Abstract
Bioorthogonal chemistry, encompasses a class of rapid, selective, non-toxic, and high yielding click reactions that can occur in living systems without interfering with native biochemical reactions. Bioorthogonal click chemistry-based stimuli-responsive hydrogels are being investigated for multiple clinical applications ranging from prodrug activation to controlled and targeted drug delivery of cytotoxic drugs. Phe-Phe motifs, popularly known as low molecular weight hydrogelators (LMWHs) and polysaccharide-based high molecular weight (HMW) polymers have been used for designing and tailoring the stimuli-responsiveness, mechanical strength and cytocompatibility of hydrogels for tissue engineering, wound healing and targeted drug delivery applications.
There is growing need for exploring and understanding the mechanisms involved in self-aggregation and degradation of the peptides and polymers used to formulate hydrogels. Insights into the types of interactions involved in self-assembly of peptides; the trigger responsiveness of self-immolative (1,4- and 1,6-) elimination mechanisms; and modification of LMWHs and polymers; will facilitate the de novo development of responsive hydrogels with improved drug/prodrug delivery properties. In addition this thesis extends the spectrum of stimuli-responsive hydrogels into systems specifically designed for triggered dissolution and spatio-temporal delivery of prodrugs utilising a trans-cyclooctene (TCO)-tetrapeptide-based hydrogel and a “click-to-target-and-release” approach.
In Chapter Two, a library of 17 azido-functionalised N-capped di/tetrapeptides (second-generation) LMWHs were designed, synthesised and then used to formulate bioorthogonal responsive sustained release “click-to-dissolve” hydrogels with improved mechanical strength and cytocompatibility of hydrogel. A 1,4-immolating N-capping group (14 and 15) was designed for conjugation to di/tetrapeptides, which were varied in order to optimise hydrogel mechanical strength and cytocompatibility. The strength of the peptide hydrogel was impacted by the substitution of –F or –OH on the phenylalanine amino acid of tetrapeptides. Substitution (second phenylalanine in tetrapeptide sequence) at the 4th (para) position with –OH (i.e., tyrosine in place of phenylalanine, 10 (GFYG)) yielded weak gels while fluorine substitution at the 4th (para) position (8 (GF(-4F)FG)) favoured the formation of strong gels. Customisation of the peptide backbone by incorporation of glycine amino acid, improved cell survival (20-fold for 35b). Triggered dissolution of 1,4-immolating hydrogels (15b) was examined and was found as hypothesised, to be slow and incomplete. Future studies to improve the injectability of the modified peptide-based hydrogel library is required in order to aid in vivo applications.
The “click-to-dissolve” approach was then investigated using thermoresponsive-bioorthogonal bipolymer-based (sodium alginate and Pluronic F-127) hydrogels (63) which could be delivered by injection (Chapter Three). Triggering for hydrogel dissolution was designed to be induced using trans-cyclooctene (TCO) and/or H2S. Following in vitro characterisation, a series of in vivo experiments were performed using dye-loaded gels to examine triggered release. The impact of varying the amount, timing and route of TCO administration, as well as the impact of a local solid tumour, on release was investigated. No significant triggered hydrogel dissolution was observed which may be due to low local concentrations of TCO and insufficient H2S production by solid tumours.
It was also demonstrated that the simple synthetic steps employed in developing the second-generation “click-to-dissolve” hydrogelators provided an opportunity for developing dual “click-to-target-and-release” prodrugs (Chapter Four). Peptide-based LMWHs were functionalised with TCO while two trigger groups were incorporated in the prodrug, the first being a tetrazine that would rapidly react with the TCO in the gel via a bioorthogonal inverse-electron demand Diels-Alder reaction, thus localising the drug at the site of hydrogel injection (click-to-target). The second trigger group was an azide that could react rapidly with both exogenous or endogenous stimulus by 1,3-dipolar cycloaddition (TCO) or azide reduction (H2S) to release active drug (click-to-release). Proof-of-concept (click-to-target-and-release) was shown with second generation prodrugs (utilising 1,6-self-elimination) and composite peptide:polymer hydrogels. However, composite hydrogel strength was poor and it was hypothesised that inclusion of the TCO moiety on the LMWHs negatively impacted the complementary N-terminal π–π interactions and self-assembly.
This thesis reports on the development of sophisticated triggerable self-assembling peptide and thermoresponsive polymer-based hydrogels with the ability to control where and when drug is released. This data provided important insights into this new and interesting area of stimuli responsive hydrogels for improved drug delivery of anticancer drugs.