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A Hydrogel Glycoarray Platform As a Replacement for Conventional Research Tools to Study Galectin Bioactivity

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A Hydrogel Glycoarray Platform As a Replacement for Conventional Research Tools to Study Galectin Bioactivity A HYDROGEL GLYCOARRAY PLATFORM AS A REPLACEMENT FOR CONVENTIONAL RESEARCH TOOLS TO STUDY GALECTIN BIOACTIVITY By INHA BAEK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2018 © 2018 Inha Baek ACKNOWLEDGEMENTS I would like to thank my research advisor, Dr. Gregory Hudalla for his guidance and support over the course of this research. His assistance and suggestions were invaluable towards the progress of this research. I would also like to thank co-advisor, Dr. Benjamin Keselowsky for his helpful insights and invaluable suggestions throughout the course of this research. I would also like to thank Eric Hill for helping me with fabrication process. 3 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ............................................................................................... 3 LIST OF FIGURES .......................................................................................................... 5 ABSTRACT ..................................................................................................................... 6 CHAPTER 1 INTRODUCTION ...................................................................................................... 7 2 OBJECTIVE AND SPECIFIC AIM .......................................................................... 11 3 MATERIALS AND METHODS ................................................................................ 12 Materials ................................................................................................................. 12 Nanofiber Fabrication.............................................................................................. 12 Fabrication of Macroscopic PEGDA-Nanofiber Hydrogels ...................................... 13 WGA Binding to Macroscopic Hydrogel .................................................................. 13 Hydrogel Array Fabrication ..................................................................................... 14 WGA Binding to Array ............................................................................................. 14 4 RESULTS ............................................................................................................... 15 Hydrogel Array on Thiol-terminated Glass Coverslip .............................................. 15 Resistant to Non-specific WGA Binding .................................................................. 15 WGA Binding to Hydrogel Array ............................................................................. 15 Challenges in Scaling Down Process ..................................................................... 15 Insufficient Reaction Between Thiol and Acrylate ............................................. 15 Alternative Silane, Acrylate-PEG-silane ........................................................... 16 Deposition of Gold/Titanium ............................................................................. 16 5 DISCUSSION AND FUTURE WORKS ................................................................... 21 LIST OF REFERENCES ............................................................................................... 23 BIOGRAPHICAL SKETCH ............................................................................................ 25 4 LIST OF FIGURES Figure page 1-1 Lectin-binding to glycopeptide nanofibers. ......................................................... 10 1-2 Schematic overview of a hydrogel microarray .................................................... 10 2-1 Schematic overview of hydrogel array fabrication. ............................................. 11 4-1 5 μL PEGDA-nanofiber hydrogel array. .............................................................. 17 4-2 Non-fouling background of thiol-terminated glass coverslip. .............................. 17 4-3 WGA binding to PEGDA hydrogel without nanofiber .......................................... 18 4-4 WGA binding to PEGDA-GQ11 .......................................................................... 19 4-5 Schematic overview of surface chemistry ........................................................... 19 4-6 Deposition of Gold/Titanium on glass coverslip .................................................. 20 5 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science A HYDROGEL GLYCOARRAY PLATFORM AS A REPLACEMENT FOR CONVENTIONAL RESEARCH TOOLS TO STUDY GALECTIN BIOACTIVITY By INHA BAEK May 2018 Chair: Gregory Hudalla Major: Biomedical Engineering Galectins are a family of carbohydrate-binding proteins that modulate functions of various cell types by recognizing cell surface glycans. Physiological and pathological roles of galectins have been identified. Galectins recognize ECM glycans, but little is understood about the biological role of these interactions because in vitro studies have had to rely on naturally-derived reagents, such as Matrigel or isolated ECM glycoproteins. Naturally-derived reagents have two distinct limitations. First, they contain undefined carbohydrate contents varying dependent on the source and preparation. Second, current studies use galectin inhibitors to investigate the galectin- ECM interactions. These inhibitors interfere with galectin-ECM and galectin-cell interactions so they cannot provide a concrete relationship between galectin-ECM interactions and galectin bioactivity. To overcome these limitations, we propose galectin-binding hydrogel array as a screening tool to investigate the regulation of galectin bioactivity via interactions with ECM glycans. These arrays are based on poly(ethylene glycol) hydrogels formed in the presence of galectin-binding peptide nanofibers. 6 CHAPTER 1 INTRODUCTION Galectins are receiving interest as therapeutic targets due to their role as extracellular signals that modulate functions of various cells types via recognition of cell surface glycans. Galectins are a 15-member protein family of soluble β-galactoside- binding lectins that can be subdivided into non-covalent homodimers with identical CRDs, tandem dimers with distinct CRDs, and galectin-3 which assembles into an oligomer.1 Galectins can modulate various cellular behaviors including adhesion2, proliferation3, apoptosis4, and differentiation5. In terms of immune cells, for example, galectin-1 promotes dendritic cell migration,6 and mediates pre-B cell/stromal cell synapse formation leading to pre-B cell receptor clustering and signal initiation7. Galectin-3 regulates the function of T cell,8 mediates alternative activation of macrophages,9 and promotes neutrophil adhesion to laminin.10 In addition, galectins can regulate a variety of functions of naïve, effector, and regulatory T cells, including apoptosis and activation. Although there is increasing understanding of the role of galectins as biological signals, much less is understood about the role of galectin interactions with ECM glycans even though ECM glycoproteins are identified as galectin-binding sites.11 This is because current studies use naturally derived reagents, such as Matrigel and isolated ECM glycoproteins. There are two practical limitations of these research tools that interfere with understanding of galectin-ECM interactions. First, naturally-derived reagents have ill-defined glycan concentration which is dependent on the source and preparation. Second, galectin inhibitors are used in in vitro studies to investigate galectin-ECM interactions on cell behavior and these inhibitors perturb galectin-ECM 7 and galectin-cell interactions. Therefore, it’s hard to get a clear relationship between galectin-ECM interactions and galectin bioactivity. So, it’s important to develop a new research tool to study the galectin bioactivity independent of cell-galectin interactions or other ECM properties. To overcome these limitation, we developed lectin-binding nanofiber based on self-assembling peptide. The advantage of our nanofibers is that we can tailor carbohydrate contents according to the intended use. For instance, peptides that have different types of carbohydrate provide different nanofiber-lectin binding specificity. Additionally, different concentration ratio between co-assembly of carbohydrate- modified and non-modified peptides provides different nanofiber lectin-binding affinity. We previously developed a peptide that self-assembles into β-sheet nanofibers, QQKFQFQFEQQ, which is modified with N-acetylglucosamine, referred to as GlcNAc- Q11 or GQ11. GQ11 preferentially binds wheat germ agglutinin, a GlcNAc-binding plant lectin as shown in Figure 1-1.13 Nanofibers were fabricated by simply mixing of GQ11 and Q11 at various molar ratios based on previous observations.14 We have also developed microgels based on nanofibers with binding affinity for the plant lectin, WGA or wheat germ agglutinin.12 A limitation of studying how galectin interactions with ECM glycans regulate galectin bioactivity is the lack of cell culture reagents with galectin-binding properties that can be tailored by intended user. To address this limitation, here we report hydrogels were formed with bio-inert polymer, PEGDA with WGA-binding nanofibers that will be scaled down to microarray format as shown in Figure 1-2. The concentration 8 of WGA bound to hydrogels can be calculated using standard curve of concentration of nanofibers and lectin within bulk culture media. 9 A B C Figure 1-113.Lectin-binding
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