Synthesis of optical probes for the visualization of human neutrophil elastase

Optical medical imaging is a new technology that uses light to improve clinical processes such as disease diagnosis and tumour margin delineation. It has the potential to produce high-resolution images in real-time, with continuously generated images on or in biological tissues at a molecular level. Imaging inside the lung can be accomplished via the use of microendoscopy, and this has been used to detect and analyse several pulmonary diseases, which traditionally have been hard to unequivocally define. Several pulmonary diseases have been linked with overactive leukocytes within the lung. A class of leukocytes, neutrophils, are used by the body to aggressively destroy potential pathogens with an arsenal of proteases including human neutrophil elastase (HNE) at its disposal. However, overexpression of unregulated HNE has been implicated in a number of pulmonary diseases, such as fibrosis, COPD and acute lung injury with irreversible damage caused to lung tissue through protein degradation. Currently in the clinic, it is difficult to identify and quantify unregulated HNE in vivo using traditional methods of diagnosis. Thus, the ability to quantify and evaluate numbers of neutrophils and levels of HNE using an in vivo technique would be very valuable. Within this work, a library of optical probes to selectively detect HNE at a molecular level were designed and evaluated. The probes consist of two parts: an inhibitor warhead designed to irreversibly bind to HNE, and a fluorophore. Two different classes of warheads have been synthesised and conjugated to a variety of fluorophores that emit over a range of wavelengths. Having optical probes over the span of several wavelength ranges is beneficial as it allows multiplex imaging so several diseases can be screened simultaneously, as well as removing the probes from possible tissue autofluorescence wavelengths. The probes were evaluated for their binding efficacy with HNE, toxicity against erythrocytes and then, subsequently used to detect and image the presence and localisation of HNE within neutrophils. It is envisioned that the use of optical probes to detect HNE overexpression will lead to quicker diagnoses for patients with debilitating pulmonary diseases