A comparison of the coronal and sagittal images from the control rat and the rat injected with microspheres () shows significant differences in the liver regions of the rats, where it is known that these non-targeted microspheres accumulate. Compared to the control rat, there is a strong negative contrast enhancement in the liver of the rat injected with the microspheres due to the presence of iron oxide. The spleen also exhibits negative contrast enhancement due to the presence of the iron oxide. There was no change in contrast in tissues that do not contain large numbers of phagocytic macrophages. This clearly demonstrates that the iron oxide-loaded microspheres serve as negative T2 contrast agents in MRI.
Fig. 2 a Coronal and b sagittal MRI slices of a control rat and a normal rat after injection with non-targeted iron oxide microspheres. The arrow-marked regions in the MRI, after injection of iron oxide protein microspheres, show strong negative T2 contrast (more ...)
The rat tumor was initially imaged with a 15 MHz transducer in B-mode and with Doppler to locate a highly vascular region of the tumor (). Using ultrasound guidance, it was also possible to differentiate necrotic areas from the more highly vascularized regions at the periphery of the tumor. After the injection of microbubbles, the ultrasound images showed an increase in scattering as the non-targeted microbubbles circulated through the tumor vasculatures (). On delivery of a rupture pulse sequence, the signal levels dramatically and transiently decreased (). Additional rupture pulse sequences were administered to ensure the clearance of microbubbles from the circulation prior to administration of the RGD-NR-SPIO protein microspheres. While continuing the ultrasound imaging of the same region, the RGD-NR-SPIO protein microspheres were administered intravenously to the rat. Using the same ultrasound image subtraction method as used with the gas-filled microbubbles, it was possible to definitively identify the presence of our microspheres in the tumor (). Ultrasound images obtained after the injection of RGD microspheres showed an increase in scattering signal, just as with the microbubbles. The dynamic scattering changes on injection of protein microspheres were unique, first increasing with the bolus, and then falling toward a new baseline scattering level (). We hypothesize this occurred because as the bolus of targeted microspheres passed through the tumor, we detected the increase, but as the microspheres circulated, reached target sites, and bound within the tumor vasculature, the scattering level dropped, until the binding sites were substantially occupied, and the new scattering baseline level was established (higher than pre-injection).
Fig. 3 High-resolution real-time in vivo ultrasound images of rat mammary tumor and microbubble transients. a Tumor imaged with 15 MHz probe. b Doppler flow imaging identifying vascular region. c Transient increase in scattering (green trace) following commercial (more ...)
Fig. 4 High-resolution real-time ultrasound imaging of tumor following injection of engineered protein microspheres. a Identified microspheres (green channel) within tumor, superimposed over grey-scale B-mode image. b Transient scattering changes (green trace (more ...)
Next, a series of 10 MHz high-intensity rupture pulse sequences were delivered from the instrument (). This time, however, rather than a rapid decrease in scattering from ruptured microbubbles, we observed (repeatedly) a rapid transient increase in scattering before returning to baseline, and gradual decrease in scattering. We hypothesize that this is due to an acoustomotive excitation of the microspheres, with possible indications of rupture. We believe this may allow us to uniquely identify the presence of our microspheres, and understand the dynamics of their binding. Following repeated rupture pulse sequences, the elevated baseline level of scattering gradually decreased, but never returned to the pre-injection level even after 45 min. We hypothesize this was due to the high-affinity binding of the targeted microspheres, the rupture of some but not all of the microspheres, and their gradual detachment and clearance by the liver over time.
The magnetomotive OCT images of the tumor () showed clear magnetomotive signal from the microspheres (green channel). A weak magnetomotive signal was observed from the liver and the bladder, and a large signal was observed from the lungs (data not shown). Further investigations are underway to determine if the size or charge of these microspheres limited their ability to pass through the pulmonary circulation. Previous studies have shown the non-targeted microspheres are finally cleared through the macrophages in liver, lungs and spleen [18
]. Histology was performed on the tumor tissue, at the site of imaging. Under fluorescence microscopy (), using filters that allow the Nile Red fluorescence emission to be selectively imaged, there appeared to be a few intact microspheres present, as well as a more diffuse appearance of dye, likely from ruptured microspheres.
Fig. 5 Multimodal imaging of multifunctional microspheres. a MM-OCT image showing both magnetomotive (green) and structural (red) signals from the tumor site. b High-magnification (63×) fluorescence microscopy of the tumor showing intact Nile red-containing (more ...)
During this study, MM-OCT was perfomed ex vivo, approximately 4 h post injection of RGD-NR-SPIO protein microspheres. Due to time constraints and multiple users, we were unable to integrate our magnetomotive coils with the ultrasound system. However, in the future, we plan to incorporate our magnetomotive coils with the ultrasound transducer so that we could modulate the magnetic microspheres externally and detect this modulation using ultrasound B-mode imaging or Doppler ultrasound.
No information is currently available regarding the life time or clearance of these microspheres in vivo, nor their long term stability and viability. Our studies indicate that the iron oxide microspheres, up to 1011 microspheres per mL, with up to 1 mL/100 g of rat body weight, are safe for short-term studies up to 24 h. Preliminary studies showed that the non-targeted microspheres accumulate in the liver, lungs, and spleen from 15 min to few hours. However, no studies have been carried out on the long term toxicity effects or clearance time of these microspheres from the circulation and system. Studies are currently being conducted on the biodistribution, binding properties, breakdown, and clearance properties of targeted microspheres in the circulation.