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<p>Sight-threatening diseases, such as age-related macular degeneration (AMD), affect the tissues of the posterior segment of the eye. Though modern classes of biomolecular based drugs are therapeutically useful, drug targeting for prolonged bioavailability to pathological sites within the eye is challenging. Current delivery approaches are invasive and lack control over drug release rates and tissue-specific localization. In this thesis, a device using microneedles embedded in a flexible platform was developed that could potentially overcome these challenges.</p> <p>New methods for microneedle fabrication were developed by co-opting simple chemical etch methods commonly used for optical probe fabrication as an alternative to current complex and expensive photolithographic technologies to produce out-of-plane, high aspect ratio microneedles which are often constrained materially to silicon and metal. Microneedles with repeatable tip and taper sizes were obtained using hydrofluoric acid, an organic phase and fused-silica capillary tubing. Microneedles with 10 um tips were made using single and batch mode methods and were then integrated into poly (dimethylsiloxane) (PDMS) for alignment using low cost micromolding approaches offering the same degree of accuracy provided by conventional photolithography<strong>. </strong></p> <p>Single microneedle-based devices successfully delivered rhodamine intrasclerally, intravitreally, suprachoroidally and to the retina. This is the first demonstration of active delivery to specific spatial regions within the posterior eye at controllable rates using a non-implantable, biocompatible device – with minimal fabrication facilities, equipment and cost. The fabricated device demonstrated a new hybrid approach of coupling a rigid microneedle with a soft and pliable substrate that could conform to biological tissues.</p> / Doctor of Philosophy (PhD)

Pseudomonas Aeruginosa is an opportunistic bacterium which is involved in nosocomial infections and which causes increasing concern in healthcare due to its high antibiotic resistance. During an infection by P.aeruginosa, the bacteria proliferate in the host’s organism by leveraging motility abilities. One of them, twitching motility, is a surface-specific translocation system. To power twitching, P.aeruginosa performs cycles of extension, attachment, and retraction of Type IV pili (T4P), which are long and thin extracellular filaments. On top of allowing cellular traction, T4P can transmit a signal induced by a contact with a solid surface leading to specific biological responses. In particular, we suspect that T4P retraction is triggered in very short timescales by the attachment of the tip of the pilus to a surface. However, the nature of the signal generated by surface contact, and how it is sensed by the cell, are unknown. The aim of this master project is to gain knowledge on how this machinery is coordinated by the cell upon solid surface contact, and particularly investigate the signal induced by surface contact.  To study T4P behavior during extension and retraction, we used label-free interferometric scattering microscopy (iSCAT), a high time and spatial resolution microscopy technology. We aimed at bringing out T4P movements by attaching bacteriophages on their body and tracking their relative position over extension and retraction events. To do so, we first fabricated microstructures in which we confined the cells in order to improve image quality and increase the odds of binding bacteriophages to T4P. Then, we were able to visualize bindings of DMS3 bacteriophages on T4P. During extension and retraction, we identified phage lateral movements around the pilus axis, which we were unable to see on non-retracting pili. Our results therefore support the hypothesis of T4P helical movements. The nature of the signal generated by tip contact and how it is sensed by the cell remains to be elucidated. Nevertheless, we elevated iSCAT to a new application by visualizing the interaction between T4P and bacteriophages.