Nucleophilic thiol-yne addition chemistry for the synthesis of tuneable and cytocompatible poly(ethylene glycol) hydrogel materials

This thesis explores the nucleophilic thiol-yne reaction as a crosslinking method for the synthesis of hydrogel materials under biologically relevant conditions. The reaction, using simple functional groups, can be carried out without the use of an external catalyst. This thesis aims to portray the immense potential this reaction has in creating hydrated polymer networks for a wide range of biomedical applications. In the review of the literature (Chapter 1), the popularity and future of hydrogels in tissue engineering has been discussed and the advantages of using alkyne functional groups to crosslink polymers has been highlighted. The main aim of this thesis is to further develop the nucleophilic thiol-yne reaction and to prepare poly(ethylene glycol) (PEG) hydrogel materials with superior performance for application as tissue engineered scaffolds (e.g. extracellular matrix (ECM) mimics or injectable scaffolds). This aim has been approached through a variety of experimental pathways in this thesis demonstrating the suitability of this reaction in the biomaterials field. In Chapter 2, the nucleophilic thiol-yne reaction has been presented as a highly efficient chemistry for producing robust, high water content hydrogels which could be repeatably compressed without hysteresis. Through a straightforward blending process of PEG thiol precursors, the material properties were easily tuned to a range of relevant biological environments. In a similar manner, using the PEG precursors to tune the resultant properties, Chapter 3 addresses the swelling profiles of the thiol-yne hydrogels. By increasing the number of hydrophobic crosslinking points within the networks, nonswelling, cytocompatible hydrogel material were created when immersed in aqueous environments. The monoaddition product of the nucleophilic thiol-yne reaction results in a vinyl thioether bond which can favour different isomers, depending on the reaction conditions. To exploit this in hydrogel synthesis, Chapter 4 describes the formation of sterecontrolled hydrogels. Significantly, an impressive range of mechanical properties was achieved, without affecting the structure or swelling behaviour of the materials. To achieve a structure with more advantageous properties (e.g. self-healing and stretchability) thiol-yne interpenetrating networks (IPNs) were synthesised through the inclusion of natural polymer hydrogels (Chapter 5). These IPNs achieved the advantageous properties required in a simple and effective manner, while retaining the characteristics already exhibited by these materials. To improve on this aim, the thiol-yne PEG hydrogels successfully encapsulated breast cancer cells with enhanced viability compared to the widely used radical thiol-ene reaction (Chapter 6). Controlled matrix degradation allowed for cell proliferation and the formation of cell clusters. Chapter 7 investigates the kinetics of the nucleophilic thiol-yne reaction with different activating groups (e.g. adjacent group to the alkyne), to reduce the toxicity of the PEG alkyne precursors and degradation of the resultant thiol-yne hydrogels. This chapter highlights key requirements of the functionalisation reaction to form alkyne and thiol precursors for successful hydrogel synthesis. Chapter 8 provides a summary of the key findings from Chapters 2-7 and Chapter 9 states the experimental procedures of this thesis

A bird's eye view: using geographic analysis to evaluate the representativeness of corvid indicators for West Nile virus surveillance

BACKGROUND: The objective of this evaluation was to determine whether reports of dead corvid sightings and submissions of dead corvids for West Nile virus testing were representative of true corvid mortality in British Columbia in 2004, a year with no West Nile virus activity, in order to ensure the system was accurately describing corvid mortality rather than reflecting regional differences in surveillance methods. RESULTS: Local Health Areas reported 0–159 (median = 3) dead corvid sightings and 0–209 (median = 5) submissions for West Nile virus testing. The expected numbers of dead corvid sightings and submissions for testing from each Local Health Area were 0–232 (median = 3) and 0–258 (median = 4), respectively. Twelve Local Health Areas reported significantly fewer sightings than expected; 21 reported significantly more. Eleven Local Health Areas submitted significantly fewer corvids than expected; 26 submitted significantly more. CONCLUSION: Some Local Health Areas were over-represented and others under-represented in terms of corvid West Nile virus surveillance indicators. Recommendations were made to improve the representativeness of corvid surveillance data. Geographic analysis can be used to evaluate the representativeness of surveillance systems and result in improvements to surveillance

Risk Factors for Cryptococcus gattii Infection, British Columbia, Canada

To determine whether particular environmental, medical, or behavioral risk factors existed among Cryptcoccus gattii–infected persons compared with the general population, we conducted a sex-matched case−control study on a subset of case-patients in British Columbia (1999–2001). Exposures and underlying medical conditions among all case-patients (1999–2007) were also compared with results of provincial population–based surveys and studies. In case−control analyses, oral steroids (matched odds ratio [MOR] 8.11, 95% confidence interval [CI] 1.74–37.80), pneumonia (MOR 2.71, 95% CI 1.05–6.98), and other lung conditions (MOR 3.21, 95% CI 1.08–9.52) were associated with infection. In population comparisons, case-patients were more likely to be >50 years of age (p<0.001), current smokers (p<0.001), infected with HIV (p<0.001), or have a history of invasive cancer (p<0.001). Although C. gattii is commonly believed to infect persons with apparently healthy immune systems, several immunosuppressive and pulmonary conditions seem to be risk factors

Challenges of Investigating Community Outbreaks of Cyclosporiasis, British Columbia, Canada

Investigations of community outbreaks of cyclosporiasis are challenged by case-patients’ poor recall of exposure resulting from lags in detection and the stealthy nature of food vehicles. We combined multiple techniques, including early consultation with food regulators, traceback of suspected items, and grocery store loyalty card records, to identify a single vehicle for a cyclosporiasis outbreak in British Columbia, Canada, in 2007

Epidemiology of Cryptococcus gattii, British Columbia, Canada, 1999–2007

Incidence is high, but the predominant strain does not seem to cause greater illness or death than do other strains

Protective Behavior Survey, West Nile Virus, British Columbia

We investigated personal protective behaviors against West Nile virus infection. Barriers to adopting these behaviors were identified, including the perception that DEET (N,N-diethyl-m-toluamide and related compounds) is a health and environmental hazard. Televised public health messages and knowing that family or friends practiced protective behaviors were important cues to action

Nonswelling thiol-yne crosslinked hydrogel materials as cytocompatible soft tissue scaffolds

A key drawback of hydrogel materials for tissue engineering applications is their characteristic swelling response, which leads to a diminished mechanical performance. However, if a solution can be found to overcome such limitations, there is a wider application for these materials. Herein, we describe a simple and effective way to control the swelling and degradation rate of nucleophilic thiol-yne poly(ethylene glycol) (PEG) hydrogel net- works using two straightforward routes: using multiarm alkyne and thiol terminated PEG precursors or introducing a thermores- ponsive unit into the PEG network while maintaining their robust mechanical properties. In situ hydrogel materials were formed in under 10 min in PBS solution at pH 7.4 without the need for an external catalyst by using easily accessible precursors. Both pathways resulted in strong tunable hydrogel materials (compressive strength values up to 2.4 MPa) which could effectively encapsulate cells, thus highlighting their potential as soft tissue scaffoldsPostprint (published version