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.

This paper presents a fully automatic segmentation system for whole-body high-frequency ultrasound (HFU) images of mouse embryos that can simultaneously segment the body contour and the brain ventricles (BVs). Our system first locates a region of interest (ROI), which covers the interior of the uterus, by sub-surface analysis. Then, it segments the ROI into BVs, the body, the amniotic fluid, and the uterine wall, using nested graph cut. Simultaneously multilevel thresholding is applied to the whole-body image to propose candidate BV components. These candidates are further truncated by the embryo mask (body+BVs) to refine the BV candidates. Finally, subsets of all candidate BVs are compared with pre-trained spring models describing valid BV structures, to identify true BV components. The system can segment the body accurately in most cases based on visual inspection, and achieves average Dice similarity coefficient of 0.8924 ± 0.043 for the BVs on 36 HFU image volumes.

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.

Real-time imaging of the embryonic murine cardiovascular system is challenging due to the small size of the mouse embryo and rapid heart rate. High-frequency, linear-array ultrasound systems designed for small-animal imaging provide high-frame-rate and Doppler modes but are limited in regards to the field of view that can be imaged at fine-temporal and -spatial resolution. Here, a plane-wave imaging method was used to obtain high-speed image data from in utero mouse embryos and multi-angle, vector-flow algorithms were applied to the data to provide information on blood flow patterns in major organs. An 18-MHz linear array was used to acquire plane-wave data at absolute frame rates >/=10 kHz using a set of fixed transmission angles. After beamforming, vector-flow processing and image compounding, effective frame rates were on the order of 2 kHz. Data were acquired from the embryonic liver, heart and umbilical cord. Vector-flow results clearly revealed the complex nature of blood-flow patterns in the embryo with fine-temporal and -spatial resolution.

PURPOSE: In this study, we evaluated a genetic approach for in vivo multimodal molecular imaging of vasculature in a mouse model of melanoma. PROCEDURES: We used a novel transgenic mouse, Ts-Biotag, that genetically biotinylates vascular endothelial cells. After inoculating these mice with B16 melanoma cells, we selectively targeted endothelial cells with (strept)avidinated contrast agents to achieve multimodal contrast enhancement of Tie2-expressing blood vessels during tumor progression. RESULTS: This genetic targeting system provided selective labeling of tumor vasculature and showed in vivo binding of avidinated probes with high specificity and sensitivity using microscopy, near infrared, ultrasound, and magnetic resonance imaging. We further demonstrated the feasibility of conducting longitudinal three-dimensional (3D) targeted imaging studies to dynamically assess changes in vascular Tie2 from early to advanced tumor stages. CONCLUSIONS: Our results validated the Ts-Biotag mouse as a multimodal targeted imaging system with the potential to provide spatio-temporal information about dynamic changes in vasculature during tumor progression.

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