Mojmir Suchy

A sensitive PARACEST contrast agent for temperature MRI: Eu3+-DOTAM-glycine (Gly)-phenylalanine (Phe)

Tissue temperature is a fundamental physiological parameter that can provide insight into pathological processes. The purpose of this study was to develop and characterize a novel paramagnetic chemical exchange saturation transfer (CEST) agent suitable for in vivo temperature mapping at 9.4T. The CEST properties of the europium (Eu3+) complex of the DOTAM‐Glycine (Gly)‐Phenylalanine (Phe) ligand were studied in vitro at 9.4T as a function of temperature, pH, and agent concentration. The transfer of magnetization (CEST effect) from the bound water to bulk water pools was ∼75% greater for Eu3+‐DOTAM‐Gly‐Phe compared to Eu3+‐DOTAM‐Gly at physiologic temperature (38°C) and pH (7.0 pH units) when using power level sufficiently low for in vivo imaging. Unlike Eu3+‐DOTAM‐Gly, whose CEST effect decreased with increasing temperature in the physiologic range, the CEST effect of Eu3+‐DOTAM‐Gly‐Phe was optimal at body temperature. A strong linear dependence of the chemical shift of the bound water pool on temperature was observed (0.3 ppm/°C), which was insensitive to pH and agent concentration. Temperature maps with SDs < 1°C were acquired at 9.4T in phantoms containing: 1) phantom A, an aqueous solution of 10 mM Eu3+‐DOTAM‐Gly‐Phe; 2) phantom B, 5% bovine serum albumin (BSA) with 15 mM Eu3+‐DOTAM‐Gly‐Phe; and 3) phantom C, mouse brain tissue with 4 mM Eu3+‐DOTAM‐Gly‐Phe. The temperature sensitivity combined with the high CEST effect observed at low concentration using low saturation power (B1) suggests this compound may be a good choice for in vivo temperature mapping at 9.4T. Magn Reson Med 59:374–381, 2008.

In vivo detection of MRI-PARACEST agents in mouse brain tumors at 9.4 T.

Paramagnetic chemical exchange saturation transfer (PARACEST) contrast agents are under development for biological target identification by magnetic resonance imaging. Image contrast associated with PARACEST agents can be generated by radiofrequency irradiation of the chemically shifted protons bound to a PARACEST contrast agent molecule or by direct irradiation of the on‐resonance bulk water protons. The observed signal change in a magnetic resonance image after the administration of a PARACEST contrast agent is due to both altered relaxation time constants and the CEST effect. Despite high sensitivity in vitro, PARACEST agents have had limited success in vivo where sensitivity is reduced by the magnetization transfer effect from endogenous macromolecules. The purpose of this study was to demonstrate the in vivo detection of a PARACEST contrast agent using the on‐resonance paramagnetic chemical exchange effect (OPARACHEE) in a mouse glioblastoma multiforme tumor model and to isolate the OPARACHEE effect from the changes in relaxation induced by the PARACEST agent. Three mice with tumors were imaged on a 9.4 T MRI scanner following tail vein injection of 150 μL 50 mM Tm3+‐DOTAM‐glycine‐lysine. A fast low angle shot pulse sequence with a low power radiofrequency pulse train (WALTZ‐16) as the preparation pulse was used to generate OPARACHEE contrast. To study the dynamics of agent uptake, reference images (without the preparation pulse) and OPARACHEE images were acquired continuously in an alternating fashion before, during and after agent injection. Signal intensity decreased by more than 10% in tumor in the control images after agent administration. Despite these changes, a clear OPARACHEE contrast of 1–5% was also observed in brain tumors after contrast agent injection and maintained in the hour following injection. This result is the first in vivo observation of OPARACHEE contrast in brain tumors with correction of T1 and T2 relaxation effects. Magn Reson Med, 2011.

Peptide-decorated gold nanoparticles via strain-promoted azide–alkyne cycloaddition and post assembly deprotection

A new method combining an interfacial strain-promoted azide–alkyne cycloaddition and post assembly deprotection (SPAAC-PAD) has been developed for the well-defined functionalization of small, water-soluble gold nanoparticles with oligopeptides.

A dual magnetic resonance imaging/fluorescent contrast agent for Cathepsin-D detection.

Currently there are no approved biomarkers for the pre-symptomatic diagnosis of Alzheimers disease (AD). Cathepsin-D (Cat-D) is a lysosomal protease that is present at elevated levels in amyloid plaques and neurons in patients with AD and is also elevated in some cancers. We have developed a magnetic resonance imaging (MRI)/fluorescent contrast agent to detect Cat-D enzymatic activity. The purpose of this study was to investigate the cellular and tissue uptake of this MRI/fluorescent contrast agent. The agent consists of an MRI probe [DOTA-caged metal ion (Gd³⁺ or Tm³⁺)] and a fluorescent probe coupled to a cell-penetrating-peptide sequence by a Cat-D recognition site. The relaxivity of Gd³⁺-DOTA-CAT(cleaved) was measured in 10% heat-treated bovine serum albumin (BSA) phantoms to assess contrast efficacy at magnetic fields ranging from 0.24 mT to 9.4 T. In vitro, Tm³⁺-DOTA-CAT was added to neuronal SN56 cells over-expressing Cat-D and live-cell confocal microscropy was performed at 30 min. Tm³⁺-DOTA-CAT was also intravenously injected into APP/PS1-dE9 Alzheimers disease mice (n = 9) and controls (n = 8). Cortical and hippocampal uptake was quantified at 30, 60 and 120 min post-injection using confocal microscopy. The liver and kidneys were also evaluated for contrast agent uptake. The relaxivity of Gd³⁺-DOTA-CAT(cleaved) was 3.3 (mM s)⁻¹ in 10% BSA at 9.4 T. In vitro, cells over-expressing Cat-D preferentially took up the contrast agent in a concentration-dependent manner. In vivo, the contrast agent effectively crossed the blood-brain barrier and exhibited a distinct time course of uptake and retention in APP/PS1-dE9 transgenic mice compared with age-matched controls. At clinical and high magnetic field strengths, Gd³⁺-DOTA-CAT produced greater T₁ relaxivity than Gd³⁺-DTPA. Tm³⁺-DOTA-CAT was taken up in a dose-dependent manner in cells over-expressing Cathepsin-D and was shown to transit the blood-brain barrier in vivo. This strategy may be useful for the in vivo detection of enzyme activity and for the diagnosis of Alzheimers disease.

Optimized MRI Contrast for On-resonance Proton Exchange Processes of PARACEST Agents in Biological Systems

Image contrast associated with paramagnetic chemical exchange saturation transfer agents can be generated by off‐resonance irradiation of agent‐bound water or amide protons or on‐resonance irradiation of bulk water. Previously, a four‐pool model was developed to describe an in vivo system. The model incorporated the magnetization transfer effect from macromolecules when using off‐resonance irradiation. In the current study, this four‐pool model is modified to describe the in vivo system when using on‐resonance irradiation. The influences of pulse power, pulse duration, the chemical shift of bound water, the proton exchange rate between bulk water and bound water, and agent concentration on the on‐resonance paramagnetic agent chemical exchange effects were simulated using a WALTZ‐16 pulse train in the absence and presence of the macromolecule pool. The results demonstrated that while contrast increases with pulse duration in aqueous solution, there is an optimal pulse duration that maximizes on‐resonance paramagnetic agent chemical exchange effects contrast in vivo. This predication was verified by experimental spectroscopic and imaging results from aqueous solution, bovine serum albumin phantoms, and a tissue phantom containing thulium‐DOTAM (1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetamide)‐glycine‐lysine. This model can be used to optimize sequence parameters to maximize in vivo on‐resonance paramagnetic agent chemical exchange effects contrast. Magn Reson Med, 2009.

Prolonged In Vivo Retention of a Cathepsin D Targeted Optical Contrast Agent in a Mouse Model of Alzheimer’s Disease

BACKGROUND Cathepsin D (CatD) is a lysosomal protease that is elevated early in Alzheimers disease (AD). We have previously developed a Targeted contrast agent (CA) to detect CatD activity in vivo, consisting of a magnetic resonance imaging/fluorescent moiety linked to a cell penetrating peptide (CPP) by means of a CatD cleavage site and have demonstrated its uptake in the brain of an AD mouse model. OBJECTIVE The purpose of this study was to characterize the in vivo retention of a near infra-red fluorescent dye labeled version of this CA. METHODS Six adult C57Bl/6 wild-type mice and six adult 5XFAD transgenic AD mice were studied using a small animal imaging system at five and twelve months of age using our novel Targeted CA, or two different control CAs; a Non-Targeted (lacking the CatD cleavage site) and a Non-Penetrating (lacking the CPP). Following intravenous CA administration, the optical signal was recorded within the brain and uptake and washout curves were measured and fitted to a one-phase exponential decay curve. RESULTS In all wild-type and 5XFAD mice, the washout of the Targeted CA that included a CPP domain was significantly slower than the washout of the Non-Penetrating and Non-Targeted CA. Furthermore, the washout of the CatD Targeted CA was significantly slower in the 5XFAD mice compared to the age matched wild-type controls (p <  0.05) at 5 and 12 months of age. Control CAs showed no differences in washout. CONCLUSIONS The prolonged retention of the CatD targeted CA in 5XFAD mice suggests this agent may be useful for AD detection.

In-Vitro and In-Vivo Study of a Magnetic Resonance Imaging (MRI)/Fluorescent Contrast Agent for Detection in Alzheimer's Disease

uptake of this MRI/fluorescent contrast agent targeting CatD. Methods: SN56 cells, a neuronal cell line, were cultured and differentiated. To simulate conditions of increased CatD, cells were transfected with a plasmid which stimulates high level expression of human CatD tagged with red fluorescent protein. Varying concentrations (5, 10, 50, 100 mM) of DOTA-CAT were added to the media of SN56 cells and confocal microscopy was performed at 30 minutes. Uptake was quantified by counting the proportion of cells which were labeled with DOTA-CAT expressed as Mean 6 SEM. Lastly, cleavage experiments on DOTA-CAT were performed using purified CatD and analyzed using electrospray-ionization mass spectrometry (ESI-MS). Results: In uptake experiments, 19 6 2% of cells exposed to the agent at 5 mM took up DOTA-CAT, and this number increased with concentration such that at 100 mM, 78 6 7% of cells took up DOTA-CAT. In contrast, a maximum of 5 6 1% of cells not over-expressing CatD took up DOTA-CAT over the entire concentration range. DOTA-CAT was observed intracellularly and in intracellular vesicles frequently co-localized with red fluorescent protein tagged-CatD within lysosomes. In cleavage experiments, incubation of DOTA-CAT with CatD produced a 1906.6 Da fragment confirming the cleavage of DOTA-CAT at the predicted CatD recognition sequence. Conclusions: These experiments demonstrate that DOTA-CAT interacts with CatD and is cleaved as predicted. Furthermore, DOTA-CAT is preferentially taken up by SN56 cells over-expressing CatD in a concentration dependant manner. Therefore, DOTA-CAT shows significant potential as a molecular imaging probe that will be further evaluated in future experiments using an in-vivo model of AD.

An Aspartyl Cathepsin Targeted PET Agent: Application in an Alzheimer’s Disease Mouse Model

BACKGROUND Early detection of Alzheimers disease (AD) pathology is a serious challenge for both diagnosis and clinical trials. The aspartyl protease, Cathepsin D (CatD), is overexpressed in AD and could be a biomarker of disease. We have previously designed a unique contrast agent (CA) for dual-optical and magnetic resonance imaging of the activity of the CatD class of enzymes. OBJECTIVE To compare the uptake and retention of a novel, more sensitive, and clinically-translatable 68Ga PET tracer targeting CatD activity in 5XFAD mice and non-Tg littermates. METHODS The targeted CA consisted of an HIV-1 Tat cell penetrating peptide (CPP) conjugated to a specialized cleavage sequence targeting aspartyl cathepsins and a DOTA conjugate chelating 68Ga. PET images were acquired using a Siemens Inveon preclinical microPET in female Tg AD mice and non-Tg age matched female littermates (n = 5-8) following intravenous CA administration at 2, 6, and 9 months of age. Additionally, 18F fluorodeoxyglucose (FDG) PET imaging was performed at 10 months to measure glucose uptake. RESULTS The Tg mice showed significantly higher relative uptake rate of the targeting CA in the forebrain relative to hindbrain at all ages compared to controls, consistent with histology. In contrast, no differences were seen in CA uptake in other organs. Additionally, the Tg mice did not show any differences in relative uptake of FDG at 10 months of age in the forebrain relative to the hindbrain compared to age matched non-Tg controls. CONCLUSIONS Elevated aspartryl cathepsin activity was detected in vivo in the 5XFAD mouse model of AD using a novel targeted PET contrast agent.

In-Vitro Cellular Uptake of a Magnetic Resonance Imaging (MRI)/Fluorescent Contrast Agent for Detection of Cathepsin-D Activity in Alzheimer's Disease

correlated moderately with change in MMSE score. Conclusions: We conclude that WBA, VE and HV rates of change from our institute are not significantly different than those from the DRC and CIND, respectively. We also observed that the measured WBA, VE and HV rate changes were significantly higher in AD subjects relative to the normal control group, showing that our methods were sensitive enough to detect changes in subjects with AD.