Semi-quantitative elemental imaging of corrosion products from bioabsorbable Mg vascular implants in vivo.

While metal materials historically have served as permanent implants and were designed to avoid degradation, next generation bioabsorbable metals for medical devices such as vascular stents are under development, which would elute metal ions and corrosion byproducts into tissues. The fate of these eluted products and their local distribution in vascular tissue largely under studied. In this study, we employ a high spatial resolution spectrometric imaging modality, laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) to map the metal distribution, (herein refered to as laser ablation mapping, or LAM) from Mg alloys within the mouse vascular system and approximate their local concentrations. We used a novel rare earth element bearing Mg alloy (WE22) wire implanted within the abdominal aorta of transgenic hypercholesterolemic mice (APOE-/-) to simulate a bioabsorbable vascular prosthesis for up to 30 days. We describe qualitatively and semi-quantitatively implant-derived corrosion product presence throughout the tissue cross sections, and their approximate concentrations within the various vessel structures. Additionally, we report the spatial changes of corrosion products, which we postulate are mediated by phagocytic inflammatory cells such as macrophages (MΦ's).

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.

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.

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.

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.

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.

Quantitative imaging approaches to understanding biological processing of metal ions.

Faster, more sensitive, and higher resolution quantitative instrumentation are aiding a deeper understanding of how inorganic chemistry regulates key biological processes. Researchers can now image and quantify metals with subcellular resolution, leading to a vast array of new discoveries in organismal development, pathology, and disease. Metals have recently been implicated in several diseases such as Parkinson's, Alzheimers, ischemic stroke, and colorectal cancer that would not be possible without these advancements. In this review, instead of focusing on instrumentation we focus on recent applications of label-free elemental imaging and quantification and how these tools can lead to a broader understanding of metals role in systems biology and human pathology.

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein.

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms.

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein.

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date 1,2 . In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes based on their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of new antibody discovery methodologies.

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.

Bioresorbable photonic devices for the spectroscopic characterization of physiological status and neural activity.

Capabilities in real-time monitoring of internal physiological processes could inform pharmacological drug-delivery schedules, surgical intervention procedures and the management of recovery and rehabilitation. Current methods rely on external imaging techniques or implantable sensors, without the ability to provide continuous information over clinically relevant timescales, and/or with requirements in surgical procedures with associated costs and risks. Here, we describe injectable classes of photonic devices, made entirely of materials that naturally resorb and undergo clearance from the body after a controlled operational lifetime, for the spectroscopic characterization of targeted tissues and biofluids. As an example application, we show that the devices can be used for the continuous monitoring of cerebral temperature, oxygenation and neural activity in freely moving mice. These types of devices should prove useful in fundamental studies of disease pathology, in neuroscience research, in surgical procedures and in monitoring of recovery from injury or illness.

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.

Synthesis and evaluation of MR probes for targeted-reporter imaging.

Visualizing disease heterogeneity remains a challenging task since most imaging agents are targeted to a single receptor. We describe the development of an MR platform able to report on multiple molecular events. Enzyme activation and enhanced cellular uptake of this modular probe make it suitable for subsequent targeted-reporter imaging applications.

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.

Exploring the Association Between Demographics, SLC30A8 Genotype, and Human Islet Content of Zinc, Cadmium, Copper, Iron, Manganese and Nickel.

A widely prevalent single nucleotide polymorphism, rs13266634 in the SLC30A8 gene encoding the zinc transporter ZnT8, is associated with an increased risk for T2DM. ZnT8 is mostly expressed in pancreatic insulin-producing islets of Langerhans. The effect of this variant on the divalent metal profile in human islets is unknown. Additionally, essential and non-essential divalent metal content of human islets under normal environmental exposure conditions has not been described. We therefore examined the correlation of zinc and other divalent metals in human islets with rs13266634 genotype and demographic characteristics. We found that the diabetes risk genotype C/C at rs13266634 is associated with higher islet Zn concentration (C/C genotype: 16792 ± 1607, n = 22, C/T genotype: 11221 ± 1245, n = 18 T/T genotype: 11543 ± 6054, n = 3, all values expressed as mean nmol/g protein ± standard error of the mean, p = 0.040 by ANOVA). A positive correlation between islet cadmium content and both age (p = 0.048, R2 = 0.09) and female gender (women: 36.88 ± 4.11 vs men: 21.22 ± 3.65 nmol/g protein, p = 0.007) was observed. Our results suggest that the T2DM risk allele C is associated with higher islet zinc levels and support prior evidence of cadmium's higher bioavailability in women and its long tissue half-life.

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.

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.

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.