Faculty Bibliography

Progress in clinical development of magnetic resonance imaging (MRI) substrate-sensors of enzymatic activity has been slow partly due to the lack of human efficacy data. We report here a strategy that may serve as a shortcut from bench to bedside. We tested ultra high-resolution 7T MRI (µMRI) of human surgical histology sections in a 3-year IRB approved, HIPAA compliant study of surgically clipped brain aneurysms. µMRI was used for assessing the efficacy of MRI substrate-sensors that detect myeloperoxidase activity in inflammation. The efficacy of Gd-5HT-DOTAGA, a novel myeloperoxidase (MPO) imaging agent synthesized by using a highly stable gadolinium (III) chelate was tested both in tissue-like phantoms and in human samples. After treating histology sections with paramagnetic MPO substrate-sensors we observed relaxation time shortening and MPO activity-dependent MR signal enhancement. An increase of normalized MR signal generated by ultra-short echo time MR sequences was corroborated by MPO activity visualization by using a fluorescent MPO substrate. The results of µMRI of MPO activity associated with aneurysmal pathology and immunohistochemistry demonstrated active involvement of neutrophils and neutrophil NETs as a result of pro-inflammatory signalling in the vascular wall and in the perivascular space of brain aneurysms.

Manganese-enhanced MRI (MRI) is a technique that allows for a noninvasive in vivo estimation of neuronal transport. It relies on the physicochemical properties of manganese, which is both a calcium analogue being transported along neurons by active transport, and a paramagnetic compound that can be detected on conventional T1-weighted images. Here, we report a multi-session MEMRI protocol that helps establish time-dependent curves relating to neuronal transport along the olfactory tract over several days. The characterization of these curves via unbiased fitting enables us to infer objectively a set of three parameters (the rate of manganese transport from the maximum slope, the peak intensity, and the time to peak intensity). These parameters, measured previously in wild type mice during normal aging, have served as a baseline to demonstrate their significant sensitivity to pathogenic processes associated with Tau pathology. Importantly, the evaluation of these three parameters and their use as indicators can be extended to monitor any normal and pathogenic processes where neuronal transport is altered. This approach can be applied to characterize and quantify the effect of any neurological disease conditions on neuronal transport in animal models, together with the efficacy of potential therapies.

Introduction Theranostics, combining diagnostic imaging with drug therapy, is promising in cancer treatment. Protein engineering may prove beneficial in theranostics development, allowing for the synthesis of rationally designed therapeutic platforms combinable with imaging probes. Here, we have engineered a theranostic agent entirely reliant on protein structure for drug binding and iron oxide (FeOx) organization and templation. FeOx is an appealing MRI probe because it is highly sensitive and well tolerated in vivo. We therefore propose that FeOx-decorated protein fibers should serve as a sensitive and biocompatible agent for tracking chemotherapeutic delivery via MRI. Our agent is based on self-assembling azide-functionalized fibers composed of a coiled-coiled protein1 with a hydrophobic pore for binding small molecules, such as anti-neoplastic curcumin. The azide moieties allow for functionalization including conjugation to an FeOx-templating peptide derived from the C-terminus of the magnetosome-associated protein Mms6 (dubbed CMms6)2. Methods Synthesis: Azide-functionalized protein was expressed in methionine auxotrophic E. coli using azidohomoalanine supplemented media. Pure protein was dialyzed into acidic pH initiating the assembly of nanofibers subsequently bound to curcumin1 forming fluorescent mesofibers then crosslinked for stability. Nanofibers and mesofibers were visualized with transmission electron microscopy (TEM) and fluorescence microscopy, respectively. Mesofibers were conjugated to alkyne-CMms6 via azide-alkyne cycloaddition and iron co-precipitation in the presence of CMms6- bound fibers yielded templated FeOx nanoparticles. Imaging: MR images and relaxometry were acquired at 7 Tesla on FeOx-bound fibers diluted in 2% agarose. 2D relaxation maps were acquired using a rapid acquisition with relaxation enhancement with variable repetition time (RAREVTR) sequence. Data was fit using T1 and T2 mono-exponential curves to determine relaxivity. T1 , T2 , and T2 *-weighted images were acquired using Look-Locker, multi-spin multi-echo, and multi-gradient echo sequences, respectively. Results TEM and fluorescence microscopy revealed fibers with an average diameter of 200nm (N=20) and 24mum (N=28) before and after curcumin binding. TEM analysis of FeOx-bound fibers revealed nanoparticles 6.6nm in diameter (N =20). A neodymium magnet confirmed the agent's magnetic responsiveness, to be further investigated by superconducting quantum interference device (SQUID) magnetometry. Relaxometry revealed a 5.7-fold higher r21 than clinical FeOx agent Feraheme, suggesting its use as a pure T2/T2* agent. Subsequent MRI confirmed the agent's potential in T2/T2 *-weighted imaging in comparison to its weak signal change when T1 -weighted. Conclusion By biologically synthesizing a coiled-coil protein and utilizing bio-inspired FeOx templation, we engineered a magnetically-functionalized drug-carrying vehicle. Initial studies suggest that this agent may have potential in magnetically-driven or implantable drug delivery visualized viaT2/T2 *-weighted MRI. (Figure Presented)

Introduction Gadolinium (Gd)-based MRI contrast agents (GBCA) are crucial for the diagnosis and monitoring of various diseases. Their use in dynamic, such as perfusion MRI (pMRI), scans provides valuable information about tissue microenvironment and function beyond conventional assessment of lesion volume or area changes^1,2. However, accurate concentration-time curves² are required to quantitatively characterize diseased tissue, monitor therapeutic efficacy, and provide insight into drug mechanisms¹. The increasing reliance on image-derived GBCA quantification is problematic as indirect signal measurement from surrounding proton exchange can be affected by non-linear relaxivity in tissue³, hardware imperfection, and motion. Direct GBCA quantification in plasma samples varies in sensitivity by method. Relaxivity achieves sub-millimolar sensitivity, requiring large sample volume, and although ICP-MS can detect nanomolar levels⁴, it is not readily available. We previously developed a technique to quantify a GBCA using Carbostyril 124 (cs124)-sensitized DTPA via energy transfer using a simple fluorescence plate reader widely available in research labs⁵. The present work extends our method to multivalent Gd complexes (see image). Methods All absorbance and fluorescence measurements were made in a standard plate reader. Amide chemistry was used to conjugate cs124 to DTPA-DPPE and Gd was chelated in water, yielding cs124-GdDTPA-DPPE. The absorption of cs124 at 330nm was used to quantify Gd. The product was dialyzed and the size distribution was assessed via dynamic light scattering (DLS). Time-resolved fluorescence, using excitation at 330nm and emission at 480nm, was integrated from 600mus-2000mus. The collision model of energy transfer was fit to fluorescence data to determine system sensitivity⁵. MRI: T1 images and relaxometry were acquired on a 7-Telsa Bruker using phantoms of cs124-GdDTPADPPE particles and clinical Gd-DTPA (Magnevist). Results DLS showed a monodisperse distribution with an average 575nm radius (Fig.1), consistent with liposomal structure expected from this lipid. The starting Gd concentration used for fluorescence studies was 35muM (Fig.2). The limit of detection was 67nM, which is comparable to ICP-MS⁴ (Fig. 3). T1-weighted MRI showed that our multivalent cs124-based T1-agent achieved a signal comparable to that of 10x higher concentration monovalent GdDTPA concentration (Fig.4). Relaxometry confirmed a 26.5x amplification in r1 and a 1.04 r2/r1 ratio (Table 1). Conclusion Incorporating cs124 into multivalent GBCAs proved effective in maintaining nanomolar quantification of Gd using a spectrophotometer. The cs124 conjugation did not hinder the agent's r1 relaxivity, suggesting its use in other novel GBCA of various sizes. This expedient system may significantly ease in-house throughput for MR agents' characterization and optimization. It may also prove useful to calibrate the arterial input function in pMRI studies. [IMAGE PRESENTED]

Introduction Theranostics is the field whereby both drug delivery and imaging are merged to promote more effective therapies¹. This is especially important in the delivery of small molecule cancer therapeutics such as doxorubicin (DOX) where clinicians must strike a balance between administering an effective dose while limiting off-target cardiotoxicity risks². In this work, an engineered fluorinated protein polymer is investigated for theranostic use as a chemotherapeutic carrier and ¹⁹F MR agent. The fluorinated protein polymer, CE2-RGD-TFL, is comprised of two functional domains: 1) a coiled-coil domain (C), flanked by two integrin targeting domains, capable of encapsulating small hydrophobic drugs and; 2) two elastin-like peptide domains (E) that impart concentration-dependent thermoresponsiveness. In this study, we have characterized CE2-RGD-TFL as a promising thermally T2-dependent MRI tracer as well as thermoresponsive to drug release for DOX delivery properties. Materials and Methods Trifluoroleucine (TFL) incorporation was achieved via recombinant expression in leucine auxotrophic E. coli. Proteins were subjected to UV-Vis spectroscopy, circular dichroism (CD), dynamic light scattering (DLS) and ¹⁹F R1 (1/T1) and R2 (1/T2) NMR relaxometry at 11.7-Tesla (-T). Water Phantoms with and without CE2-RGD-TFL were imaged using a 7-T Bruker micro-MRI using a homemade broadband coil tunable to ¹⁹F or ¹H. DOX-bound protein was separated from free drug by size exclusion chromatography (SEC). Results Fluorination imparts interesting thermoresponsive properties upon CE2-RGD. While the CD analysis reveals that CE2-RGD-TFL is less structured than the wild-type variant, CE2-RGD-TFL coacervates in the physiological range for hyperthermic treatment (39-42degreeC). Relaxometric characterization revealed a remarkable R2 linear dependence as a function of concentrations and temperatures and very little change in R1 (Fig. 1, plots). The predominant R2 linear sensitivity based on relative r2 /r1 ratio within physiological temperature range supports the prospect for using this protein as a T2-nano-thermometer. MRI water Phantoms with and without CE2-RGD-TFL were expectedly visible with 1H MRI while only CE2-RGD-TFL was visualized in 19F imaging. Importantly, CE2-RGD-TFL exhibited a 2.75 times increase in DOX loading (49.1%) compared to CE2-RGD with only 17.8% loading. Conclusion Incorporation of TFL in CE2-RGD yields a drug carrier nanoparticle that can undergo temperature dependent structural changes associated with R2 linear variation, making this compound a great potential candidate as a nanothermometric tracer. Furthermore, preliminary ¹⁹F MRI confirmed our ability to visualize CE²-RGD-TFL. Finally, fluorination imparts both greater thermoresponsiveness and greater loading of doxorubicin which provides further value to CE2-RGD-TFL as a theranostic agent. Future work includes ultra-short echo time MRI to increase sensitivity and testing of the targeting capability to integrin +/- breast cell lines for effective DOX delivery and release upon hyperthermic induction

Solid tumor microstructure is related to the aggressiveness of the tumor, interstitial pressure and drug delivery pathways, which are closely associated with treatment response, metastatic spread and prognosis. In this study, we introduce a novel diffusion MRI data analysis framework, pulsed and oscillating gradient MRI for assessment of cell size and extracellular space (POMACE), and demonstrate its feasibility in a mouse tumor model. In vivo and ex vivo POMACE experiments were performed on mice bearing the GL261 murine glioma model (n = 8). Since the complete diffusion time dependence is in general non-analytical, the tumor microstructure was modeled in an appropriate time/frequency regime by impermeable spheres (radius Rcell , intracellular diffusivity Dics ) surrounded by extracellular space (ECS) (approximated by constant apparent diffusivity Decs in volume fraction ECS). POMACE parametric maps (ECS, Rcell , Dics , Decs ) were compared with conventional diffusion-weighted imaging metrics, electron microscopy (EM), alternative ECS determination based on effective medium theory (EMT), and optical microscopy performed on the same samples. It was shown that Decs can be approximated by its long time tortuosity limit in the range [1/(88 Hz)-31 ms]. ECS estimations (44 +/- 7% in vivo and 54 +/- 11% ex vivo) were in agreement with EMT-based ECS and literature on brain gliomas. Ex vivo, ECS maps correlated well with optical microscopy. Cell sizes (Rcell = 4.8 +/- 1.3 in vivo and 4.3 +/- 1.4 microm ex vivo) were consistent with EM measurements (4.7 +/- 1.8 microm). In conclusion, Rcell and ECS can be quantified and mapped in vivo and ex vivo in brain tumors using the proposed POMACE method. Our experimental results support the view that POMACE provides a way to interpret the frequency or time dependence of the diffusion coefficient in tumors in terms of objective biophysical parameters of neuronal tissue, which can be used for non-invasive monitoring of preclinical cancer studies and treatment efficacy

Introduction Abnormal changes in the neurovascular architecture are associated with numerous conditions including tumors, Alzheimer's disease, and diabetes. Pre-clinical mouse models are invaluable in our understanding, diagnosis and treatment of such conditions, but we have yet to see a longitudinal assessment of neurovascular changes in wild type (WT) control mice. Contrast enhanced-magnetic resonance angiography (CE-MRA) utilizes an exogenous contrast agent to study neurovasculature clinically and pre-clinically with little hemodynamic-dependence. Here, we implemented 3D in vivo CE-MRA to longitudinally study the neurovasculature of aging WT mice. This study provides insight into the normal aging process in WT mice and could serve as a baseline for future studies of neurovascular disease models. Materials and Methods Gadolinium (Gd)-bound micelles were synthesized to serve as a blood pool agent via a previously described thin-film method¹ combining Gd-DPTA, polyethylene glycol, and Rhodamine B-bound lipids. Assessment of size, relaxivity, and plasma half-life confirmed the imaging potential of this compound. Gd-micelles were administered via femoral injection into female WT C57BL/6 mice; the most widely used inbred strain for models of human disease². Twentyseven micelle-administered mice were imaged between ages 2-to-26 months (mo). A subset of mice was aged and imaged at 2-4mo, 14-16mo, and 24-26mo to assess variability in neurovascular changes of individual mice. Angiograms were acquired on a 7-Tesla Bruker micro-MRI system with an 87-minute (100mum) ³ isotropic resolution scan. Neurovascular analysis was applied to anatomically identifiable regions following brain alignment with software by the Mouse Imaging Center (Toronto, Canada)3 and tools developed by the Montreal Neurological Institute (Montreal, Canada). Neurovascular changes were quantified using intensity-based vascular thresholding and segmentation (see figure). Results Quantification of the whole brain showed a significant decrease in detectable neurovasculature between the 2-4mo and 14-16mo groups (p<0.01, one-way ANOVA plus Bonferroni test). A reduction was again seen in the second year of aging. We also quantified the neurovascular changes of the cortex, circle of willis, and sagittal midline and found a significant reduction during the first year of aging and further reduction in the second year. However, the hippocampus showed no significant neurovascular changes. Conclusion Gd micelle-enhanced MRA allowed for the detection of an overall decline in the neurovascular volume of aging C57BL/6 mice. To our knowledge, this is the first report of an age-dependent neurovascular reduction in longitudinally monitored WT animals. These unexpected results stress the need to establish a baseline using control animals of the same background when studying transgenic models of neurovascular diseases. Such reductions may also explain age-dependent changes in cerebral blood flow and function. (Figure Presented)

PURPOSE: To disentangle the free diffusivity (D0 ) and cellular membrane restrictions, by means of their surface-to-volume ratio (S/V), using the frequency-dependence of the diffusion coefficient D(omega), measured in brain tumors in the short diffusion-time regime using oscillating gradients (OGSE). METHODS: In vivo and ex vivo OGSE experiments were performed on mice bearing the GL261 murine glioma model (n = 10) to identify the relevant time/frequency (t/omega) domain where D(omega) linearly decreases with omega-1/2 . Parametric maps (S/V, D0 ) are compared with conventional DWI metrics. The impact of frequency range and temperature (20 degrees C versus 37 degrees C) on S/V and D0 is investigated ex vivo. RESULTS: The validity of the short diffusion-time regime is demonstrated in vivo and ex vivo. Ex vivo measurements confirm that the purely geometric restrictions embodied in S/V are independent from temperature and frequency range, while the temperature dependence of the free diffusivity D0 is similar to that of pure water. CONCLUSION: Our results suggest that D(omega) in the short diffusion-time regime can be used to uncouple the purely geometric restriction effect, such as S/V, from the intrinsic medium diffusivity properties, and provides a nonempirical and objective way to interpret frequency/time-dependent diffusion changes in tumors in terms of objective biophysical tissue parameters. Magn Reson Med, 2015. (c) 2015 Wiley Periodicals, Inc.

Expression of myeloperoxidase (MPO) activity in the blood vessel wall by cells participating in the innate immune response is a known indicator of downstream acute vascular events (heart attacks, strokes). MPO activity associates with increased risk of rupture of brain aneurysms in humans [1]. Consequently, non-invasive imaging of MPO activity using a clinically acceptable imaging substrate would greatly assist in performing differential diagnosis in patients facing potential risk of aneurysm rupture. We devised an imaging setup in which a novel paramagnetic probe based on highly water-soluble, negatively charged 5-hydroxytryptamide (5HT) of DOTAGAGd could be tested ex vivo in human aneurysmal tissue. In the presence of DOTAGAGd-5HT substrate and serum albumin, we observed an increase in molar longitudinal relaxivity of Gd (r1; 2.3 fold at 0.47 T) as a result of MPO-driven catalysis. In contrast to bis-tryptamides of DTPA [2], DOTAGAGd-based MPO substrate showed better solubility and a higher enzyme-mediated molar relaxivity increase due to the combined effect of a lower relaxivity of DOTAGA-chelated gadolinium (III) and lower binding to plasma proteins of mono-5HT substrates. Furthermore, we tested DOTAGAGd- 5HT for 1) mapping MPO activity in tissue regions using thick frozen histology sections and 2) corroborating micro- MRI (muMRI) images with ultrathin human sections examined using fluorescent MPO substrate (Cy3-5HT). Surgical samples of human saccular brain aneurysm clippings were obtained under the approved UMMS IRB protocol with patient consent (n=23). Frozen aneurysm sections (8 and 50 mum thick) were fixed with acetone. Red fluorescence of Cy3-5HT substrate was localized in several areas in the adventitia of the ruptured aneurysm section (R) as well as within the partially thrombosed lumina of human samples. Parallel thick non-consecutive sections were subjected to muMRI imaging following incubation with 0.5 mM DOTAGAGd-5HT and hydrogen peroxide. Direct muMRI of tissue sections was performed using a set of homebuilt histological coils tuned to operate in a 7T Bruker muMRI system interfaced to a 200 mm horizontal bore magnet [3]. The highly detailed muMR images (57 mum in-plane resolution) of thin tissue sections acquired in less than 8-hours revealed the presence of high focal T1w-enhancement corresponding to the blood vessel wall (R) and the plaque (UR) areas of the same sections. The set of tissue samples examined so far confirm positive correlation between normalized MPO enzymatic activity found in the tissue and the increased risk of developing a rupture within a 5-year period. Therefore, low molecular weight, highly stable DOTAGA-based chelates are promising molecular imaging probes for further clinical translation. The initial assessment of a potential link between brain aneurysm instability and vascular wall inflammation is further evidenced by our imaging setup enabling the direct muMRI analysis of human histology samples and the testing of novel MR enzyme-specific probes in situ. (Figure Presented)