We have developed a modular architecture for preparing high-relaxivity multiplexed probes utilizing click chemistry. Our system incorporates azide bearing Gd(III) chelates and a trialkyne scaffold with a functional group for subsequent modification. In optimizing the relaxivity of this new complex we undertook a study of the linker length between a chelate and the scaffold to determine its effect on relaxivity. The results show a strong dependence on flexibility between the individual chelates and the scaffold with decreasing linker length leading to significant increases in relaxivity. Nuclear magnetic resonance dispersion (NMRD) spectra were obtained to confirm a tenfold increase in the rotational correlation time from 0.049 ns to 0.60 ns at 310 K. We have additionally obtained a crystal structure demonstrating that modification with an azide does not impact the coordination of the lanthanide. The resulting multinuclear center has a 500% increase in per Gd (or ionic) relaxivity at 1.41 T versus small molecule contrast agents and a 170% increase in relaxivity at 9.4 T.

Multiple imaging modalities are often required for in vivo imaging applications that require both high probe sensitivity and excellent spatial and temporal resolution. In particular, MR and optical imaging are an attractive combination that can be used to determine both molecular and anatomical information. Herein, we describe the synthesis and in vivo testing of two multimeric NIR–MR contrast agents that contain three Gd(III) chelates and an IR-783 dye moiety. One agent contains a PEG linker and the other a short alkyl linker. These agents label cells with extraordinary efficacy and can be detected in vivo using both imaging modalities. Biodistribution of the PEGylated agent shows observable fluorescence in xenograft MCF7 tumors and renal clearance by MR imaging.

We describe the design, synthesis and in vitro evaluation of a multimodal and multimeric contrast agent. The agent consists of three macrocyclic Gd(III) chelates conjugated to a fluorophore and possesses high relaxivity, water solubility, and is nontoxic. The modular synthesis is amenable for the incorporation of a variety of fluorophores to generate molecular constructs for a number of applications.

Our goal was to develop strategies to quantify the accumulation of model therapeutics in small brain metastases using multimodal imaging, in order to enhance the potential for successful treatment. Human melanoma cells were injected into the left cardiac ventricle of immunodeficient mice. Bioluminescent, MR and PET imaging were applied to evaluate the limits of detection and potential for contrast agent extravasation in small brain metastases. A pharmacokinetic model was applied to estimate vascular permeability. Bioluminescent imaging after injecting D-Luciferin (molecular weight (MW) 320D) suggested tumor cell extravasation had already occurred at week 1, which was confirmed by histology. 7T T1w MRI at week 4 was able to detect non-leaky 100 μm sized lesions and leaky tumors with diameters down to 200 μm after contrast injection at week 5. PET imaging showed that 18F-FLT (MW 244D) accumulated in the brain at week 4. Gadolinium-based MRI tracers (MW 559D and 2.066kD) extravasated after 5 weeks (tumor diameter 600 μm), and the lower MW agent cleared more rapidly from the tumor (mean apparent permeabilities 2.27×10-5 cm/s versus 1.12×10-5 cm/s). PET imaging further demonstrated tumor permeability to 64Cu-BSA (MW 65.55kD) at week 6 (tumor diameter 700 μm). In conclusion, high field T1w MRI without contrast may improve the detection limit of small brain metastases, allowing for earlier diagnosis of patients, although the smallest lesions detected with T1w MRI were permeable only to D-Luciferin and the amphipathic small molecule 18F-FLT. Different-sized MR and PET contrast agents demonstrated the gradual increase in leakiness of the blood tumor barrier during metastatic progression, which could guide clinicians in choosing tailored treatment strategies.

Purpose To demonstrate a new MR imaging approach that unambiguously identifies and quantitates contrast agents based on intrinsic agent properties such as r1, r2, r2*, and magnetic susceptibility. The approach is referred to as Magnetic Barcode Imaging (MBI). Methods Targeted and bio-responsive contrast agents were imaged in agarose phantoms to generate T1, T2, T2*, and quantitative susceptibility maps. The parameter maps were processed by a machine learning algorithm trained to recognize the contrast agents based on these parameters. The output is a quantitative map of contrast agent concentration, identity, and functional state. Results MBI allowed the quantitative interpretation of intensities, removed confounding backgrounds, enabled contrast agent multiplexing, and unambiguously detected the activation and binding states of bio-responsive and targeted contrast agents. Conclusion MBI has the potential to overcome significant limitations in the interpretation, quantitation, and multiplexing of contrast enhancement by MR imaging probes.

As part of a long-range effort involving the design and fabrication of electrodes for in vivo use, we describe herein a method for the production of conducting polymer fibers with diameters on the order of a few micrometers. This method has been shown to be applicable to several different combinations of monomers ͓pyrrole, N-methylpyrrole, 3-methylthiophene, poly͑3,4-ethylene dioxythiophene͔͒, deposition substrates ͑Pt, stainless steel͒, and dopant ions ͑dodecyl benzene sulfonate, perchlorate, chloride, polymethylmethacrylate͒. Cyclic voltammetric characterization shows that the background current is minimal, and normal behavior is seen for the ferri/ferrocyanide couple with the exception of a slightly increased peak separation. An ancillary benefit of these studies is the production of extremely smooth films of these polymers, even in cases where the films are up to 20 m thick.

Introduction: Efficient drug delivery to brain metastases is difficult due to an often intact blood-brain barrier (BBB) around smaller lesions, and very few therapeutic drugs penetrate an intact BBB. Relevant experimental brain metastatic models are needed to study biological mechanisms and therapeutic responses of early brain lesions in relation to the BBB. Previous studies injecting small molecules such as sodium fluorescein (MW 376D) have indicated that the BBB is disrupted in experimental brain metastasis larger than 0.25 mm (1). However, little is known regarding at which time point in experimental brain metastasis development the BBB is disrupted, and to what extent larger sized molecules at that time are able to penetrate tumor tissue. We addressed these issues by performing multimodal imaging studies, after injecting contrast agents of various sizes into an established melanoma brain metastasis model (2, 3). Materials and Methods: All studies were approved by the UCD Animal Care and Use Committee. As in (3), we injected 5X105 human melanoma brain metastasis cells harboring the Luciferase and GFP genes, intracardially into 32 NOD/SCID mice. All animals were followed by bioluminescence imaging (BLI) weekly for 6 weeks. MRI (Bruker Biospec 70/30, RARE sequence, RARE factor=2, TE/TR/ST=9 ms/750 ms/1mm, FOV=2x2 cm, Matrix=256x256, NA=4) was performed at weeks 3, 4, 5 and 6 after tumor cell inoculation, before and after i.v. injections of either Gd-HPDO3A (Prohance, MW 559D, 0.5 μmol/g, 20 mice) or a newly synthetized Gadolinium contrast agent with 3 Gd(III) chelates, termed C3, (MW 2kD, 0.167 μmol/g, 5 mice). 64Cu-Albumin (MW 66.5kD) and 18F-FLT (MW 244D) was also injected i.v. prior to PET imaging at weeks 4 and 6. Results and Discussion: BLI visualized tumor cell spread in all animal brains 15mins after injections, and a gradual increase in tumor burden between weeks 1 to 6. T1w MRI at week 3 did not show any tumors, however BLI confirmed presence of metastasis. MRI detected tumors at week 4, and the mean, total number of tumors increased from 26 tumors at week 4, to 89 tumors at week 6. There was an exponential increase in tumor burden from week 4 to 6. The number of leaky tumors also increased exponentially, with mean diameters increasing from around 600 μm to around 700 μm. The smallest leaky tumors detectable by MRI were ~200 μm (week 5). The number of non-leaky tumors peaked at 5 weeks, and their mean diameters increased from 260 μm to 450 μm. Metastatic tumor cell proliferation was shown by 18F-FLT, which accumulated in the animal brains at weeks 4 and 6. 64Cu-Albumin PET also showed leakage of albumin into the metastatic lesions at week 6. Conclusions: Our study shows that T1w 7T MRI can detect leaky brain metastases down to ~200 μm in diameter. Molecules up to 2kD in size may penetrate brain tumor tissue early in tumor development. Our PET study indicates that larger molecules such as albumin may leak out of the metastatic lesions later in tumor development. References (1) Fidler IJ et al. Lancet Oncol 3:53-57, 2002. (2) Sundstrøm T et al, Cancer Res 2012 (In Press). (3) Wang J et al. Neuropathol Appl Neurobiol 37:189-205, 2011. Citation Format: Frits Thorsen, Brett Fite, Lisa Mahakian, Victoria Harrison, Sarah Johnson, Elizabeth Ingham, Shengping Qin, Jai W. Seo, Thomas Meade, Terje Sundstrøm, Katherine W. Ferrara. Multimodal imaging of blood-brain barrier disruption during brain metastatic progression in a relevant experimental mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A39.