Lanthanide Nanoparticles as Contrast Agents for In Vivo Dual Energy Microcomputed Tomography of the Mouse Vasculature

Dual energy (DE) computed tomography (CT) has the capability to influence medicine and pre-clinical research by providing quantitative information that can detect nascent lesions, identify perfusion restoration or inhomogeneities within tissues, and recognize the presence of calcium deposits. A wide variety of instrumentation techniques and scan protocols have been developed for DE CT, with a common goal of acquiring a pair of images that reports the attenuation of a given volume to two different x-ray distributions. While DE image acquisition has benefitted from technical advancements in CT, the contrast agents that are used are still predominantly composed of iodinated small molecules, which first appeared in the 1970s. Recent work has demonstrated that lanthanide-based contrast agents have optimized properties for DE decomposition, specifically when using in vivo micro-CT scanners. By adopting nanoparticle design strategies that were developed for disease therapeutics and diagnosis, this thesis takes advantage of existing technical advancements in nanotechnology and polymer science to develop a long-circulating contrast agent that can be used for in vivo micro‑CT and DE micro‑CT imaging of the mouse vasculature. The contrast agents that were developed provided a high loading of 100 mg/mL of lanthanide for intravenous injections of mice, and introduced CT contrast enhancements of at least 245 HU. The contrast was maintained for at least 30 minutes, and for as long as one hour, which exceeds the in vivo micro-CT scan time requirements. Furthermore, although the synthesis techniques and in vivo scans were demonstrated using model lanthanides such as gadolinium and erbium, they can easily be substituted by any other lanthanide. By using a fast-filter switcher to obtain interleaved scans, the feasibility of an in vivo DE CT technique that produces decomposed quantitative images of soft tissue, bone and gadolinium-enhanced vessels was demonstrated, which can be used with any pre-clinical, gantry-based micro-CT scanner. When used in combination with the DE CT technique presented, the long-circulating lanthanide contrast agents that were developed in this thesis have the potential to become powerful tools for pre-clinical research on the microvasculature

Polymer assembly encapsulation of lanthanide nanoparticles as contrast agents for in vivo micro-CT

Despite recent technological advancements in microcomputed tomography (micro-CT) and contrast agent development, pre-clinical contrast agents are still predominantly iodine-based. Higher contrast can be achieved when using elements with higher atomic numbers, such as lanthanides; lanthanides also have x-ray attenuation properties that are ideal for spectral CT. However, the formulation of lanthanide-based contrast agents at the high concentrations required for vascular imaging presents a significant challenge. In this work, we developed an erbium-based contrast agent that meets micro-CT imaging requirements, which include colloidal stability upon redispersion at high concentrations, evasion of rapid renal clearance, and circulation times of tens of minutes in small animals. Through systematic studies with poly(ethylene glycol) (PEG)-poly(propylene glycol), PEG-polycaprolactone, and PEG-poly(l-lactide) (PLA) block copolymers, the amphiphilic block copolymer PEG114-PLA53 was identified to be ideal for encapsulating oleate-coated lanthanide-based nanoparticles for in vivo intravenous administration. We were able to synthesize a contrast agent containing 100 mg/mL of erbium that could be redispersed into colloidally stable nanoparticles in saline after lyophilization. Contrast enhancement of over 250 HU was achieved in the blood pool for up to an hour, thereby meeting the requirements of live animal micro-CT

3D vessel-wall virtual histology of whole-body perfused mice using a novel heavy element stain

© 2019, The Author(s). Virtual histology – utilizing high-resolution three-dimensional imaging – is becoming readily available. Micro-computed tomography (micro-CT) is widely available and is often coupled with x-ray attenuating histological stains that mark specific tissue components for 3D virtual histology. In this study we describe a new tri-element x-ray attenuating stain and perfusion protocol that provides micro-CT contrast of the entire vasculature of an intact mouse. The stain – derived from an established histology stain (Verhoeff’s) – is modified to enable perfusion through the vasculature; the attenuating elements of the stain are iodine, aluminum, and iron. After a 30-minute perfusion through the vasculature (10-minute flushing with detergent-containing saline followed by 15-minute perfusion with the stain and a final 5-minute saline flush), animals are scanned using micro-CT. We demonstrate that the new staining protocol enables sharp delineation of the vessel walls in three dimensions over the whole body; corresponding histological analysis verified that the CT stain is localized primarily in the endothelial cells and media of large arteries and the endothelium of smaller vessels, such as the coronaries. The rapid perfusion and scanning protocol ensured that all tissues are available for further analysis via higher resolution CT of smaller sections or traditional histological sectioning

High-concentration gadolinium nanoparticles for pre-clinical vascular imaging

© 2018 SPIE. Gadolinium-based contrast agents that have long circulation times in small animals have always been of interest in preclinical imaging. Although gadolinium-based contrast media are used clinically in MRI, these agents are composed of small molecules; by renal clearance, these molecules exit the blood pool of small animals before imaging can be completed. Long circulation times that are appropriate for microimaging - in the order of tens of minutes - can be achieved by using nanoparticles that are large enough to evade rapid renal clearance (i.e. over 10 nm in diameter). The encapsulation of nanoparticles within polymers is also required to minimize their detection by the immune system, thus delaying hepatic clearance. Hence, the objective of our work was to develop a gadolinium-based contrast agent that will circulate long enough for pre-clinical computed tomography (CT) while maintaining blood pool detectability in the image (i.e. initial gadolinium content of around 100 mg/mL). We synthesized a contrast agent in the form of polymerencapsulated gadolinium nanoparticles by following a method that our group has reported. The nanoparticles in the contrast agent were characterized to have an average diameter of 110 ± 3 nm, and contains 94 ± 7 mg/mL of gadolinium. Our in vivo results in 2 mice show blood pool contrast enhancements of 220 ± 22 HU and circulates for up to an hour after tail vein injection. Given that the contrast agent stays in the blood pool of mice for up to an hour, the contrast agent has promising utility in pre-clinical vascular research

Blood-pool contrast agent for pre-clinical computed tomography

© 2017 SPIE. Advances in nanotechnology have led to the development of blood-pool contrast agents for micro-computed tomography (micro-CT). Although long-circulating nanoparticle-based agents exist for micro-CT, they are predominantly based on iodine, which has a low atomic number. Micro-CT contrast increases when using elements with higher atomic numbers (i.e. lanthanides), particularly at higher energies. The purpose of our work was to develop and evaluate a lanthanide-based blood-pool contrast agent that is suitable for in vivo micro-CT. We synthesized a contrast agent in the form of polymer-encapsulated Gd nanoparticles and evaluated its stability in vitro. The synthesized nanoparticles were shown to have an average diameter of 127 ± 6 nm, with good size dispersity. Particle size distribution - evaluated by dynamic light scattering over the period of two days - demonstrated no change in size of the contrast agent in water and saline. Additionally, our contrast agent was stable in a mouse serum mimic for up to 30 minutes. CT images of the synthesized contrast agent (containing 27 mg/mL of Gd) demonstrated an attenuation of over 1000 Hounsfield Units. This approach to synthesizing a Gd-based blood-pool contrast agent promises to enhance the capabilities of micro-CT imaging