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Hedayati Colostate 0053A 15720.Pdf (6.821Mb) DISSERTATION DYNAMICS OF PROTEIN INTERACTIONS WITH NEW BIOMIMETIC INTERFACES: TOWARD BLOOD-COMPATIBLE BIOMATERIALS Submitted by Mohammadhasan Hedayati Department of Chemical and Biological Engineering In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Fall 2019 Doctoral Committee: Advisor: Matt J. Kipper Diego Krapf Melissa Reynolds Travis Bailey Copyright by Mohammadhasan Hedayati 2019 All Rights Reserved ABSTRACT DYNAMICS OF PROTEIN INTERACTIONS WITH NEW BIOMIMETIC INTERFACES: TOWARD BLOOD-COMPATIBLE BIOMATERIALS Nonspecific blood protein adsorption on the surfaces is the first event that occurs within seconds when a biomaterial comes into contact with blood. This phenomenon may ultimately lead to significant adverse biological responses. Therefore, preventing blood protein adsorption on biomaterial surfaces is a prerequisite towards designing blood-compatible artificial surfaces. This project aims to address this problem by engineering surfaces that mimic the inside surface of blood vessels, which is the only known material that is completely blood-compatible. The inside surface of blood vessels presents a carbohydrate-rich, gel-like, dynamic surface layer called the endothelial glycocalyx. The polysaccharides in the glycocalyx include polyanionic glycosaminoglycans (GAGs). This polysaccharide-rich surface has excellent and unique blood compatibility. We developed a technique for preparing and characterizing dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. The glycocalyx-mimetic surfaces were prepared by adsorbing heparin- or chondroitin sulfate-containing polyelectrolyte complex nanoparticles (PCNs) to chitosan-hyaluronan polyelectrolyte multilayers (PEMs). We then studied in detail the interactions of two important blood proteins (albumin and fibrinogen) with these glycocalyx mimics. Surface plasmon resonance (SPR) is a common ensemble averaging technique for detection of biomolecular interactions. SPR was used to quantify the amount of protein adsorption on these surfaces. Moreover, single-molecule microscopy along ii with advanced particle tracking were used to directly study the interaction of single molecule proteins with synthetic surfaces. Finally, we developed a groundwork for a kinetic model of long- term protein adsorption on biomaterial surfaces. In the first chapter, we thoroughly summarize the important blood-material interactions that regulate blood compatibility, organize recent developments in this field from a materials perspective, and recommend areas for future research. In the second chapter, we report preparation and characterization of dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. In the third chapter, we investigate how combining surface plasmon resonance, X-ray spectroscopy, atomic force microscopy, and single-molecule total internal reflection fluorescence microscopy provides a more complete picture of protein adsorption on ultralow fouling polyelectrolyte multilayer and polymer brush surfaces, over different regimes of protein concentration. In the fourth chapter, the interactions of two important proteins from blood (albumin and fibrinogen) with glycocalyx-mimetic surfaces are revealed in detail using surface plasmon resonance and single-molecule microscopy. Finally in the fifth chapter, the long-term protein interactions with different biomaterial surfaces are studied with single-molecule microscopy and iii ACKNOWLEDGMENTS I would like to express my sincere gratitude to all amazing scientists, students, staff, and coworkers during my Ph.D. First, I would like to recognize my advisor Dr. Matt J. Kipper for his exceptional role as a mentor and friend who never stopped me from working in different fields. Matt’s passion for science has inspired and given me the motivation and strength to follow my goals and overcome challenges. His endless patience and dedication to listen and improve my ideas helped me learn many valuable skills. Thank you for mentoring me and making my experience as your research assistant fun, enriching, and fulfilling. Second, I would like to thank my committee members, Dr. Diego Krapf, Dr. Melissa Reynolds, and Dr. Travis Bailey for their support and helpful insights. I am extremely grateful for having had the opportunity to work with these exceptional scientists. Diego, thank you being wonderful co-advisor and collaborator, for always making time for meetings and designing my TIRFM experiments. Melissa, thank you for your input and advice for designing surfaces. Travis, thank you for the great time that we had during polymers science and engineering course which I learnt a lot. Third, I thank my research colleagues past and present who assisted with the research or gave feedback on research presentations including Drs. Raimundo Romero, Xinran Xu, Jessi Vlcek, Tara Wigmosta, and Dafu Wang. I would especially like to thank undergraduate students Sarah Igli, Anugrah Mathew, and Natalie Rapp who worked patiently in the lab to prepare my samples. Fourth, I thank several CSU faculty and staff for their discussions, administrative assistance, equipment training, and use which were invaluable in this work. These include: Drs. iv Pat McCurdy and Karolien Denef from the Central Instrument Facility in the Department of Chemistry; Dr. Arun Kota from the Department of Mechanical Engineering; Dr. Ellen Brennan- Pierce, Tim Gonzalez, Claire Lavelle, and Denise Morgan from the Department of Chemical and Biological Engineering. Fifth, I must thank my funding sources which supported me during my time in graduate school which included the National Science Foundation, NTU foundation, and CSU Vice President for Research. The special acknowledgements go to my parents who always have taught me about science and instilled in me an appreciation for the education and opportunities I have received in my life. It has been very tough to be far away from them, but I know they are now very happy that their son is a scientist. Finally, I would like to thank my wife, for being there for me when I needed her, for her patience, for her positive energy, and for making every experience we live together. v DEDICATION To my family vi TABLE OF CONTENTS ABSTRACT .......................................................................................................................... ii ACKNOWLEDGMENTS ..................................................................................................... iv DEDICATION ...................................................................................................................... vi CHAPTER 1: THE QUEST FOR BLOOD-COMPATIBLE MATERIALS: RECENT ADVANCES AND FUTURE TECHNOLOGIES......................................................... 1 1.1. INTRODUCTION .................................................................................................. 2 1.1.1 THE BLOOD-MATERIAL INTERFACE IN MEDICAL DEVICES ................ 2 1.1.2 MATERIALS AT THE BLOOD-MATERIAL INTERFACE ……………..…... 4 1.2. THROMBOSIS AND INFLAMMATION ………………………………………. 5 1.2.1 COSTS, CONSEQUENCES, AND COMPLICATED MANAGEMENT OF THROMBOSIS ……………………………………………………………….………. 6 1.2.2 BIOLOGICAL MECHANISMS OF BLOOD CLOTTING ........................…… 8 1.2.2.1 PLATELETS …………………………………………………………………. 9 1.2.2.2 THE COAGULATION CASCADE ……………………………………..…… 13 1.2.3 INFLAMMATION ……………………………………………………………... 14 1.2.4. RED BLOOD CELLS AND HEMOLYSIS …………………………………… 19 1.3. PROTEIN INTERACTIONS WITH BIOMATERIALS ………………………... 19 1.3.1 FIBRINOGEN ………………………………………………………………….. 21 1.3.2 ALBUMIN ……………………………………………………………………… 22 1.3.3 VWF …………………………………………………………………………….. 22 1.3.4 COMPLEMENT ………………………………………………………………... 25 1.3.5 DRIVING FORCES OF PROTEIN ADSORPTION AND DENATURATION . 25 1.3.5.1 PROTEIN CHARACTERISTICS INFLUENCING ADSORPTION ………... 27 1.3.5.2 SOLUTION CHARACTERISTICS INFLUENCING PROTEIN ADSORPTION 28 1.3.5.3 SURFACE CHARACTERISTICS INFLUENCING PROTEIN ADSORPTION . 29 1.4. STRATEGIES FOR IMPROVING BLOOD COMPATIBILITY OF BIOMATERIALS …………………………………………………………………….. 31 1.4.1 POLYMER BRUSHES ………………………………………………………… 32 vii 1.4.1.1 PHYSICAL STRUCTURE AND CHEMISTRIES OF POLYMER BRUSHES ... 32 1.4.1.2 MECHANISMS OF PROTEIN RESISTANCE ……………………………… 34 1.4.1.3 TUNING PEG/PEO BRUSHES TO OPTIMIZE PROTEIN AND CELL RESISTANCE ………………………………………………………………………... 35 1.4.1.4 POXS AND POLY(2-OXAZINE)S ………………………………………….. 39 1.4.1.5 OTHER SYNTHETIC APPROACHES ……………………………………… 42 1.4.2 ZWITTERIONIC INTERFACES ………………………………………………. 44 1.4.2.1 MECHANISM OF PROTEIN RESISTANCE ……………………………….. 44 1.4.2.2 TUNING ZWITTERIONIC SURFACES TO OPTIMIZE BLOOD COMPATIBILITY …………………………………………………………………… 45 1.4.3 BIOACTIVE SURFACES ……………………………………………………… 47 1.4.3.1 HEPARIN AND HEPARIN MIMICS ……………………………………….. 48 1.4.3.2 DIRECT THROMBIN INHIBITORS AND IMMUNOMODULATORS …... 54 1.4.3.3 DRUG RELEASE ……………………………………………………………. 56 1.4.3.4 OTHER BIOPOLYMERS …………………………………………………… 59 1.4.4 BIOINSPIRED SURFACES …………………………………………………… 62 1.4.4.1 SUPERHYDROPHOBIC (SH) SURFACES ………………………………… 64 1.4.4.2 LUBRICANT-INFUSED SURFACES ………………………………………. 67 1.4.5 GLYCOCALYX-MIMETIC SURFACES ……………………………………... 71 1.4.6 DELIVERY OF THE SMALL MOLECULE NO ……………………………... 77 1.4.6.1 NO IN THE CARDIOVASCULAR SYSTEM ………………………………. 78 1.4.6.2 MATERIALS WITH NO-DONATING GROUPS …………………………... 82 1.4.6.3 CATALYTIC GENERATION OF NO ………………………………………. 88 1.5. SUMMARY AND OUTLOOK FOR FUTURE RESEARCH ………………….. 96 REFERENCES …………………………………………………………………................
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