Magnetic resonance imaging of tumor-associated-macrophages (TAMs) with a nanoparticle contrast agent.

In the tumor micro-environment, tumor associated macrophages (TAMs) represent a predominant component of the total tumor mass, and TAMs play a complex and diverse role in cancer pathogenesis with potential for either tumor suppressive, or tumor promoting biology. Thus, understanding macrophage localization and function are essential for cancer diagnosis and treatment. Typically, tissue biopsy is used to evaluate the density and polarization of TAMs, but provides a limited "snapshot" in time of a dynamic and potentially heterogeneous tumor immune microenvironment. Imaging has the potential for three-dimensional mapping; however, there is a paucity of macrophage-targeted contrast agents to specifically detect TAM subtypes. We have previously found that sulfated-dextran coated iron oxide nanoparticles (SDIO) can target macrophage scavenger receptor A (SR-A, also known as CD204). Since CD204 (SR-A) is considered a biomarker for the M2 macrophage polarization, these SDIO might provide M2-specific imaging probes for MRI. In this work, we investigate whether SDIO can label M2-polarized cells in vitro. We evaluate the effect of degree of sulfation on uptake by primary cultured bone marrow derived macrophages (BMDM) and found that a higher degree of sulfation led to higher uptake, but there were no differences across the subtypes. Further analysis of the BMDM showed similar SR-A expression across stimulation conditions, suggesting that this classic model for macrophage subtypes may not be ideal for definitive M2 subtype marker expression, especially SR-A. We further examine the localization of SDIO in TAMs in vivo, in the mammary fat pad mouse model of breast cancer. We demonstrate that uptake by TAMs expressing SR-A scales with degree of sulfation, consistent with the in vitro studies. The TAMs demonstrate M2-like function and secrete Arg-1 but not iNOS. Uptake by these M2-like TAMs is validated by immunohistochemistry. SDIO show promise as a valuable addition to the toolkit of imaging probes targeted to different biomarkers for TAMs.

A nephrotoxicity-free, iron-based contrast agent for magnetic resonance imaging of tumors.

Gadolinium-based contrast agents (GBCAs) are the most widely used T1 contrast agents for magnetic resonance imaging (MRI) and have achieved remarkable success in clinical cancer diagnosis. However, GBCAs could cause severe nephrogenic systemic fibrosis to patients with renal insufficiency. Nevertheless, GBCAs are quickly excreted from the kidneys, which shortens their imaging window and prevents long-term monitoring of the disease per injection. Herein, a nephrotoxicity-free T1 MRI contrast agent is developed by coordinating ferric iron into a telodendritic, micellar nanostructure. This new nano-enabled, iron-based contrast agent (nIBCA) not only can reduce the renal accumulation and relieve the kidney burden, but also exhibit a significantly higher tumor to noise ratio (TNR) for cancer diagnosis. In comparison with Magnevist (a clinical-used GBCA), Magnevist induces obvious nephrotoxicity while nIBCA does not, indicating that such a novel contrast agent may be applicable to renally compromised patients requiring a contrast-enhanced MRI. The nIBCA could precisely image subcutaneous brain tumors in a mouse model and the effective imaging window lasted for at least 24 h. The nIBCA also precisely highlights the intracranial brain tumor with high TNR. The nIBCA presents a potential alternative to GBCAs as it has superior biocompatibility, high TNR and effective imaging window.

Two-way magnetic resonance tuning and enhanced subtraction imaging for non-invasive and quantitative biological imaging.

Distance-dependent magnetic resonance tuning (MRET) technology enables the sensing and quantitative imaging of biological targets in vivo, with the advantage of deep tissue penetration and fewer interactions with the surroundings as compared with those of fluorescence-based Förster resonance energy transfer. However, applications of MRET technology in vivo are currently limited by the moderate contrast enhancement and stability of T1-based MRET probes. Here we report a new two-way magnetic resonance tuning (TMRET) nanoprobe with dually activatable T1 and T2 magnetic resonance signals that is coupled with dual-contrast enhanced subtraction imaging. This integrated platform achieves a substantially improved contrast enhancement with minimal background signal and can be used to quantitatively image molecular targets in tumours and to sensitively detect very small intracranial brain tumours in patient-derived xenograft models. The high tumour-to-normal tissue ratio offered by TMRET in combination with dual-contrast enhanced subtraction imaging provides new opportunities for molecular diagnostics and image-guided biomedical applications.

Antioxidant Sensing by Spiropyrans: Substituent Effects and NMR Spectroscopic Studies.

The development of stimuli-responsive small molecules for probing biologically active antioxidants such as glutathione (GSH) has important ramifications in the detection of oxidative stress. An ideal sensor for biological applications should exhibit sufficient sensitivity and selectivity for detection at physiological concentrations and be reversible to allow continuous and dynamic monitoring of antioxidant levels. Designing a suitable sensor thus requires a detailed understanding of activation properties and mechanism of action. In this work, we report a new set of GSH-responsive spiropyrans and demonstrate how changes in the electronic structure of spiropyrans influence GSH sensing with high specificity versus other structurally similar and biologically relevant redox-active molecules. The sensitivity, selectivity, kinetics, binding constant, and reversibility of GSH-responsive-substituted spiropyrans were investigated using UV-vis spectroscopy and laser irradiation experiments. Detailed studies of the mechanism of interaction between spiropyrans with GSH were investigated using NMR spectroscopy. Understanding how electronic effects impact the sensing ability of spiropyrans toward antioxidants and elucidating the mechanism of the spiropyran-GSH interaction will facilitate the design of more effective sensors for detection of antioxidants in vivo.

In Vivo MRI of Functionalized Iron Oxide Nanoparticles for Brain Inflammation.

Microglia are intrinsic components of the brain immune system and are activated in many central nervous system disorders. The ability to noninvasively image these cells would provide valuable information for both research and clinical applications. Today, most imaging probes for activated microglia are mainly designed for positron emission tomography (PET) and target translocator proteins that also reside on other cerebral cells. The PET images obtained are not specific for microglia-driven inflammation. Here, we describe a potential PET/MRI multimodal imaging probe that selectively targets the scavenger receptor class A (SR-A) expressed on activated microglia. These sulfated dextran-coated iron oxide (SDIO) nanoparticles are avidly taken up by microglia and appear to be nontoxic when administered intravenously in a mouse model. Intravenous administration of this SDIO demonstrated visualization by T2∗ -weighted MRI of microglia activated by intracerebral administration of tumor necrosis factor alpha (TNF-α). The contrast was significantly enhanced by SDIO, whereas there was little to no contrast change in animals treated with nontargeted nanoparticles or untreated controls. Thus, SR-A targeting represents a promising strategy to image activated microglia in the brain.

Biological effects of MRI contrast agents: gadolinium retention, potential mechanisms and a role for phosphorus.

No discussion of challenges for chemistry in molecular imaging would be complete without addressing the elephant in the room-which is that the purest of chemical compounds needs to interact with a biological system in a manner that does not perturb normal biology while still providing efficacious feedback to assist in diagnosis of disease. In the past decade, magnetic resonance imaging (MRI) agents long considered inert have produced adverse effects in certain patient populations under certain treatment regimens. More recently, inert blood pool agents have been found to deposit in the brain. Release of free metal is often suspected as the culprit but that hypothesis has yet to be validated. In addition, even innocuous agents can cause painful side effects during injection in some patients. In this brief review, we summarize known biological effects for gadolinium- and iron-based MRI contrast agents, and discuss some of the potential mechanisms for the observed biological effects, including the potential role of phosphorus imbalance, related to kidney disease or cancer, in destabilizing gadolinium-based chelates and precipitating free gadolinium.This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

Preparation of a conjugation-ready thiol responsive molecular switch.

In this work we synthesize molecular switches that are responsive to cysteine, homocysteine, and glutathione; three redox systems that make up the majority of the body's antioxidant defenses. Synthesized spiropyran isomers with conjugation-ready linkages showed good selectivity of response to these major antioxidant thiols over nucleophilic amino acids; however the position of the linking group can affect selectivity and reversibility of the switching response. An isomer with selectivity for cysteine against GSH and Hcy was identified.

Quantitative assessment of binding affinities for nanoparticles targeted to vulnerable plaque.

Recent successes in targeted immune and cell-based therapies have driven new directions for pharmaceutical research. With the rise of these new therapies there is an unfilled need for companion diagnostics to assess patients' potential for therapeutic response. Targeted nanomaterials have been widely investigated to fill this niche; however, in contrast to small molecule or peptide-based targeted agents, binding affinities are not reported for nanomaterials, and to date there has been no standard, quantitative measure for the interaction of targeted nanoparticle agents with their targets. Without a standard measure, accurate comparisons between systems and optimization of targeting behavior are challenging. Here, we demonstrate a method for quantitative assessment of the binding affinity for targeted nanoparticles to cell surface receptors in living systems and apply it to optimize the development of a novel targeted nanoprobe for imaging vulnerable atherosclerotic plaques. In this work, we developed sulfated dextran-coated iron oxide nanoparticles with specific targeting to macrophages, a cell type whose density strongly correlates with plaque vulnerability. Detailed quantitative, in vitro characterizations of (111)In(3+) radiolabeled probes show high-affinity binding to the macrophage scavenger receptor A (SR-A). Cell uptake studies illustrate that higher surface sulfation levels result in much higher uptake efficiency by macrophages. We use a modified Scatchard analysis to quantitatively describe nanoparticle binding to targeted receptors. This characterization represents a potential new standard metric for targeted nanomaterials.

Multimodality PET/MRI agents targeted to activated macrophages.

The recent emergence of multimodality imaging, particularly the combination of PET and MRI, has led to excitement over the prospect of improving detection of disease. Iron oxide nanoparticles have become a popular platform for the fabrication of PET/MRI probes owing to their advantages of high MRI detection sensitivity, biocompatibility, and biodegradability. In this article, we report the synthesis of dextran-coated iron oxide nanoparticles (DIO) labeled with the positron emitter (64)Cu to generate a PET/MRI probe, and modified with maleic anhydride to increase the negative surface charge. The modified nanoparticulate PET/MRI probe (MDIO-(64)Cu-DOTA) bears repetitive anionic charges on the surface that facilitate recognition by scavenger receptor type A (SR-A), a ligand receptor found on activated macrophages but not on normal vessel walls. MDIO-(64)Cu-DOTA has an average iron oxide core size of 7-8 nm, an average hydrodynamic diameter of 62.7 nm, an r1 relaxivity of 16.8 mM(-1) s(-1), and an r 2 relaxivity of 83.9 mM(-1) s(-1) (37 °C, 1.4 T). Cell studies confirmed that the probe was nontoxic and was specifically taken up by macrophages via SR-A. In comparison with the nonmodified analog, the accumulation of MDIO in macrophages was substantially improved. These characteristics demonstrate the promise of MDIO-(64)Cu-DOTA for identification of vulnerable atherosclerotic plaques via the targeting of macrophages.

Rapid microwave-assisted synthesis of dextran-coated iron oxide nanoparticles for magnetic resonance imaging.

Currently, magnetic iron oxide nanoparticles are the only nanosized magnetic resonance imaging (MRI) contrast agents approved for clinical use, yet commercial manufacturing of these agents has been limited or discontinued. Though there is still widespread demand for these particles both for clinical use and research, they are difficult to obtain commercially, and complicated syntheses make in-house preparation unfeasible for most biological research labs or clinics. To make commercial production viable and increase accessibility of these products, it is crucial to develop simple, rapid and reproducible preparations of biocompatible iron oxide nanoparticles. Here, we report a rapid, straightforward microwave-assisted synthesis of superparamagnetic dextran-coated iron oxide nanoparticles. The nanoparticles were produced in two hydrodynamic sizes with differing core morphologies by varying the synthetic method as either a two-step or single-step process. A striking benefit of these methods is the ability to obtain swift and consistent results without the necessity for air-, pH- or temperature-sensitive techniques; therefore, reaction times and complex manufacturing processes are greatly reduced as compared to conventional synthetic methods. This is a great benefit for cost-effective translation to commercial production. The nanoparticles are found to be superparamagnetic and exhibit properties consistent for use in MRI. In addition, the dextran coating imparts the water solubility and biocompatibility necessary for in vivo utilization.

Development of iron-doped silicon nanoparticles as bimodal imaging agents.

We demonstrate the synthesis of water-soluble allylamine-terminated Fe-doped Si (Si(xFe)) nanoparticles as bimodal agents for optical and magnetic imaging. The preparation involves the synthesis of a single-source iron-containing precursor, Na(4)Si(4) with x% Fe (x = 1, 5, 10), and its subsequent reaction with NH(4)Br to produce hydrogen-terminated Si(xFe) nanoparticles. The hydrogen-capped nanoparticles are further terminated with allylamine via thermal hydrosilylation. Transmission electron microscopy indicates that the average particle diameter is ∼3.0 ± 1.0 nm. The Si(5Fe) nanoparticles show strong photoluminescence quantum yield in water (∼10%) with significant T(2) contrast (r(2)/r(1) value of 4.31). Electron paramagnetic resonance and Mössbauer spectroscopies indicate that iron in the nanoparticles is in the +3 oxidation state. Analysis of cytotoxicity using the resazurin assay on HepG2 liver cells indicates that the particles have minimal toxicity.

Rapid size-controlled synthesis of dextran-coated, 64Cu-doped iron oxide nanoparticles.

Research into developing dual modality probes enabled for magnetic resonance imaging (MRI) and positron emission tomography (PET) has been on the rise recently due to the potential to combine the high resolution of MRI and the high sensitivity of PET. Current synthesis techniques for developing multimodal probes is largely hindered in part by prolonged reaction times during radioisotope incorporation--leading to a weakening of the radioactivity. Along with a time-efficient synthesis, the resulting products must fit within a critical size range (between 20 and 100 nm) to increase blood retention time. In this work, we describe a novel, rapid, microwave-based synthesis technique to grow dextran-coated iron oxide nanoparticles doped with copper (DIO/Cu). Traditional methods for coprecipitation of dextran-coated iron oxide nanoparticles require refluxing for 2 h and result in approximately 50 nm diameter particles. We demonstrate that microwave synthesis can produce 50 nm nanoparticles with 5 min of heating. We discuss the various parameters used in the microwave synthesis protocol to vary the size distribution of DIO/Cu and demonstrate the successful incorporation of (64)Cu into these particles with the aim of future use for dual-mode MR/PET imaging.

Receptor-targeted iron oxide nanoparticles for molecular MR imaging of inflamed atherosclerotic plaques.

In a number of literature reports iron oxide nanoparticles have been investigated for use in imaging atherosclerotic plaques and found to accumulate in plaques via uptake by macrophages, which are critical in the process of atheroma initiation, propagation, and rupture. However, the uptake of these agents is non-specific; thus the labeling efficiency for plaques in vivo is not ideal. We have developed targeted agents to improve the efficiency for labeling macrophage-laden plaques. These probes are based on iron oxide nanoparticles coated with dextran sulfate, a ligand of macrophage scavenger receptor type A (SR-A). We have sulfated dextran-coated iron oxide nanoparticles (DIO) with sulfur trioxide, thereby targeting our nanoparticle imaging agents to SR-A. The sulfated DIO (SDIO) remained mono-dispersed and had an average hydrodynamic diameter of 62 nm, an r(1) relaxivity of 18.1 mM(-1) s(-1), and an r(2) relaxivity of 95.8 mM(-1) s(-1) (37 °C, 1.4 T). Cell studies confirmed that these nanoparticles were nontoxic and specifically targeted to macrophages. In vivo MRI after intravenous injection of the contrast agent into an atherosclerotic mouse injury model showed substantial signal loss on the injured carotid at 4 and 24 h post-injection of SDIO. No discernable signal decrease was seen at the control carotid and only mild signal loss was observed for the injured carotid post-injection of non-sulfated DIO, indicating preferential uptake of the SDIO particles at the site of atherosclerotic plaque. These results indicate that SDIO can facilitate MRI detection and diagnosis of vulnerable plaques in atherosclerosis.

Paramagnetic, silicon quantum dots for magnetic resonance and two-photon imaging of macrophages.

Quantum dots (QDs) are an attractive platform for building multimodality imaging probes, but the toxicity for typical cadmium QDs limits enthusiasm for their clinical use. Nontoxic, silicon QDs are more promising but tend to require short-wavelength excitations which are subject to tissue scattering and autofluorescence artifacts. Herein, we report the synthesis of paramagnetic, manganese-doped, silicon QDs (Si(Mn) QDs) and demonstrate that they are detectable by both MRI and near-infrared excited, two-photon imaging. The Si(Mn) QDs are coated with dextran sulfate to target them to scavenger receptors on macrophages, a biomarker of vulnerable plaques. TEM images show that isolated QDs have an average core diameter of 4.3 +/- 1.0 nm and the hydrodynamic diameters of coated nanoparticles range from 8.3 to 43 nm measured by dynamic light scattering (DLS). The Si(Mn) QDs have an r(1) relaxivity of 25.50 +/- 1.44 mM(-1) s(-1) and an r(2) relaxivity of 89.01 +/- 3.26 mM(-1) s(-1) (37 degrees C, 1.4 T). They emit strong fluorescence at 441 nm with a quantum yield of 8.1% in water. Cell studies show that the probes specifically accumulate in macrophages by a receptor-mediated process, are nontoxic to mammalian cells, and produce distinct contrast in both T(1)-weighted magnetic resonance and single- or two-photon excitation fluorescence images. These QDs have promising diagnostic potential as high macrophage density is associated with atherosclerotic plaques vulnerable to rupture.

Size-controlled synthesis of dextran sulfate coated iron oxide nanoparticles for magnetic resonance imaging.

In the generation of nanoparticles for biological applications, the control over synthetic parameters influencing the particles' physicochemical properties are of great interest due to the strong influence of particle size and surface properties on cellular uptake and biodistribution. We have synthesized dextran sulfate coated particles and systematically evaluated synthetic parameters that may influence the properties of these nanoparticles as potential magnetic resonance (MR) contrast agents. The amount of base, polysaccharide content, ratio of iron salts, and reaction time were optimized to yield approximately 30 nm particles as determined by dynamic light scattering with good MR properties (r(1) = 14.46 mM(-1) s(-1) and r(2) = 72.55 mM(-1) s(-1)) and in good yield (50%). Particle sizes and relaxivities are compared with clinically available dextran coated particles and the resulting physical properties of the dextran sulfate coated particles show these particles could be used as potential MR contrast agents for cardiovascular imaging.

Development of contrast agents targeted to macrophage scavenger receptors for MRI of vascular inflammation.

Atherosclerosis is a leading cause of death in the U.S. Because there is a potential to prevent coronary and arterial disease through early diagnosis, there is a need for methods to image arteries in the subclinical stage as well as clinical stage using various noninvasive techniques, including magnetic resonance imaging (MRI). We describe a development of a novel MRI contrast agent targeted to plaques that will allow imaging of lesion formation. The contrast agent is directed to macrophages, one of the earliest components of developing plaques. Macrophages are labeled through the macrophage scavenger receptor A, a macrophage specific cell surface protein, using an MRI contrast agent derived from scavenger receptor ligands. We have synthesized and characterized these contrast agents with a range of relaxivities. In vitro studies show that the targeted contrast agent accumulates in macrophages, and solution studies indicate that micromolar concentrations are sufficient to produce contrast in an MR image. Cell toxicity and initial biodistribution studies indicate low toxicity, no detectable retention in normal blood vessels, and rapid clearance from blood. The promising performance of this contrast agent targeted toward vascular inflammation opens doors to tracking of other inflammatory diseases such as tumor immunotherapy and transplant acceptance using MRI.

Receptor mediated uptake of a radiolabeled contrast agent sensitive to beta-galactosidase activity.

Radiolabeling of the MRI contrast agent 1-[2-(beta-galactopyranosyloxy)propyl]-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane with (111)In, and its evaluation is reported. Radiolabeling was performed in acetate buffer with 50-78% radiochemical yield. In vitro studies revealed that the asialoglycoprotein receptor-poor cell line MH1C1 has low uptake, while the receptor-rich cell lines BNL-CL2 and Hep G2 have higher uptake. In vivo, the uptake of the compound in receptor-rich organ liver was very high. Blocking the receptor in vivo, reduced liver uptake by 90% suggesting that the compound localizes in receptor-enriched tissues by binding to the asialoglycoprotein receptor.