Multifunctional Mitochondrial Imaging Agents

Overview

Fluorescent molecules provide a highly sensitive technique for imaging cells and are widely used to study biological processes. These molecular probes are typically limited to a single function of fluorescence. Designing novel probes that are multifunctional is a desirable prospect that could find applications in research and therapeutics. It is our aim to synthesise probes that target the mitochondria with the ability for (i) in vivo and in vitro fluorescence reporting, (ii) small molecule delivery and (iii) inclusion of a radiolabel for signalling in positron emission tomography (PET).

Methodology

Healthy cells have a negative mitochondrial membrane potential (MMP) of 150 to 170 mV. When cell dysfunction occurs, through cancer or ischaemic heart disease, this potential can be significantly disrupted, such that the accumulation of MMP dependent compounds can increase tenfold. Cell death through apoptosis or necrosis results in complete membrane depolarisation and no uptake is observed. These phenomena can therefore be utilised to target the mitochondria.
Work Package 1

Lipophilic cations can traverse membrane lipid bilayers and accumulate in the mitochondria in a Nernstian manner. Phosphonium salts have been used to target the mitochondria in this way since 1960s and are now commercialised products (e.g. MitoQ). The Higham group has recently prepared phosphonium-based lipophilic cations containing the fluorophore boron dipyrromethene (Bodipy) and an alkyl fluoride pendent chain suitable for exchange with 18F for PET1. The compounds prepared so far have already shown promising MMP dependent uptake. The aim of this work package is to create a library of Bodipy-phosphonium salts by systematically varying the steric, electronic and lipophilic properties of the phosphonium moiety and the identity of the negatively charged counterion. These salts will then be submitted for biological testing to understand how the structure can be optimised for MMP uptake.

Work Package 2

The knowledge gained from work package 1 will be used to optimise another Bodipy-phosphonium salt synthesised by the Higham group containing an alkyne function2. The alkyne function is amenable towards copper catalysed click chemistry. Therefore, attaching a biomolecule, such as RNA, to an azide function and carrying out a click reaction would enable the fluorescent product to deliver cargo in an MMP dependent way. This process will require optimisation to find the best solvent, catalyst, ligand and reaction temperature.

Work Package 3

Although Bodipy has excellent photophysical properties and biocompatibility its light absorption and emission profile is at shorter wavelengths than are optimal for tissue penetration. Living tissues are most transparent at 800nm and above, whereas most Bodipy structures emit at 600nm and below. Fortunately adjusting the Bodipy core can lead to red-shifted fluorescence. Therefore, developing new core structures that can be tuned to longer wavelengths is highly desirable. This will be investigated and the new Bodipy cores combined with the optimised structures synthesised in work packages 1 and 2 to deliver highly effective, red-shifted, multifunctional mitochondrial imaging agents.

References

1. S. Nigmam, B.P. Burke, L.H. Davies, J. Domarkas, J. F. Wallis, P.G. Waddell, J.S. Waby, D.M. Benoit, A.M. Seymour, C. Cawthorne, L.J. Higham and S.J. Archibald, Chem Comm., 2016, S2, 7114
2. J.F. Wallis, Synthesis of Fluorescent Phosphorus Ligands and their Applications in Medical Imaging and Catalysis, p111, Newcastle University 2017.