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

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

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)

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

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

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

Nanodiscs as a Modular Platform for Multimodal MR-Optical Imaging

Nanodiscs are monodisperse, self-assembled discoidal particles that consist of a lipid bilayer encircled by membrane scaffold proteins (MSP). Nanodiscs have been used to solubilize membrane proteins for structural and functional studies and deliver therapeutic phospholipids. Herein, we report on tetramethylrhodamine (TMR) tagged nanodiscs that solubilize lipophilic MR contrast agents for generation of multimodal nanoparticles for cellular imaging. We incorporate both multimeric and monomeric Gd­(III)-based contrast agents into nanodiscs and show that particles containing the monomeric agent (<b>ND2</b>) label cells with high efficiency and generate significant image contrast at 7 T compared to nanodiscs containing the multimeric agent (<b>ND1</b>) and Prohance, a clinically approved contrast agent

Graphene Oxide Enhances Cellular Delivery of Hydrophilic Small Molecules by Co-incubation

The delivery of bioactive molecules into cells has broad applications in biology and medicine. Polymer-modified graphene oxide (GO) has recently emerged as a <i>de facto</i> noncovalent vehicle for hydrophobic drugs. Here, we investigate a different approach using native GO to deliver hydrophilic molecules by co-incubation in culture. GO adsorption and delivery were systematically studied with a library of 15 molecules synthesized with Gd(III) labels to enable quantitation. Amines were revealed to be a key chemical group for adsorption, while delivery was shown to be quantitatively predictable by molecular adsorption, GO sedimentation, and GO size. GO co-incubation was shown to enhance delivery by up to 13-fold and allowed for a 100-fold increase in molecular incubation concentration compared to the alternative of nanoconjugation. When tested in the application of Gd(III) cellular MRI, these advantages led to a nearly 10-fold improvement in sensitivity over the state-of-the-art. GO co-incubation is an effective method of cellular delivery that is easily adoptable by researchers across all fields

Nanodiamond–Gadolinium(III) Aggregates for Tracking Cancer Growth In Vivo at High Field

The ability to track labeled cancer cells in vivo would allow researchers to study their distribution, growth, and metastatic potential within the intact organism. Magnetic resonance (MR) imaging is invaluable for tracking cancer cells in vivo as it benefits from high spatial resolution and the absence of ionizing radiation. However, many MR contrast agents (CAs) required to label cells either do not significantly accumulate in cells or are not biologically compatible for translational studies. We have developed carbon-based nanodiamond–gadolinium­(III) aggregates (NDG) for MR imaging that demonstrated remarkable properties for cell tracking in vivo. First, NDG had high relaxivity independent of field strength, a finding unprecedented for gadolinium­(III) [Gd­(III)]–nanoparticle conjugates. Second, NDG demonstrated a 300-fold increase in the cellular delivery of Gd­(III) compared to that of clinical Gd­(III) chelates without sacrificing biocompatibility. Further, we were able to monitor the tumor growth of NDG-labeled flank tumors by <i>T</i>₁- and <i>T</i>₂-weighted MR imaging for 26 days in vivo, longer than was reported for other MR CAs or nuclear agents. Finally, by utilizing quantitative maps of relaxation times, we were able to describe tumor morphology and heterogeneity (corroborated by histological analysis), which would not be possible with competing molecular imaging modalities

Gd(III)-Labeled Peptide Nanofibers for Reporting on Biomaterial Localization <i>in Vivo</i>

Bioactive supramolecular nanostructures are of great importance in regenerative medicine and the development of novel targeted therapies. In order to use supra­molecular chemistry to design such nanostructures, it is extremely important to track their fate <i>in vivo</i> through the use of molecular imaging strategies. Peptide amphiphiles (PAs) are known to generate a wide array of supramolecular nanostructures, and there is extensive literature on their use in areas such as tissue regeneration and therapies for disease. We report here on a series of PA molecules based on the well-established β-sheet amino acid sequence V₃A₃ conjugated to macrocyclic Gd(III) labels for magnetic resonance imaging (MRI). These conjugates were shown to form cylindrical supra­molecular assemblies using cryogenic transmission electron microscopy and small-angle X-ray scattering. Using nuclear magnetic relaxation dispersion analysis, we observed that thermal annealing of the nanostructures led to a decrease in water exchange lifetime (τ_m) of hundreds of nanoseconds only for molecules that self-assemble into nanofibers of high aspect ratio. We interpret this decrease to indicate more solvent exposure to the paramagnetic moiety on annealing, resulting in faster water exchange within angstroms of the macrocycle. We hypothesize that faster water exchange in the nanofiber-forming PAs arises from the dehydration and increase in packing density on annealing. Two of the self-assembling conjugates were selected for imaging PAs after intra­muscular injections of the PA C₁₆V₃A₃E₃-NH₂ in the <i>tibialis anterior</i> muscle of a murine model. Needle tracts were clearly discernible with MRI at 4 days post­injection. This work establishes Gd(III) macrocycle-conjugated peptide amphiphiles as effective tracking agents for peptide amphiphile materials <i>in vivo</i> over the timescale of days