The design, synthesis and evaluation of selective, non invasive imaging agents for atherosclerotic plaque.

The aim of this research study was the synthesis of an imaging agent, which could be used to non-invasively image atherosclerotic plaques via MRI. In order to achieve this, a library of peptide substrates designed to be substrate specific for either legumain or MMP-2/9 were synthesized. This was achieved via a combination of both solid and solution phase peptide synthesis. The first phase of the research study focused on the synthesis of a targeted MRI contrast agent designed to image legumain expressing atherosclerotic plaques. This was achieved by using a Fluorescent Resonance Energy Transfer (FRET) active system to evaluate the substrate specificity of synthesized peptide sequences for legumain. The self quenched FRET substrates were engineered to carry a legumain sensitive bond in between the donor (5(6)-carboxyfluorescein) and quencher (aminoanthraquinone) moiety. Legumain mediated activation of the FRET substrates was measured via fluorescence spectroscopic analysis which showed the release in fluorescence that was initially quenched by the anthraquinone moiety. Optimized peptide sequences were then covalently coupled to the gadolinium chelate (Gd-DOTA) so that, upon enzymatic cleavage, the contrast agent could be released at the site of activation. Mass spectrometric analysis on the post enzyme digested samples (incubated with recombinant human legumain) showed that the substrates were fragmented by the activated enzyme in vitro. The targeted MRI contrast agent and FRET substrates were successfully synthesized and then characterized by low/high resolution mass spectrometry. Additionally cytotoxic studies on selected compounds and a model of the enzyme-digested product [Lys(DOTA-Gd)-Sp-AQ] via MTT assay demonstrated that the compounds did not affect the cell viability of MDA-MB-231 cell line. The second phase of this project was the synthesis of an aminoanthraquinone peptide substrate coupled to a contrast agent (CA). This substrate was designed to be specific to MMP-2/9. Although the synthetic strategy was successful, subsequent reaction optimization will be required (increasing yield) before in vitro CA release could be evaluated. Thus, the targeted MRI CAs synthesized during this research study provide a starting point for novel imaging agents. These agents, which upon successful in vitro and in vivo trials could potentially be used to image legumain expression or MMP (2/9) expression or atherosclerotic plaques.

Name dc:type.qualificationname

MPhil

Level dc:type.qualificationlevel

Masters

Grantor dc:publisher.institution

Robert Gordon University

Year dc:date.issued

2017

Author dc:creator

• Jesudoss, Jesintha Rose

Advisor dc:contributor.advisor

• Graeme Kay, Alberto Di Salvo and Cherry L. Wainwright

• Atherosclerosis
• MMPs
• Contrast agent
• FRET
• Legumain
• GdDOTA

Language dc:language

en

Identifier

oai:rgu-repository.worktribe.com:248939

OAI identifier oai:identifier

oai:rgu-repository.worktribe.com:248939