Depolymerizing self-immolative polymeric lanthanide chelates for vascular imaging.

Medical imaging is widely used clinically and in research to understand disease progression and monitor responses to therapies. Vascular imaging enables the study of vascular disease and therapy, but exogenous contrast agents are generally needed to distinguish the vasculature from surrounding soft tissues. Lanthanide-based agents are commonly employed in MRI, but are also of growing interest for micro-CT, as the position of their k-edges allows them to provide enhanced contrast and also to be employed in dual-energy micro-CT, a technique that can distinguish contrast-enhanced blood vessels from tissues such as bone. Small molecule Gd3+ chelates are available, but are excreted too rapidly. At the same time, a lack of rapid clearance from the body for long-circulating agents presents toxicity concerns. To address these challenges, we describe here the use of self-immolative polymers for the development of new degradable chelates that depolymerize completely from end-to-end following the cleavage of a single end-cap from the polymer terminus. We demonstrate that tuning the end-cap allows the rate of depolymerization to be controlled, while tuning the polymer length enables the polymer to exhibit long circulation times in the blood of mice. After successfully providing one hour of blood contrast, depolymerization led to excretion of the resulting small molecule chelates into the bladder. Despite the high doses required for micro-CT, the agents were well tolerated in mice. Thus, these self-immolative polymeric chelates provide a new platform for the development of medical imaging contrast agents. STATEMENT OF SIGNIFICANCE: Vascular imaging is used clinically to diagnose and monitor vascular disease and in research to understand the progression of disease and study responses to new therapies. For techniques such as magnetic resonance imaging and x-ray computed tomography (CT), long circulating contrast agents are needed to differentiate the vasculature from surrounding tissues. However, if these agents are not rapidly excreted from the body, they can lead to toxicity. We present here a new polymeric system that can chelate hundreds of lanthanide ions for imaging contrast and can circulate for one hour in the blood, but then after end-cap cleavage breaks down completely into small molecules for excretion. The successful application of this system in micro-CT in mice is demonstrated.

PEG-modified gadolinium nanoparticles as contrast agents for in vivo micro-CT.

Vascular research is largely performed in rodents with the goal of developing treatments for human disease. Micro-computed tomography (micro-CT) provides non-destructive three-dimensional imaging that can be used to study the vasculature of rodents. However, to distinguish vasculature from other soft tissues, long-circulating contrast agents are required. In this study, we demonstrated that poly(ethylene glycol) (PEG)-coated gadolinium nanoparticles can be used as a vascular contrast agent in micro-CT. The coated particles could be lyophilized and then redispersed in an aqueous solution to achieve 100 mg/mL of gadolinium. After an intravenous injection of the contrast agent into mice, micro-CT scans showed blood pool contrast enhancements of at least 200 HU for 30 min. Imaging and quantitative analysis of gadolinium in tissues showed the presence of contrast agent in clearance organs including the liver and spleen and very low amounts in other organs. In vitro cell culture experiments, subcutaneous injections, and analysis of mouse body weight suggested that the agents exhibited low toxicity. Histological analysis of tissues 5 days after injection of the contrast agent showed cytotoxicity in the spleen, but no abnormalities were observed in the liver, lungs, kidneys, and bladder.

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

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.

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, preclinical 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 particles 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.

Size-Dependent Gold Nanoparticle Interaction at Nano-Micro Interface Using Both Monolayer and Multilayer (Tissue-Like) Cell Models.

Gold nanoparticles (GNPs) are emerging as a novel tool to improve existing cancer therapeutics. GNPs are being used as radiation dose enhancers in radiation therapy as well as anticancer drugs carriers in chemotherapy. However, the success of GNP-based therapeutics depends on their ability to penetrate tumor tissue. GNPs of 20 and 50 nm diameters were used to elucidate the effects of size on the GNP interaction with tumor cells at monolayer and multilayer level. At monolayer cell level, smaller NPs had a lower uptake compared to larger NPs at monolayer cell level. However, the order was reversed at tissue-like multilayer level. The smaller NPs penetrated better compared to larger NPs in tissue-like materials. Based on our study using tissue-like materials, we can predict that the smaller NPs are better for future therapeutics due to their greater penetration in tumor tissue once leaving the leaky blood vessels. In this study, tissue-like multilayer cellular structures (MLCs) were grown to model the post-vascular tumor environment. The MLCs exhibited a much more extensive extracellular matrix than monolayer cell cultures. The MLC model can be used to optimize the nano-micro interface at tissue level before moving into animal models. This would accelerate the use of NPs in future cancer therapeutics.

Elucidating the Uptake and Distribution of Nanoparticles in Solid Tumors via a Multilayered Cell Culture Model.

Multicellular layers (MCLs) have previously been used to determine the pharmacokinetics of a variety of different cancer drugs including paclitaxel, doxorubicin, methotrexate, and 5-fluorouracil across a number of cell lines. It is not known how nanoparticles (NPs) navigate through the tumor microenvironment once they leave the tumor blood vessel. In this study, we used the MCL model to study the uptake and penetration dynamics of NPs. Gold nanoparticles (GNPs) were used as a model system to map the NP distribution within tissue-like structures. Our results show that NP uptake and transport are dependent on the tumor cell type. MDA-MB-231 tissue showed deeper penetration of GNPs as compared to MCF-7 one. Intracellular and extracellular distributions of NPs were mapped using CytoViva imaging. The ability of MCLs to mimic tumor tissue characteristics makes them a useful tool in assessing the efficacy of particle distribution in solid tumors.

ROC analysis in patient specific quality assurance.

PURPOSE: This work investigates the use of receiver operating characteristic (ROC) methods in patient specific IMRT quality assurance (QA) in order to determine unbiased methods to set threshold criteria for γ-distance to agreement measurements. METHODS: A group of 17 prostate plans was delivered as planned while a second group of 17 prostate plans was modified with the introduction of random multileaf collimator (MLC) position errors that are normally distributed with σ ≈ ± 0.5, ± 1.0, ± 2.0, and ± 3.0 mm (a total of 68 modified plans were created). All plans were evaluated using five different γ-criteria. ROC methodology was applied by quantifying the fraction of modified plans reported as "fail" and unmodified plans reported as "pass." RESULTS: γ-based criteria were able to attain nearly 100% sensitivity/specificity in the detection of large random errors (σ > 3 mm). Sensitivity and specificity decrease rapidly for all γ-criteria as the size of error to be detected decreases below 2 mm. Predictive power is null with all criteria used in the detection of small MLC errors (σ < 0.5 mm). Optimal threshold values were established by determining which criteria maximized sensitivity and specificity. For 3%/3 mm γ-criteria, optimal threshold values range from 92% to 99%, whereas for 2%/2 mm, the range was from 77% to 94%. CONCLUSIONS: The optimal threshold values that were determined represent a maximized test sensitivity and specificity and are not subject to any user bias. When applied to the datasets that we studied, our results suggest the use of patient specific QA as a safety tool that can effectively prevent large errors (e.g., σ > 3 mm) as opposed to a tool to improve the quality of IMRT delivery.