HYALURONAN POLYMER BRUSH STRUCTURE, TOPOGRAPHICAL CONTROL, AND ITS RESPONSE TO THE ENVIRONMENT: CHARACTERIZING A NOVEL ULTRA-THICK BIOMATERIAL
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Polymer brushes are dense assemblies of end-grafted polymers which have a wide range of applications in lubrication, colloidal stabilization, surface functionalization, and fundamental polymer physics. This thesis focuses on developing and leveraging a new class of polymer brush generated by the enzyme, hyaluronan synthase. The hyaluronan brushes are tunable and can reach heights of up to ~22 µm – 2 orders of magnitude thicker than most brushes and more than one order of magnitude thicker than any previously reported brush. These ultra-thick brushes enable unprecedented characterization through direct visualization with confocal microscopy, as well as manipulation for future applications. In this thesis, I first establish control over brush synthesis, the ability to stop and start the brush growth, and demonstrate the inherent regenerative capability of the brushes. Then, building on those results, my focus is to elucidate and manipulate the internal brush structure and response to stimuli by exploiting the rapid characterization capabilities of confocal microscopy. Techniques to fluorescently label the brush were developed to acquire high-resolution concentration profiles of the hyaluronan brush versus brush height. The profiles are consistently convex and decay in an exponential-like fashion consistent with theoretical predictions for polydisperse brushes. When coupled with experimentally acquired molecular weight distribution, these profiles can be used to directly test theoretical polymer physics, especially in the domain of polyelectrolytes where theory is still being established. Next, spatial manipulation of the local brush grafting density was developed to enable precision patterning and sculpting of the topography of the thick brush. Careful studies revealed that the mechanism behind the grafting density alteration arises from the indirect laser deactivation of the HA synthase enzymes via the generation of reactive oxygen species from light-substrate interactions, specifically the bacterial membrane fragments containing the HA synthase. The patterning is most efficient at shorter wavelengths (405nm), but also can be achieved using wavelengths in the visible spectrum. The same technique can also be used to make binary-brush landscapes consisting of brush-rich and brush-free regions. The stimulus-responsiveness of the brush was explored as both an exercise in polymer physics, as well as for future materials applications. In response to varying salt concentration (NaNO3), the hyaluronan brushes reversibly traverse through the osmotic and salted brush regimes, while irreversibly collapsing in the presence of 90% ethanol, a poor solvent. Groundwork for future studies of the time-dependent nature of the stimulus response and of the dynamics of flow over the brushes has been established. These studies will be integral for potential applications such as anti-microbial implant coatings, where understanding the stimulus response time and flow-dependent decay of the brush will be key. The knowledge and tools gained in this work will aid in a spectrum of rich research arenas ranging from polymer physics to cell biophysics to materials science.