Rational Design, Synthesis, and Biological Evaluation of Progesterone-Modified MRI Contrast Agents

SummaryA series of contrast agents for magnetic resonance imaging (MRI) aimed at noninvasively determining the hormone receptor status of cancer in vitro was developed. These MRI contrast agents were prepared by conjugating progesterone to clinically used Gd(III) chelates. These agents exhibited higher progesterone receptor binding affinities in the nanomolar range and intracellular accumulation. High logP values of the modified compounds suggested that the lipophilicity of the steroid conjugates may have contributed to membrane permeability. Synchrotron radiation X-ray fluorescence microscopy and magnetic resonance images revealed that the synthesized conjugates showed the greatest cellular accumulation and significant increase in relaxivity in vitro compared to the previously developed steroid-modified agent. Transcriptional assays using the progesterone response element linked to luciferase indicated that the contrast agents entered the cell, interacted with the biological target, and drove specific progesterone-mediated transcription

Quantitative Imaging of Cell-Permeable Magnetic Resonance Contrast Agents Using X-Ray Fluorescence

The inability to transduce cellular membranes is a limitation of current magnetic resonance imaging probes used in biologic and clinical settings. This constraint confines contrast agents to extracellular and vascular regions of the body, drastically reducing their viability for investigating processes and cycles in developmental biology. Conversely, a contrast agent with the ability to permeate cell membranes could be used in visualizing cell patterning, cell fate mapping, gene therapy, and, eventually, noninvasive cancer diagnosis. Therefore, we describe the synthesis and quantitative imaging of four contrast agents with the capability to cross cell membranes in sufficient quantity for detection. Each agent is based on the conjugation of a Gd(III) chelator with a cellular transduction moiety. Specifically, we coupled Gd(III)–diethylenetriaminepentaacetic acid DTPA and Gd(III)–1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid with an 8–amino acid polyarginine oligomer and an amphipathic stilbene molecule, 4-amino-4'-( N,N -dimethylamino)stilbene. The imaging modality that provided the best sensitivity and spatial resolution for direct detection of the contrast agents is synchrotron radiation x-ray fluorescence (SR-XRF). Unlike optical microscopy, SR-XRF provides two-dimensional images with resolution 10 3 better than 153 Gd gamma counting, without altering the agent by organic fluorophore conjugation. The transduction efficiency of the intracellular agents was evaluated by T 1 analysis and inductively coupled plasma mass spectrometry to determine the efficacy of each chelate-transporter combination

Gd(III)-Dithiolane Gold Nanoparticles for <i>T</i>₁‑Weighted Magnetic Resonance Imaging of the Pancreas

Pancreatic adenocarcinoma has a 5 year survival of approximately 3% and median survival of 6 months and is among the most dismal of prognoses in all of medicine. This poor prognosis is largely due to delayed diagnosis where patients remain asymptomatic until advanced disease is present. Therefore, techniques to allow early detection of pancreatic adenocarcinoma are desperately needed. Imaging of pancreatic tissue is notoriously difficult, and the development of new imaging techniques would impact our understanding of organ physiology and pathology with applications in disease diagnosis, staging, and longitudinal response to therapy in vivo. Magnetic resonance imaging (MRI) provides numerous advantages for these types of investigations; however, it is unable to delineate the pancreas due to low inherent contrast within this tissue type. To overcome this limitation, we have prepared a new Gd­(III) contrast agent that accumulates in the pancreas and provides significant contrast enhancement by MR imaging. We describe the synthesis and characterization of a new dithiolane-Gd­(III) complex and a straightforward and scalable approach for conjugation to a gold nanoparticle. We present data that show the nanoconjugates exhibit very high per particle values of <i>r</i>₁ relaxivity at both low and high magnetic field strengths due to the high Gd­(III) payload. We provide evidence of pancreatic tissue labeling that includes MR images, post-mortem biodistribution analysis, and pancreatic tissue evaluation of particle localization. Significant contrast enhancement was observed allowing clear identification of the pancreas with contrast-to-noise ratios exceeding 35:1

High Relaxivity Gd(III)–DNA Gold Nanostars: Investigation of Shape Effects on Proton Relaxation

Gadolinium(III) nanoconjugate contrast agents (CAs) have distinct advantages over their small-molecule counterparts in magnetic resonance imaging. In addition to increased Gd(III) payload, a significant improvement in proton relaxation efficiency, or relaxivity (<i>r</i>₁), is often observed. In this work, we describe the synthesis and characterization of a nanoconjugate CA created by covalent attachment of Gd(III) to thiolated DNA (Gd(III)–DNA), followed by surface conjugation onto gold nanostars (DNA–Gd@stars). These conjugates exhibit remarkable <i>r</i>₁ with values up to 98 mM^–1 s^–1. Additionally, DNA–Gd@stars show efficient Gd(III) delivery and biocompatibility <i>in vitro</i> and generate significant contrast enhancement when imaged at 7 T. Using nuclear magnetic relaxation dispersion analysis, we attribute the high performance of the DNA–Gd@stars to an increased contribution of second-sphere relaxivity compared to that of spherical CA equivalents (DNA–Gd@spheres). Importantly, the surface of the gold nanostar contains Gd(III)–DNA in regions of positive, negative, and neutral curvature. We hypothesize that the proton relaxation enhancement observed results from the presence of a unique hydrophilic environment produced by Gd(III)–DNA in these regions, which allows second-sphere water molecules to remain adjacent to Gd(III) ions for up to 10 times longer than diffusion. These results establish that particle shape and second-sphere relaxivity are important considerations in the design of Gd(III) nanoconjugate CAs

Multimeric Near IR–MR Contrast Agent for Multimodal <i>In Vivo</i> Imaging

Multiple imaging modalities are often required for <i>in vivo</i> 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 <i>in vivo</i> 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 <i>in vivo</i> using both imaging modalities. Biodistribution of the PEGylated agent shows observable fluorescence in xenograft MCF7 tumors and renal clearance by MR imaging

Cell Labeling via Membrane-Anchored Lipophilic MR Contrast Agents

Cell tracking <i>in vivo</i> with MR imaging requires the development of contrast agents with increased sensitivity that effectively label and are <i>retained</i> by cells. Most clinically approved Gd­(III)-based contrast agents require high incubation concentrations and prolonged incubation times for cellular internalization. Strategies to increase contrast agent permeability have included conjugating Gd­(III) complexes to cell penetrating peptides, nanoparticles, and small molecules which have greatly improved cell labeling but have not resulted in improved cellular retention. To overcome these challenges, we have synthesized a series of lipophilic Gd­(III)-based MR contrast agents that label cell membranes <i>in vitro</i>. Two of the agents were synthesized with a multiplexing strategy to contain three Gd­(III) chelates (<b>1</b> and <b>2</b>) while the third contains a single Gd­(III) chelate (<b>3</b>). 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 (<b>4</b> and Prohance)

Progesterone-Targeted Magnetic Resonance Imaging Probes

Determination of progesterone receptor (PR) status in hormone-dependent diseases is essential in ascertaining disease prognosis and monitoring treatment response. The development of a noninvasive means of monitoring these processes would have significant impact on early detection, cost, repeated measurements, and personalized treatment options. Magnetic resonance imaging (MRI) is widely recognized as a technique that can produce longitudinal studies, and PR-targeted MR probes may address a clinical problem by providing contrast enhancement that reports on PR status without biopsy. Commercially available MR contrast agents are typically delivered via intravenous injection, whereas steroids are administered subcutaneously. Whether the route of delivery is important for tissue accumulation of steroid-modified MRI contrast agents to PR-rich tissues is not known. To address this question, modification of the chemistry linking progesterone with the gadolinium chelate led to MR probes with increased water solubility and lower cellular toxicity and enabled administration through the blood. This attribute came at a cost through lower affinity for PR and decreased ability to cross the cell membrane, and ultimately it did not improve delivery of the PR-targeted MR probe to PR-rich tissues or tumors in vivo. Overall, these studies are important, as they demonstrate that targeted contrast agents require optimization of delivery and receptor binding of the steroid and the gadolinium chelate for optimal translation in vivo

Synthesis and Biological Evaluation of Water-Soluble Progesterone-Conjugated Probes for Magnetic Resonance Imaging of Hormone Related Cancers

Progesterone receptor (PR) is strongly associated with disease prognosis and therapeutic efficacy in hormone-related diseases such as endometriosis and breast, ovarian, and uterine cancers. Receptor status is currently determined by immunohistochemistry assays. However, noninvasive PR imaging agents could improve disease detection and help elucidate pathological molecular pathways, leading to new therapies and animal disease models. A series of water-soluble PR-targeted magnetic resonance imaging (MRI) probes were synthesized using Cu(I)-catalyzed click chemistry and evaluated <i>in vitro</i> and <i>in vivo</i>. These agents demonstrated activation of PR <i>in vitro</i> and preferential accumulation in PR(+) compared to PR(-) human breast cancer cells with low toxicity. In xenograft tumor models, the agents demonstrated enhanced signal intensity in PR(+) tumors compared to PR(-) tumors. The results suggest that these agents may be promising MRI probes for PR(+) diseases