Investigations into the Chemistry of Functional Cyanine Dyes as Synthetic Platforms for the Multimodal Imaging of Prostate Cancer

  • Ruediger M. Exner

Student thesis: Doctoral ThesisPhD


New investigations into the chemistry and optical properties of sulfonated indocyanine dyes have been performed and are described. At the heart of the investigations is their use as imaging agents for living prostate cancer cells. The synthesis and investigation of different groups of agents is described in each chapter. As-synthesized probes were assessed for their potential to inhibit cellular metabolism and investigated by time-correlated single photon counting (TCSPC) measurements in different solvent systems, fluorescence lifetime imaging (FLIM) in living cell lines and, where possible, confocal microscopy and fluorescence-assisted cell sorting.

The majority of the synthesized, water-soluble near-infrared (NIR) dyes showed slow cellular uptake, even when conjugated to peptides synthesized in-house and can hardly be recommended for further development, at least with small molecule-based targeting agents. Synthesized far-red probes, on the other hand, appeared to be promising imaging agents. FLIM results suggest cellular trafficking in agreement with lysosomal uptake and long fluorescence lifetimes. In some cases, multi-component fitting was necessary, either suggesting two distinct (bio)chemical environments, or intermolecular quenching.

Chapter 1 describes the broader context of this work, discussing the necessity of improved diagnosis and therapeutic modalities for prostate cancer, one of the most common cancers in men in the developed world. It highlights various imaging modalities of relevance to prostate cancer and cancer in general, which are of relevance in the context of this work. In it, new small-molecule agents used for radio-imaging and optical imaging, and some aspects of the chemistry and properties of far-red and near-infrared dyes are reviewed.

Chapter 2 describes the synthesis and characterization of water-soluble far-red and near-infrared dyes used as part of this work. Inaccuracies in the state-of-the-art are discussed and well-reproducible methodologies for their synthesis and purification are described. The as-synthesized fluorophores were assessed in living cancer cells, and results from MTT-assays, TCSPC and FLIM experiments are presented. Computational work using the Amsterdam Density Functional Suite (ADF) is presented which highlights the problems associated with treating these molecules theoretically and investigates the influence of applying a dielectric continuum and non-equilibrium solvation on these molecules.

Chapter 3 describes initial attempts at peptide bond-formation with meso-Cl dyes and an investigation into the side-reactions that make the desired products difficult to purify and obtain. The main side-product resulting from these reactions was identified to be the corresponding keto-polymethines. Subsequent work resulted in the synthesis and isolation of the first keto-polymethine conjugates, which were assessed for their potential as far-red imaging agents with large Stokes shift. The results suggest that highly water-soluble keto-polymethines are not readily taken up in cells. In the course of these investigations, changes to the optical properties in biological media were observed, which appear to be caused by supramolecular interactions with proteins.

Chapter 4 discusses the synthesis of meso-substituted cyanine dyes from a combined experimental/theoretical point-of-view. Selected attempts at the synthesis of selected meso-substituted dyes are presented, some of which resulted in the production of radiolabelled tricarbocyanine derivatives. The optical properties of the synthesized probes are put into the context of medical imaging. The synthesized dyes are assessed in MTT assays and by TCSPC and FLIM, revealing a highly localized uptake pattern, and, in some cases, multiple decay components in living cell lines.

Chapter 5 describes the synthesis of small-molecule conjugates of a far-red pentamethine cyanine dye. Following some initial optimization, these could be obtained in straightforward fashion, in appreciable yields and purity. Conjugation to small molecules, either peptides, glucosamine or nitroimidazoles or ligands for metals, such as deferoxamine (DFO), did not produce noticeable spectral shifts. Mass spectrometry evidence suggests that radiolabeling with the DFO-derivative may be possible. Uptake in prostate cancer cell lines was affected drastically by conjugation. The uptake was also found to be drastically affected by the medium in which the cells were incubated. TCSPC and FLIM results are presented and gave results similar to those obtained with tricarbocyanine dyes.

Chapter 6 contains a chapter-by-chapter conclusion to this thesis, along with an outlook and recommendations for future work.

Chapter 7 provides detailed experimental methods and procedures, along with characterization data for the synthesized compounds.

The appendix contains supporting information, xyz-coordinates of computed (optimized) geometries, screenshots showing the fitting of FLIM data, and data of single crystal X-ray diffraction experiments.
Date of Award16 Nov 2022
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorSofia Pascu (Supervisor) & Charareh Pourzand (Supervisor)

Cite this