Luis M. De León-Rodríguez

A New Gadolinium-Based MRI Zinc Sensor

The properties of a novel Gd(3+)-based MRI zinc sensor are reported. Unlike previously reported Gd(3+)-based MRI contrast agents, this agent (GdL) differs in that the agent alone binds only weakly with human serum albumin (HSA), while the 1:2 GdL:Zn(2+) ternary complex binds strongly to HSA resulting in a substantial, 3-fold increase in water proton relaxivity. The GdL complex is shown to have a relatively strong binding affinity for Zn(2+) (K(D) = 33.6 nM), similar to the affinity of the Zn(2+) ion with HSA alone. The agent detects as little as 30 microM Zn(2+) in the presence of HSA by MRI in vitro, a value slightly more than the total Zn(2+) concentration in blood (approximately 20 microM). This combination of binding affinity constants and the high relaxivity of the agent when bound to HSA suggests that this new agent may be useful for detection of free Zn(2+) ions in vivo without disrupting other important biological processes involving Zn(2+).

Noninvasive MRI of β-cell function using a Zn2+-responsive contrast agent.

Elevation of postprandial glucose stimulates release of insulin from granules stored in pancreatic islet β-cells. We demonstrate here that divalent zinc ions coreleased with insulin from β-cells in response to high glucose are readily detected by MRI using the Zn2+-responsive T1 agent, GdDOTA-diBPEN. Image contrast was significantly enhanced in the mouse pancreas after injection of a bolus of glucose followed by a low dose of the Zn2+ sensor. Images of the pancreas were not enhanced by the agent in mice without addition of glucose to stimulate insulin release, nor were images enhanced in streptozotocin-treated mice with or without added glucose. These observations are consistent with MRI detection of Zn2+ released from β-cells only during glucose-stimulated insulin secretion. Images of mice fed a high-fat (60%) diet over a 12-wk period and subjected to this same imaging protocol showed a larger volume of contrast-enhanced pancreatic tissue, consistent with the expansion of pancreatic β-cell mass during fat accumulation and progression to type 2 diabetes. This MRI sensor offers the exciting potential for deep-tissue monitoring of β-cell function in vivo during development of type 2 diabetes or after implantation of islets in type I diabetic patients.

Multimeric system of 99mTc-labeled gold nanoparticles conjugated to c[RGDfK(C)] for molecular imaging of tumor α(v)β(3) expression.

Integrin α(V)β(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic RGD peptides can be used for noninvasive imaging of α(V)β(3) expression. The aim of this research was to prepare a multimeric system of technetium-99m-labeled gold nanoparticles conjugated to c[RGDfK(C)] and to evaluate its biological behavior as a potential radiopharmaceutical for molecular imaging of tumor angiogenesis. Hydrazinonicotinamide-GGC (HYNIC-GGC) and c[RGDfK(C)] peptides were synthesized and conjugated to gold nanoparticles (AuNP, 20 nm) by means of spontaneous reaction of the thiol groups of cysteine. The nanoconjugate was characterized by TEM, FT-IR, UV-vis, XPS, and Raman spectroscopy. To obtain (99m)Tc-HYNIC-GGC-AuNP-c[RGDfK(C)] ((99m)Tc-AuNP-RGD), the (99m)Tc-HYNIC-GGC radiopeptide was first prepared and added to 1.5 mL of AuNP solution (1 nM) followed by c[RGDfK(C)] (10 μL, 50 μM) at 18 °C with stirring for 15 min. Radiochemical purity (RP) was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in α(V)β(3) receptor-positive C6 glioma cancer cells. Biodistribution studies were accomplished in athymic mice with C6-induced tumors with blocked and nonblocked receptors, and images were obtained using a micro-SPECT/CT. TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with peptides. RP was 96 ± 2% without postlabeling purification. (99m)Tc-AuNP-RGD showed specific recognition for α(V)β(3) integrins expressed in C6 cells, and 3 h after i.p. administration in mice, the tumor uptake was 8.18 ± 0.57% ID/g. Micro-SPECT/CT images showed evident tumor uptake. (99m)Tc-AuNP-RGD demonstrates properties suitable for use as a target-specific agent for molecular imaging of tumor α(V)β(3) expression.

Simultaneous determination of methylxanthines in coffees and teas by UV-Vis spectrophotometry and partial least squares

This work describes a method to simultaneously determine caffeine (CF) and theobromine (TB) in coffee and tea samples using partial least squares (PLS-1). Sample preparation was required to eliminate strong interfering components. High-performance liquid chromatography (HPLC)-found concentrations of caffeine and theobromine (theophylline was not found in any analyzed sample) were used to construct universal calibration matrixes for coffee and tea. Due to the low levels of theobromine when compared to caffeine (up to 1000:1), theobromine addition standard was required to dramatically improve method performance. The method developed did not show statistically significant differences with an HPLC standard technique.

MRI Detection of VEGFR2 in Vivo Using a Low Molecular Weight Peptoid−(Gd)8-Dendron for Targeting

The synthesis of a polylysine dendron containing eight GdDOTA units conjugated to a peptoid dimer known to have a high affinity for the vascular endothelial growth factor receptor 2 (VEGFR2) is described. This simple low molecular weight system with a molecular r(1) relaxivity of ∼48 mM(-1) s(-1) is shown to enhance MR images of tumors grown in mice in vivo.

Multi-Frequency PARACEST Agents Based on Europium(III)-DOTA-Tetraamide Ligands†

Magnetic resonance imaging (MRI) is one of the most versatile and powerful diagnostic tools in modern medicine. Recently, a conceptually different approach to contrast enhancement based on chemical exchange saturation transfer (CEST) has emerged that takes advantage of slow-to-intermediate exchange conditions between two or more pools of protons (kex ≤ Δω).[1] While the first reported CEST agents were diamagnetic molecules containing exchangeable NH and OH groups (Δω ≤ 5 ppm), it was later shown that the slow water exchange characteristics of certain paramagnetic Ln3+ complexes of DOTA-tetraamide ligands allows selective saturation of a hyperfine shifted Ln3+-bound water pool (Δω > 50 ppm) for creating CEST contrast.[2] Radio frequency (RF) saturation of highly shifted exchange resonances in paramagnetic systems offer significant advantages over diamagnetic CESTagents with small Δω values.[3]

Basic MR relaxation mechanisms and contrast agent design

The diagnostic capabilities of magnetic resonance imaging (MRI) have undergone continuous and substantial evolution by virtue of hardware and software innovations and the development and implementation of exogenous contrast media. Thirty years since the first MRI contrast agent was approved for clinical use, a reliance on MR contrast media persists, largely to improve image quality with higher contrast resolution and to provide additional functional characterization of normal and abnormal tissues. Further development of MR contrast media is an important component in the quest for continued augmentation of diagnostic capabilities. In this review we detail the many important considerations when pursuing the design and use of MR contrast media. We offer a perspective on the importance of chemical stability, particularly kinetic stability, and how this influences ones thinking about the safety of metal–ligand‐based contrast agents. We discuss the mechanisms involved in MR relaxation in the context of probe design strategies. A brief description of currently available contrast agents is accompanied by an in‐depth discussion that highlights promising MRI contrast agents in the development of future clinical and research applications. Our intention is to give a diverse audience an improved understanding of the factors involved in developing new types of safe and highly efficient MR contrast agents and, at the same time, provide an appreciation of the insights into physiology and disease that newer types of responsive agents can provide. J. Magn. Reson. Imaging 2015;42:545–565.

Effect of antimicrobial peptides derived from human cathelicidin LL-37 on Entamoeba histolytica trophozoites.

The human cathelicidin hCAP18/LL-37 is an antimicrobial protein consisting of a conserved N-terminal prosequence called the cathelin-like domain and a C-terminal peptide called LL-37. This peptide contains 37 amino acid residues, and several truncated variants obtained from natural sources or by chemical synthesis differ in their capability to damage Gram positive and Gram negative bacteria as well as Candida albicans. KR-12 is the shortest peptide (12 amino acids) of LL-37 that has conserved antibacterial activity. In addition to LL-37, other active cathelicidin-derived peptides have been reported; for instance, the peptides KR-20, a 20-aa derivative of LL-37, and KS-30, a 30-aa derivative of LL-37, have been found in human sweat. Both peptides exhibit an overall increased antibacterial and antifungal activity when compared with LL-37. We investigated the effect of LL-37 and three peptides derived from this antimicrobial molecule, KR-12, KR-20 and KS-30, on the integrity of Entamoeba histolytica trophozoites. The four peptides showed effects on E. histolytica integrity and viability in the concentration range of 10-50 μM. The peptides KR-12, KR-20, KS-30 and LL-37 differed in their capability to damage the parasite integrity, with KR-20 being the most effective and with KR-12 and LL-37 being less active. These results demonstrate the ability of antimicrobial peptides derived from human cathelicidin to damage Entamoeba trophozoites. Moreover, it was shown that the integrity of the peptides is altered in the presence of an ameba soluble fraction with cysteine protease activity.

Zinc-sensitive MRI contrast agent detects differential release of Zn(II) ions from the healthy vs. malignant mouse prostate.

Significance The normal prostate gland contains the most Zn(II) of all mammalian tissues, and there are marked differences in Zn(II) content between the healthy, malignant, and benign hyperplastic prostate. Given that multiparametric MRI does not always reliably distinguish between these tissue conditions, the release of Zn(II) ions from the prostate in response to an external stimulus may prove valuable as a specific biomarker of prostate cancer progression. In this work, we show that glucose stimulates the release of Zn(II) from intracellular stores in healthy prostate tissue and that Zn(II) secretion is reduced in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Many secretory tissues release Zn(II) ions along with other molecules in response to external stimuli. Here we demonstrate that secretion of Zn(II) ions from normal, healthy prostate tissue is stimulated by glucose in fasted mice and that release of Zn(II) can be monitored by MRI. An ∼50% increase in water proton signal enhancement is observed in T1-weighted images of the healthy mouse prostate after infusion of a Gd-based Zn(II) sensor and an i.p. bolus of glucose. Release of Zn(II) from intracellular stores was validated in human epithelial prostate cells in vitro and in surgically exposed prostate tissue in vivo using a Zn(II)-sensitive fluorescent probe known to bind to the extracellular surface of cells. Given the known differences in intracellular Zn(II) stores in healthy versus malignant prostate tissues, the Zn(II) sensor was then evaluated in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model in vivo. The agent proved successful in detecting small malignant lesions as early as 11 wk of age, making this noninvasive MR imaging method potentially useful for identifying prostate cancer in situations where it may be difficult to detect using current multiparametric MRI protocols.

A second generation MRI contrast agent for imaging zinc ions in vivo

A Zn2+ specific GdDOTA derivative containing two bis-(3-pyrazolyl) units was prepared and characterized. Unlike a previously reported Zn2+ binding agent, the new agent binds to human albumin both in the presence and absence of Zn2+.