UUnniivveerrssiittyy ooff MMaassssaacchhuusseettttss AAmmhheerrsstt SScchhoollaarrWWoorrkkss@@UUMMaassss AAmmhheerrsstt Doctoral Dissertations Dissertations and Theses March 2015 CCeellll AAddhheessiioonn BBiioopphhyyssiiccss oonn DDyynnaammiicc PPoollyymmeerr CCoonnssttrruuccttss Andreas Kourouklis University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/dissertations_2 Part of the Biology and Biomimetic Materials Commons, Biomaterials Commons, Cell Biology Commons, and the Polymer Science Commons RReeccoommmmeennddeedd CCiittaattiioonn Kourouklis, Andreas, "Cell Adhesion Biophysics on Dynamic Polymer Constructs" (2015). Doctoral Dissertations. 309. https://doi.org/10.7275/6285925.0 https://scholarworks.umass.edu/dissertations_2/309 This Open Access Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks@UMass Amherst. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. CELL ADHESION BIOPHYSICS ON DYNAMIC POLYMER CONSTRUCTS A Dissertation Presented by ANDREAS P. KOUROUKLIS Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY February 2015 Chemical Engineering (cid:13)c Copyright by Andreas P. Kourouklis 2015 All Rights Reserved CELL ADHESION BIOPHYSICS ON DYNAMIC POLYMER CONSTRUCTS A Dissertation Presented by ANDREAS P. KOUROUKLIS Approved as to style and content by: Harry Bermudez, Chair David Hoagland, Member Shelly Peyton, Member John Collura, Interim Department Head Chemical Engineering To those who remained loyal ACKNOWLEDGMENTS This thesis was completed under the mentorship of Professor Bermudez. I am grateful to him for his ongoing support and advice in academic and personal issues. I appreciate his time, ideas and funding that made my Ph.D. experience a constructive and stimulating journey. As characteristic of our interaction, I recollect our amusing discussion on block copolymer thermodynamics during our flight to the spring MRS at San Francisco. I am also thankful to my research committee members, Professor Dave Hoagland and Professor Shelly Peyton. They generously shared their feedback toward the measurement of film mobility and the characterization of cell adhesion responses. Moreover, I am grateful for their academic support toward my future professional goals. I would like to acknowledge the Department of Chemical Engineering, the Depart- ment of Polymer Science and Engineering and the Graduate School of the University of Massachusetts at Amherst for the opportunities they provided to me. I am par- ticularly indebted to Professor McCarthy’s group for their generosity to use their lab facilities toward the creation of supported polymer films. The measurements of film mobility were performed at the central microscopy facility upon the constructive comments and discussion of Dale Callaham. In addition, I would like to thank my past and present colleagues in Bermudez groupwithwhomIdevelopedstrongprofessionalandfriendshiprelationships. Irecall with gratitude the days I spent with Ronald Lerum, Lang Chen, Phapanin Charoen- phol (Ploy), Stephen Strassburg, Sandipan Dawn, Purnendu Kumar Nayak, Laura v Lanier, Jung Won, Adam Hathorne and Treniece Terry sharing our plans, concerns and joys. I would like to specifically acknowledge Ronald Lerum who was an excel- lent mentor and lab partner. Together, we worked for a couple of years toward the development of mobile polymer films and biophysical protocols. I would also like to express my gratitude to Sandipan Dawn, Jacob John and Professor Ken Carter for their encouragement and critical role toward the fabrication of substrates with spatially arranged fibronectin dots. In addition, I would like to thank Alison Douglas (Barker group, Georgia Tech) and Katrina Adlerz (Aranda-Espinoza group, Univer- sity of Maryland) for additional cell-film experiments that enhanced my intuition on the mechano-sensing of film mobility. Furthermore, I wish to express my sincere gratitude to those who shared parts of their life with me in Amherst and USA. I own them the feeling of being surrounded by people who care about me. I am especially grateful to Professor Mountziaris for his generosity and mentorship in various aspects of my life. Finally, I want to acknowledge my extended family back home and specifically my parents and my sister for their altruistic love. Their constant support and presence gives me the priceless right to keep pursuing my distant Ithaca. vi ABSTRACT CELL ADHESION BIOPHYSICS ON DYNAMIC POLYMER CONSTRUCTS FEBRUARY 2015 ANDREAS P. KOUROUKLIS DIPLOMA, NATIONAL TECHNICAL UNIVERSITY OF ATHENS Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Harry Bermudez The biophysical characteristics of cell adhesion from single protein to cell length scales have primarily been studied using purely elastic substrates. However, natu- ral extracellular matrix (ECM) is viscoelastic and contains mobile components. In this work, we combined chemistry and cell biology tools to design and characterize laterally mobile viscoelastic polymer films that promote receptor-specific cell adhe- sion. Moreover, we used amphiphilic block copolymers that are end-labeled with RGD peptide ligands to allow for integrin-mediated cell adhesion. The addition of a trace hydrophobic homopolymer in the supported bilayer block-copolymer films is used to tune the lateral mobility of the films. NIH 3T3 fibroblasts demonstrate a non- linear spreading response against the mobility of the RGD-displaying polymer films. Employing immunostaining and adhesion strength assays, we decoupled the partial contributions of focal adhesions (FA) and integrin-RGD complexes on cell adhesion. vii Furthermore, we employed these biomimetic polymer platforms to investigate the im- portanceofviscousdissipationwithintheextracellularsubstrateanditsconnectionto cell-surface receptors. Our results suggest that cells preferentially use α β and α β v 3 5 1 integrins to control spreading and polarization in response to mechanical properties of their substrate. In order to further control the spatial presentation of biochemical molecules on mechanically-tunable polymer substrates, we successfully transferred fi- bronectin patterns on bilayer polymer films. We showed that NIH 3T3 fibroblasts spreading and adhesion features depend on the mechanical properties of these hy- brid materials even in the presence of spatially and chemically identical biochemical signals. Overall, the present work demonstrates the potential of amphiphilic block copolymers to form artificial substrates that can capture a key feature of cell-ECM interactions: specifically, the ability of cells to induce changes in the substrate over time. Furthermore, it highlights the need for future studies on cell-substrate interac- tions that simultaneously consider the time-dependent mechanical properties of the ECM, the spatial characteristics of ligand presentation, and the receptor-mediated intracellular signaling. viii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ............................................. v ABSTRACT......................................................... vii LIST OF TABLES .................................................... xi LIST OF FIGURES.................................................. xii CHAPTER 1. INTRODUCTION ................................................. 1 1.1 Extracellular matrix (ECM) and cell adhesion ........................1 1.2 Biomaterials for artificial ECM mimics ..............................3 1.3 Block copolymers.................................................9 1.4 Fabrication and characterization of supported polymer films ............9 1.5 Outline ........................................................11 2. CELL ADHESION ON MOBILE POLYMER FILMS ............. 14 2.1 Introduction ....................................................14 2.2 Results.........................................................17 2.2.1 Interfacial studies .........................................17 2.2.2 Measurement of lateral mobility in supported polymer films..................................................20 2.2.3 Cell spreading response to substrate mobility .................24 2.2.4 Focal adhesion responses to substrate mobility ................26 2.2.5 Contractility-independent mechanisms of cell adhesion .........28 2.3 Discussion ......................................................31 2.3.1 Substrate mobility as a step towards mimicry of the ECM ......31 2.3.2 Distinct modes of cell spreading due to substrate mobility ......32 2.4 Conclusions.....................................................33 ix