Abstract
Photodynamic therapy (PDT) is a minimally invasive approach for the treatment of cancer and various other human disorders, based on the selective activation of photosensitizers (PSs) with light. At present, one of the most promising strategies for PDT and also fluorescence photodiagnosis (PDD) is to use 5-aminolevulinic acid (ALA) as a prodrug to increase intracellular levels of the endogenous PS, protoporphyrin IX (PpIX). Although ALA-PDT has been shown to be a very promising clinical approach, the physicochemical properties and chemical reactivity of ALA present some challenges. These may be addressed by incorporation of ALA units into a variety of prodrug systems, which also offer a way to improve the selectivity of ALA delivery, leading to enhanced PpIX accumulation and PDT effects. In this study, two novel and easy to assemble prodrug systems are described in which the delivery of ALA to specific cell types may be achieved using targeting with tumour-homing peptides.The first prodrug approach was based on a molecular core structure to which multiple ALA units (ALA dendron derivatives) are attached, and with ALA itself connected by an ester bond. The core structure was also linked to a targeting peptide prepared by solid phase synthesis, with selective peptide attachment to the core being achieved via Cu-catalysed click chemistry. This combines the concept of ALA dendrimers and ALA-peptide prodrugs. As proof of concept of this particular approach, prodrugs were investigated containing a bombesin-derived peptide that allows selective targeting of the GRP receptor (GRPR) which is overexpressed in a variety of tumours. A new generation of peptide-targeted ALA dendritic prodrug was successfully synthesized containing six ALA units, and the structure was subsequently optimized with respect to the attachment of the ALA dendrons and targeting peptide. Targeted ALA delivery and PpIX production with these prodrugs in GRPR-expressing PC3 cells was investigated by fluorescence spectroscopy and confocal fluorescence microscopy, and red and blue light-activated cell killing was evaluated using cell viability (MTT) assays. The effectiveness and selectivity of ALA delivery was demonstrated by comparison with parallel studies in HaCaT cells which have low GRPR expression. The generality of this prodrug system was also subsequently investigated and the efficient attachment of various tumour-homing peptide sequences to the optimised prodrug structure could be achieved, as well as novel ALA dendrons which increased the ALA payload to 18 units.
The second prodrug approach involved the incorporation of ALA into a cyclic peptide system. This consisted of three parts: a targeting peptide, an ALA unit, and a non-peptide bifunctional linker. ALA was connected to the linker through an ester bond, and the N-terminus of the targeting peptide was connected to a carboxylic acid function of the linker through an amide bond. The structure was then cyclized between the C-terminus of the peptide and the amino function of ALA to form the cyclic prodrug. A prototype cyclic peptide ALA prodrug was thus successfully synthesized, and the ALA delivery and PpIX production from this prodrug in PC3 and HaCaT cells were confirmed by fluorescence spectroscopy. The subsequent successful synthesis of a cyclic peptide prodrug based on an integrin-targeting RGD peptide proved the generality of this system and its potential as a means of enhanced ALA delivery for cancer detection and targeted therapy.
| Date of Award | 11 Sept 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Ian Eggleston (Supervisor) & Charareh Pourzand (Supervisor) |