Molecular Imaging Probes
A goal of the Meade lab is the development of magnetic resonance imaging (MRI) probes capable of reporting on anatomical and physiological function of cellular processes in whole animals. MRI has proven to be an effective modality for molecular imaging applications by providing excellent spatial and temporal resolution and unlimited depth of penetration without the use of ionizing radiation. The bioactivated Gd(III) complexes prepared in this research are designed to report on a biological event of interest and transduce this metabolic signal and/or cellular status as a change in MR contrast. Additionally, targeted Gd(III) probes are being prepared to report cellular status through signal enhancement as a function of receptor expression. Our lab addresses the inherent sensitivity issues associated with MRI by developing probes that increase cellular uptake, thereby maximizing Gd(III) loading using both molecular and nano-based approaches. Development of new MR probes addresses an unmet need for monitoring specific tissue types and environments relevant to dynamic biological processes.
Our approach begins with computation and synthesis and proceeds to biological assays, in vivo imaging and processing.
q-modulated MR Probes
Bioactivated q-modulated contrast agents alter the coordination environment of the Gd(III) center as a means to modulate contrast. These probes allow for real-time in vivo imaging of dynamic cellular processes such as gene expression and ion fluxes.
The Meade lab pioneered the development of q-modulated Gd(III) contrast agents with Egad. In the absence of the reporter protein β-galactosidas (β-gal), the pendant sugar blocks water access to the Gd(III) center. Hydrolysis of the glycosidic bond by β-gal results in a change in the coordination environment (q) resulting in enhanced image contrast. Several generations of enzyme-activated agents have been developed with increased changes in contrast and improved kinetics of activation.
Embryos were injected at the two-cell stage. Both cells received EgadMe; one cell which represents the right (R) side of the animal also received mRNA for β-gal. (A) GFP florescence image of a living embryo. (B) MR image of the same living embryo in (A) with signal from water made transparent. (C) same embryo stained for
The current generation of β-gal-activated agents incorporate a self-immolative prodrug electron cascade and a back-binding carboxylate. This design has two major advantages: 1) the self-immolative electron cascade allows for rapid kinetics of activation and 2) the back-binding moiety makes the agent q=0 thus the “off” state is darker.
Ion-activated agents: Zn(II) and Ca(II)
We have developed q-modulated Gd(III) contrast agents responding selectively and specifically to Zn(II) and Ca(II). Calcium is the most important intracellular secondary messenger in the body. Spatial and temporal detection of Ca(II) fluxes provides insight into many biological mechanisms. The Meade lab developed the first ion sensing q-modulated contrast agent. The Ca(II) agent is comprised of a aminopolycarboxylic acid Ca(II)-binding ligand known as BAPTA. In the absence of Ca(II), the acetate groups of BAPTA are believed to bind to the Gd(III) center, restricting water access. Ca(II) concentrations in the millimolar range are bound by the BAPTA domain resulting in an increase in MR signal.
Zn(II) is the second most abundant transition metal in the body with known structural and signaling roles, similar to Ca(II). A structurally optimized Zn(II) responsive agent has been developed to respond to changes in millimolar concentrations of Zn(II).
The receptor enhanced magnetization enhancement (RIME) effect is a common strategy to increase contrast in an MR image. RIME relies on the coupling of CA motion to a macromolecule or protein, thereby increasing its rotational correlation time; lengthening of this value at low magnetic fields translates to an increase in image brightness. To this end, a haloalkane dehalogenase (HaloTag gene reporter protein) targeted MR probe was developed. HaloTag selectively and irreversibly binds this agent’s distal haloalkane, enabling MR molecular imaging of gene expression with high specificity and sensitivity.
Determination of progesterone (PR) status in hormone-dependent diseases such as ovarian and breast cancer is essential for assessing disease states and subsequent decisions regarding treatment. The Meade lab is developing noninvasive PR-targeted MR probes to monitor PR status, precluding the need for biopsy. The first generation (ProGlo) of this agent showed selective accumulation in PR+ tumors, but was limited due to its low solubility. We are currently working towards water-soluble agents that maintain the same selective accumulation.
A series of lipophilic MR CAs have been developed for cell-tracking in vivo by labeling the cells ex vivo. Rationale design of these agents incorporates the fatty acids commonly incorporated in cellular membranes conjugated to a Gd-DO3A chelate. These new agents exhibit significantly enhanced labeling and retention in HeLa and MDA-MB-231-mcherry cells compared to agents that are internalized by cells.
Multimeric MR-optical CAs
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.
Signal Amplification using Nanotechnology
Nanotechnology provides a diverse set of multifunctional platforms for the development of MR contrast agents. The Meade lab uses two biologically-compatible nanoconstructs based on either carbon or gold. These constructs allow for high Gd(III) loading in addition to targeting moieties.
13 nm Gd(III)-enriched DNA-Au nanoparticle (AuNP) conjugates represent a new class of MR CAs that demonstrate efficient cell penetration and accumulation allowing for imaging small cell populations with μM Gd(III). These AuNPs also bear a pendant fluorophore that allows for histological analysis.
In collaboration with the Odom group at Northwestern we are developing Gd(III)-DNA Au Nanostars. Using the same synthetic scheme as the spherical AuNPs, these faceted particles enable large second-sphere water contributions to further enhance contrast efficiency.
Gd(III) associated with graphene relaxes water proton spins at an effectiveness that approaches or exceeds the theoretical limit for a single bound water molecule. These Gd(III)-labeled materials represent a potential breakthrough in sensitivity for Gd(III)-based contrast agents used in MRI. A library of Gd(III) chelates have been evaluated for their relaxivity and cellular uptake properties.
Gd(III)-nanodiamond conjugates have been prepared and exhibit a 10-fold increase in relaxivity relative to the corresponding molecular species. Nanodiamonds provide an easily functionalized material with tunable therapeutic loading and release profiles.
Visualization and Volume Rendering of Images
The images generated using these probes are displayed and interrogated in the Center for Advanced Molecular Imaging adjacent to our laboratory (http://cami.northwestern.edu/). The 3D “Wall” is based on graphics Processing Units (GPUs) that were originally designed for the sole purpose of rendering computer generated imagery onto a digital display. Over the last decade, however, these increasingly powerful processors have evolved to solve problems they were not originally intended to solve. A growing number of commercial software products, including the digital artist tools used by Northwestern Visualization to generate animated movies, now integrate these GPUs into their workflow. Faculty, graduate students, and postdoctoral fellows have at their disposal an immersive, highly-interactive means of visually interrogating their data while simultaneously serving as a medium on which to present their three-dimensional data to colleagues, students, and the general public.