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.

Size-stable solid lipid nanoparticles loaded with Gd-DOTA for magnetic resonance imaging.

Solid lipid nanoparticles (SLNs) have recently emerged as nontoxic, versatile alternatives to traditional carriers (liposomes, polymeric nanoparticles) for drug delivery. Because SLNs are composed of a solid lipid core, they offer significant protection against chemical degradation of their drug cargo and offer the potential for controlled release. SLNs also hold promise for use as targeted agents and multimodal imaging agents. Here we report the synthesis and characterization of SLNs loaded with gadolinium (1,4,7,10-tetraazacyclododecane)-1,4,7,10-tetraacetate (Gd-DOTA) in order to produce a new category of stable T1-weighted (T1w) magnetic resonance imaging (MRI) contrast agents. Systematically varying components in the SLN synthesis, we demonstrated an increase in Gd-DOTA incorporation and an increase in longitudinal relaxivity (r1) through optimizing the amount of surfactant (Span 80) in the "oil" phase. These highly monodisperse SLNs confirm stable loading of Gd-DOTA and a stable size distribution (∼150 nm) over time in aqueous solution. Relaxivity measurements (1.4T, 37 °C) demonstrate that the r1 of Gd-DOTA does not strongly decrease following encapsulation in SLNs, demonstrating an advantage over liposomes. These Gd-loaded SLNs demonstrate enhanced contrast in vivo at 7T using T1w MRI and in the future can be loaded with other cargo (hydrophilic or hydrophobic) to enable functionality with other imaging modalities such as optical or positron emission tomography.

Strategies for the development of gadolinium-based 'q'-activatable MRI contrast agents.

The emergence and rapid development of activatable contrast agents (CAs), whose relaxivity changes in response to the variation of a specific marker in the surrounding physiological microenvironment, have expanded the scope of MRI beyond anatomical and functional imaging to also convey information at the cellular and molecular level. The essence of an activatable MRI CA is the difference in relaxivity before and after a change in a physiological variable: the larger the difference, the better the CA. In this review, strategies for the design of activatable gadolinium CAs, with a switching mechanism based on the modulation of hydration (q), sensitive to common variables in the physiological microenvironment, such as pH, light, redox and metal ions, are illustrated and discussed.

Reversible low-light induced photoswitching of crowned spiropyran-DO3A complexed with gadolinium(III) ions.

Photoswitchable spiropyran has been conjugated to the crowned ring system DO3A, which improves its solubility in dipolar and polar media and stabilizes the merocyanine isomer. Adding the lanthanide ion gadolinium(III) to the macrocyclic ring system leads to a photoresponsive magnetic resonance imaging contrast agent that displays an increased spin-lattice relaxation time (T1) upon visible light stimulation. In this work, the photoresponse of this photochromic molecule to weak light illumination using blue and green light emitting diodes was investigated, simulating the emission spectra from bioluminescent enzymes. Photon emission rate of the light emitting diodes was changed, from 1.75 × 10¹6 photons·s⁻¹ to 2.37 × 10¹² photons·s⁻¹. We observed a consistent visible light-induced isomerization of the merocyanine to the spiropyran form with photon fluxes as low as 2.37 × 10¹² photons·s⁻¹ resulting in a relaxivity change of the compound. This demonstrates the potential for use of the described imaging probes in low light level applications such as sensing bioluminescence enzyme activity. The isomerization behavior of gadolinium(III)-ion complexed and non-complexed spiropyran-DO3A was analyzed in water and ethanol solution in response to low light illumination and compared to the emitted photon emission rate from over-expressed Gaussia princeps luciferase.

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.

Nanoformulations for molecular MRI.

Nanoscale contrast agents have shown the ability to increase the detection sensitivity of magnetic resonance imaging (MRI) by several orders of magnitude, endowing this traditionally macroscopic modality with the ability to observe unique molecular signatures. Herein, we describe three types of nanoparticulate contrast agents: iron oxide nanoparticles, gadolinium-based nanoparticles, and bio-essential manganese, cobalt, nickel, and copper ion-containing nanoformulations. Some of these agents have been approved for clinical use, but more are still under development for medical imaging. The advantages and disadvantages of each nanoformulation, in terms of intrinsic magnetism, ease of synthesis, biodistribution, etc. are discussed.

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.

PET Imaging and Biodistribution of Silicon Quantum Dots in Mice.

Investigation of nanomaterial disposition and fate in the body is critical before such material can be translated into clinical application. Herein a new macrocyclic ligand-(64)Cu(2+) complex was synthesized and used to label dextran-coated silicon quantum dots (QD), with an average hydrodynamic diameter of 15.1 ± 7.6 nm. The chelate showed exceptional stability, demonstrated by no loss radiolabel under a ligand competition reaction with EDTA. The QDs' biodistribution in mice was quantitatively evaluated by in vivo positron emission tomography (PET) imaging and ex vivo gamma counting. Results showed that they were excreted via renal filtration shortly postinjection and also accumulated in the liver.

Activatable T1 and T2 magnetic resonance imaging contrast agents.

Magnetic resonance imaging (MRI) has become one of the most important diagnosis tools available in medicine. Typically MRI is not capable of sensing biochemical activities. However, recently emerged activatable MRI contrast agents (CAs), whose relaxivity is variable in response to a specific parameter change in the surrounding physiological microenvironment, potentially allow for MRI to indicate biological processes. Among the various factors influencing the relaxivity of a CA, the number of inner-sphere water molecules (q) directly coordinated to the metal center, the residence time of the coordinated water molecule (τ (m)), and the rotational correlation time representing the molecular tumbling time of a complex (τ (R)) contribute strongly to the relaxivity of an activatable CA. Tuning the ligand structure and properties has been the subject of intensive research for activatable MR CA designs. This review summarizes a variety of activatable MRI CAs sensitive to common variables in microenvironment in vivo, i.e., pH, luminescence, metal ions, redox, and enzymes, etc., with emphasis on the influence of ligand design on parameters q, τ (m), and τ (R).

Modulation of T2 relaxation time by light-induced, reversible aggregation of magnetic nanoparticles.

A reversible T2 contrast agent consisting of cross-linked anionic dextran coated iron oxide nanoparticles covalently coupled to a light-sensitive spiropyran (SP)/merocyanine (MC) motif was synthesized and characterized. In aqueous solution, light induced isomerization of the molecular switches between the hydrophobic SP isomer and hydrophilic MC isomer directs the aggregation and dispersion of the nanoparticles, respectively. When in the dark, where the MC form dominates, the probe has a T2 relaxation time of 37.09 ms (60 MHz, 37 degrees C) and two size populations at 70 and 540 nm. After irradiation with visible light, the T2 relaxation time is shortened 33.7%, and the size correspondingly shifts to a single population at 520 nm upon aggregation. This "smart" T2 agent provides the advantage of reversibility which may enable dynamic monitoring with MRI. In addition, the light responsiveness of this agent suggests the potential to employ them as MRI gene reporters for the luciferase expression system.

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.

Synthesis and characterization of a redox- and light-sensitive MRI contrast agent.

A redox- and light-sensitive, T(1)-weighted magnetic resonance imaging (MRI) contrast agent which tethers a spiropyran(SP)/merocyanine(MC) motif to a Gd-DO3A moiety was synthesized and characterized. When in the dark, the probe is in its MC form which has an r(1) relaxivity of 2.51 mM(-1)s(-1) (60MHz, 37°C). After irradiation with visible light or mixing with NADH, the probe experiences an isomerization and the r(1) relaxivity decreased 18% and 26%, respectively. Additionally, the signal intensity in MRI showed an observable decrease after the compound was mixed with NADH.

Photochromically-controlled, reversibly-activated MRI and optical contrast agent.

The contrast agent which tethers a spiropyran group to a Gd-DO3A moiety has higher relaxivity and fluorescence intensity in the dark; the relaxivity and fluorescence intensity decrease after irradiation with visible light.

Di-ionizable p-tert-butylcalix[4]arene-1,2-crown-5 and -crown-6 compounds in the cone conformation: synthesis and alkaline earth metal cation extraction.

Di-ionizable p-tert-butylcalix[4]arene-1,2-crown-5 and -crown-6 ethers in the cone conformation were prepared and their conformations and regioselectivities were verified by NMR spectroscopy. The metal ion-complexing properties of these ligands were evaluated by competitive solvent extractions of alkaline earth metal cations from water into chloroform. The ligands were found to be efficient extractants with selectivity for Ba(2+). The maximal loadings were 95-100% as calculated for formation of 1 : 1 ionized ligand-metal ion complexes. With the variation of proton-ionizable groups, which were oxyacetic acid moieties and N-(X)sulfonyl oxyacetamide units with X = methyl, phenyl, 4-nitrophenyl, and trifluoromethyl, "tunable" acidity was obtained.