the imaging experiments were conducted in situ, we expect to be able to obtain these images in vivo and in real-time with the aid of a catheter. In the future, GNRs could also be used as a multifunctional probe for both diagnostic and therapeutic purposes.