Determination of metal ion transport rate of human ZIP4 using stable zinc isotopes.

The essential microelement zinc is absorbed in the small intestine mainly by the zinc transporter ZIP4, a representative member of the Zrt/Irt-like protein (ZIP) family. ZIP4 is reportedly upregulated in many cancers, making it a promising oncology drug target. To date, there have been no reports on the turnover number of ZIP4, which is a crucial missing piece of information needed to better understand the transport mechanism. In this work, we used a nonradioactive zinc isotope, 70Zn, and inductively coupled plasma mass spectrometry to study human ZIP4 (hZIP4) expressed in Human embryonic kidney 293 cells. Our data showed that 70Zn can replace the radioactive 65Zn as a tracer in kinetic evaluation of hZIP4 activity. This approach, combined with the quantification of the cell surface expression of hZIP4 using biotinylation or surface-bound antibody, allowed us to estimate the apparent turnover number of hZIP4 to be in the range of 0.08 to 0.2 s-1. The turnover numbers of the truncated hZIP4 variants are significantly smaller than that of the full-length hZIP4, confirming a crucial role for the extracellular domain in zinc transport. Using 64Zn and 70Zn, we measured zinc efflux during the cell-based transport assay and found that it has little effect on the zinc import analysis under these conditions. Finally, we demonstrated that use of laser ablation inductively coupled plasma-TOF-mass spectrometry on samples applied to a solid substrate significantly increased the throughput of the transport assay. We envision that the approach reported here can be applied to the studies of metal transporters beyond the ZIP family.

Self-Immolative Activation of ß-Galactosidase-Responsive Probes for In Vivo MR Imaging in Mouse Models.

Our lab has developed a new series of self-immolative MR agents for the rapid detection of enzyme activity in mouse models expressing ß-galactosidase (ß-gal). We investigated two molecular architectures to create agents that detect ß-gal activity by modulating the coordination of water to GdIII . The first is an intermolecular approach, wherein we designed several structural isomers to maximize coordination of endogenous carbonate ions. The second involves an intramolecular mechanism for q modulation. We incorporated a pendant coordinating carboxylate ligand with a 2, 4, 6, or 8 carbon linker to saturate ligand coordination to the GdIII ion. This renders the agent ineffective. We show that one agent in particular (6-C pendant carboxylate) is an extremely effective MR reporter for the detection of enzyme activity in a mouse model expressing ß-gal.

Water-Soluble Nanoconjugate for Enhanced Cellular Delivery of Receptor-Targeted Magnetic Resonance Contrast Agents.

ProGlo is an efficient steroid receptor-targeted magnetic resonance (MR) imaging contrast agent (CA). It has been shown to bind to the progesterone receptor (PR) and produce enhanced image contrast in PR-positive cells and tissues in vitro and in vivo. However, the hydrophobicity of the steroid targeting domain of ProGlo (logP = 1.4) limits its formulation and delivery at clinically relevant doses. In this work, a hydrophobic moiety was utilized to drive efficient adsorption onto nanodiamond (ND) clusters to form a water-soluble nanoconstruct (logP = -2.4) with 80% release in 8 h under biological conditions. In cell culture, the ND-ProGlo construct delivered increased concentrations of ProGlo to target cells compared to ProGlo alone. Importantly, these results were accomplished without the use of solvents such as DMSO, providing a significant advance toward formulating ProGlo for translational applications. Biodistribution studies confirm the delivery of ProGlo to PR(+) tissues with enhanced efficacy over untargeted controls. These results demonstrate the potential for a noncovalent ND-CA construct as a general strategy for solubilizing and delivering hydrophobic targeted MR CAs.

A Markedly Improved Synthetic Approach for the Preparation of Multifunctional Au-DNA Nanoparticle Conjugates Modified with Optical and MR Imaging Probes.

We describe a new, and vastly superior approach for labeling spherical nucleic acid conjugates (SNAs) with diagnostic probes. SNAs have been shown to provide the unique ability to traverse the cell membrane and deliver surface conjugated DNA into cells while preserving the DNA from nuclease degradation. Our previous work on preparing diagnostically labeled SNAs was labor intensive, relatively low yielding, and costly. Here, we describe a straightforward and facile preparation for labeling SNAs with optical and MR imaging probes with significantly improved physical properties. The synthesis of Gd(III) labeled DNA Au nanoparticle conjugates is achieved by sequential conjugation of 3'-thiol-modified oligonucleotides and cofunctionalization of the particle surface with the subsequent addition of 1,2 diothiolate modified chelates of Gd(III) (abbreviated: DNA-GdIII@AuNP). This new generation of SNA conjugates has a 2-fold increase of DNA labeling and a 1.4-fold increase in Gd(III) loading compared to published constructs. Furthermore, the relaxivity ( r1) is observed to increase 4.5-fold compared to the molecular dithiolane-Gd(III) complex, and 1.4-fold increase relative to previous particle constructs where the Gd(III) complexes were conjugated to the oligonucleotides rather than directly to the Au particle. Importantly, this simplified approach (2 steps) exploits the advantages of previous Gd(III) labeled SNA platforms; however, this new approach is scalable and eliminates modification of DNA for attaching the contrast agent, and the particles exhibit improved cell labeling.

Gd(III)-Gold Nanoconjugates Provide Remarkable Cell Labeling for High Field Magnetic Resonance Imaging.

In vivo cell tracking is vital for understanding migrating cell populations, particularly cancer and immune cells. Magnetic resonance (MR) imaging for long-term tracking of transplanted cells in live organisms requires cells to effectively internalize Gd(III) contrast agents (CAs). Clinical Gd(III)-based CAs require high dosing concentrations and extended incubation times for cellular internalization. To combat this, we have devised a series of Gd(III)-gold nanoconjugates (Gd@AuNPs) with varied chelate structure and nanoparticle-chelate linker length, with the goal of labeling and imaging breast cancer cells. These new Gd@AuNPs demonstrate significantly enhanced labeling compared to previous Gd(III)-gold-DNA nanoconstructs. Variations in Gd(III) loading, surface packing, and cell uptake were observed among four different Gd@AuNP formulations suggesting that linker length and surface charge play an important role in cell labeling. The best performing Gd@AuNPs afforded 23.6 ± 3.6 fmol of Gd(III) per cell at an incubation concentration of 27.5 µM-this efficiency of Gd(III) payload delivery (Gd(III)/cell normalized to dose) exceeds that of previous Gd(III)-Au conjugates and most other Gd(III)-nanoparticle formulations. Further, Gd@AuNPs were well-tolerated in vivo in terms of biodistribution and clearance, and supports future cell tracking applications in whole-animal models.

Catalytically Active Silicon Oxide Nanoclusters Stabilized in a Metal-Organic Framework.

Post-synthetic modification of the zirconium-based metal-organic framework (MOF) NU-1000 by atomic layer deposition (ALD), using tetramethoxysilane (Si(OMe)4 ) as a precursor, led to the incorporation and stabilization of silicon oxide clusters composed of only a few silicon atoms in the framework's pores. The resulting SiOx functionalized material (Si-NU-1000) was found to be catalytically active despite the inactivity of related bulk silicon dioxide (SiO2 ), thus demonstrating the positive effects of having nanosized clusters of SiOx . Moreover, Si-NU-1000 showed activity greater than that found for aluminum oxide based catalysts-oxides known for their high acidity-such as an aluminum oxide functionalized MOF (Al-NU-1000) and bulk γ-Al2 O3 . X-ray photoelectron spectroscopy and infrared spectroscopy measurements unmasked the electron donating nature of Si-NU-1000, explaining the unusual electronic properties of the nanosized SiOx clusters and supporting their high catalytic activity.

Gd(III)-Dithiolane Gold Nanoparticles for T1-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 r1 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.

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 T1- and T2-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.

Cell-Permeable Esterase-Activated Ca(II)-Sensitive MRI Contrast Agent.

Calcium [Ca(II)] is a fundamental transducer of electrical activity in the central nervous system (CNS). Influx of Ca(II) into the cytosol is responsible for action potential initiation and propagation, and initiates interneuronal communication via release of neurotransmitters and activation of gene expression. Despite the importance of Ca(II) in physiology, it remains a challenge to visualize Ca(II) flux in the central nervous system (CNS) in vivo. To address these challenges, we have developed a new generation, Ca(II)-activated MRI contrast agent that utilizes ethyl esters to increase cell labeling and prevent extracellular divalent Ca(II) binding. Following labeling, the ethyl esters can be cleaved, thus allowing the agent to bind Ca(II), increasing relaxivity and resulting in enhanced positive MR image contrast. The ability of this probe to discriminate between extra- and intracellular Ca(II) may allow for spatiotemporal in vivo imaging of Ca(II) flux during seizures or ischemia where large Ca(II) fluxes (1-10 µM) can result in cell death.

Multimeric Near IR-MR Contrast Agent for Multimodal In Vivo Imaging.

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

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 (ND2) label cells with high efficiency and generate significant image contrast at 7 T compared to nanodiscs containing the multimeric agent (ND1) and Prohance, a clinically approved contrast agent.

Water-soluble lipophilic MR contrast agents for cell membrane labeling.

Long-term cell tracking using MR imaging necessitates the development of contrast agents that both label and are retained by cells. One promising strategy for long-term cell labeling is the development of lipophilic Gd(III)-based contrast agents that anchor into the cell membrane. We have previously reported the efficacy of monomeric and multimeric lipophilic agents and showed that the monomeric agents have improved labeling and contrast enhancement of cell populations. Here, we report on the synthesis, characterization, and in vitro testing of a series of monomeric lipophilic contrast agents with varied alkyl chain compositions. We show that these agents disperse in water, localize to the cell membrane, and label HeLa and MCF7 cells effectively. Additionally, these agents have up to tenfold improved retention in cells compared to clinically available ProHance(®).

Cell labeling via membrane-anchored lipophilic MR contrast agents.

Cell tracking in vivo with MR imaging requires the development of contrast agents with increased sensitivity that effectively label and are retained 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 in vitro. Two of the agents were synthesized with a multiplexing strategy to contain three Gd(III) chelates (1 and 2) while the third contains a single Gd(III) chelate (3). 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 (4 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.

In vivo micro-computed tomography evaluation of radiopaque, polymeric device degradation in normal and inflammatory environments.

Polymeric biomedical implants are an important clinical tool, but degradation remains difficult to determine post-implantation. Computed tomography (CT) could be a powerful tool for device monitoring, but polymers require incorporation of radiopaque contrast agents to be distinguishable from tissue. In addition, immune response to radiopaque devices must be characterized as it modulates device function. Radiopaque devices and films were produced by incorporating 0-20 wt% TaOx nanoparticles into polymers: polycaprolactone (PCL) and poly(lactide-co-glycolide) (PLGA). In vitro inflammatory responses of mouse bone marrow-derived macrophages to polymer matrix incorporating TaOx nanoparticles was determined by monitoring cytokine secretion. Nanoparticle addition stimulated a slight inflammatory reaction, increasing TNFα secretion, mediated by changes in polymer matrix properties. Subsequently, devices (PLGA 50:50 + 20 wt% TaOx) were implanted subcutaneously in a mouse model of chronic inflammation, that featured a sustained increase in inflammatory response local to the implant site over 12 weeks. No changes to device degradation rates or foreign body response were noted between a normal and chronically stimulated inflammatory environment. Serial CT device monitoring post-implantation provided a detailed timeline of device collapse, with no rapid, spontaneous release of nanoparticles that occluded matrix visualization. Importantly, repeat CT sessions did not ablate the immune system or alter degradation kinetics. Thus, polymer devices incorporating radiopaque nanoparticles can be used for in situ monitoring and be readily combined with other medical imaging techniques, for a dynamic view biomaterial and tissue interactions. STATEMENT OF SIGNIFICANCE: A growing number of implantable devices are in use in the clinic, exposing patients to inherent risks of implant movement, collapse, and infection. The ability to monitor implanted devices would enable faster diagnosis of failure and open the door for personalized rehabilitation therapies - both of which could vastly improve patient outcomes. Unfortunately, polymeric materials which make up most biomedical devices are not radiologically distinguishable from tissue post-implantation. The introduction of radiopaque nanoparticles into polymers allows for serial monitoring via computed tomography, without affecting device degradation. Here we demonstrate for the first time that nanoparticles do not undergo burst release from devices post-implantation and that inflammatory responses - a key determinant of device function in vivo - are also unaffected by nanoparticle addition.

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or "nanodrug") administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Multi-modal investigation of the bone micro- and ultrastructure, and elemental distribution in the presence of Mg-xGd screws at mid-term healing stages.

Magnesium (Mg) - based alloys are becoming attractive materials for medical applications as temporary bone implants for support of fracture healing, e.g. as a suture anchor. Due to their mechanical properties and biocompatibility, they may replace titanium or stainless-steel implants, commonly used in orthopedic field. Nevertheless, patient safety has to be assured by finding a long-term balance between metal degradation, osseointegration, bone ultrastructure adaptation and element distribution in organs. In order to determine the implant behavior and its influence on bone and tissues, we investigated two Mg alloys with gadolinium contents of 5 and 10 wt percent in comparison to permanent materials titanium and polyether ether ketone. The implants were present in rat tibia for 10, 20 and 32 weeks before sacrifice of the animal. Synchrotron radiation-based micro computed tomography enables the distinction of features like residual metal, degradation layer and bone structure. Additionally, X-ray diffraction and X-ray fluorescence yield information on parameters describing the bone ultrastructure and elemental composition at the bone-to-implant interface. Finally, with element specific mass spectrometry, the elements and their accumulation in the main organs and tissues are traced. The results show that Mg-xGd implants degrade in vivo under the formation of a stable degradation layer with bone remodeling similar to that of Ti after 10 weeks. No accumulation of Mg and Gd was observed in selected organs, except for the interfacial bone after 8 months of healing. Thus, we confirm that Mg-5Gd and Mg-10Gd are suitable material choices for bone implants.

Structural optimization of Zn(II)-activated magnetic resonance imaging probes.

We report the structural optimization and mechanistic investigation of a series of bioactivated magnetic resonance imaging contrast agents that transform from low relaxivity to high relaxivity in the presence of Zn(II). The change in relaxivity results from a structural transformation of the complex that alters the coordination environment about the Gd(III) center. Here, we have performed a series of systematic modifications to determine the structure that provides the optimal change in relaxivity in response to the presence of Zn(II). Relaxivity measurements in the presence and absence of Zn(II) were used in conjunction with measurements regarding water access (namely, number of water molecules bound) to the Gd(III) center and temperature-dependent (13)C NMR spectroscopy to determine how the coordination environment about the Gd(III) center is affected by the distance between the Zn(II)-binding domain and the Gd(III) chelate, the number of functional groups on the Zn(II)-binding domain, and the presence of Zn(II). The results of this study provide valuable insight into the design principles for future bioactivated magnetic resonance probes.

Rational engineering of an elevator-type metal transporter ZIP8 reveals a conditional selectivity filter critically involved in determining substrate specificity.

Engineering of transporters to alter substrate specificity as desired holds great potential for applications, including metabolic engineering. However, the lack of knowledge on molecular mechanisms of substrate specificity hinders designing effective strategies for transporter engineering. Here, we applied an integrated approach to rationally alter the substrate preference of ZIP8, a Zrt-/Irt-like protein (ZIP) metal transporter with multiple natural substrates, and uncovered the determinants of substrate specificity. By systematically replacing the differentially conserved residues with the counterparts in the zinc transporter ZIP4, we created a zinc-preferring quadruple variant (Q180H/E343H/C310A/N357H), which exhibited largely reduced transport activities towards Cd2+, Fe2+, and Mn2+ whereas increased activity toward Zn2+. Combined mutagenesis, modeling, covariance analysis, and computational studies revealed a conditional selectivity filter which functions only when the transporter adopts the outward-facing conformation. The demonstrated approach for transporter engineering and the gained knowledge about substrate specificity will facilitate engineering and mechanistic studies of other transporters.

Synthesis, Characterization, and in vitro Testing of a Bacteria-Targeted MR Contrast Agent.

A bacteria-targeted MR contrast agent, Zn-1, consisting of two Zn-dipicolylamine (Zn-dpa) groups conjugated to a GdIII chelate has been synthesized and characterized. In vitro studies with S. aureus and E. coli show that Zn-1 exhibits a significant improvement in bacteria labeling efficiency vs. control. Studies with a structural analogue, Zn-2, indicate that removal of one Zn-dpa moiety dramatically reduces the agent's affinity for bacteria. The ability of Zn-1 to significantly reduce the T1 of labeled vs. unlabeled bacteria, resulting in enhanced MR image contrast, demonstrates its potential for visualizing bacterial infections in vivo.