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DESIGN AND DEVELOPMENT OF SILK-ELASTINLIKE PROTEIN POLYMER LIQUID EMBOLICS FOR TREATMENT OF HEPATOCELLULAR CARCINOMA by Azadeh Poursaid A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Bioengineering The University of Utah August 2016 Copyright © Azadeh Poursaid 2016 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Azadeh Poursaid has been approved by the following supervisory committee members: Hamidreza Ghandehari Chair 04.25.16 Date Approved Joseph Cappello Member 04.25.16 Date Approved Jindrich Kopecek Member 04.25.16 Date Approved Eugene Huo Member 04.25.16 Date Approved Bruce Gale Member 04.25.16 Date Approved and by Patrick Tresco Chair/Dean of the Department/College/School o f ________________ Bioengineering and by David B. Kieda, Dean of The Graduate School. ABSTRACT Globally, hepatocellular carcinoma (HCC) of the liver is diagnosed in over 700,000 people annually and trends indicate increasing prevalence. The majority of cases, >80%, are detected at advanced stages where systemic chemotherapies have little efficacy. The primary curative treatment is liver transplant, but if a donor liver is not available, only palliative care such as transarterial chemoembolization (TACE) is possible. TACE targets the tumor blood supply. An embolic containing a chemotherapeutic agent is injected into the tumor's vasculature via an endovascular catheter, subsequently shutting down blood flow while delivering localized chemotherapy. A presently approved product, Lipiodol®, is an oily emulsion mixed with a chemotherapeutic used in conjunction with gelatin particles or synthetic polymer beads that act as emboli. Calibrated spherical drug eluting beads are now gaining favor for this procedure, replacing the multistep oil emulsion system. These beads, however, have shortcomings: aggregation of smaller diameter beads, fracturing of beads while under strain in the catheter, off target embolization particularly in pulmonary circulation, elution of only charged small molecule therapeutics, nondegradability, limited tumor depth penetration, and revascularization induced by a hypoxic state. To address these limitations, a genetically engineered silk-elastinlike protein polymer (SELP) system was developed to create a liquid-to-solid embolic agent capable of retaining and releasing a wider range of therapeutics, controlled degradation into nontoxic amino acids, and soluble until injected into the body where they transition irreversibly to a solid hydrogel network. This provides potential for ideal injectability as a low viscosity fluid at room temperature followed by optimal embolization by a highly stable hydrogel at body temperature. The proposed research involved engineering a SELP formulation with suitable viscosity for injection into the tumor vasculature via a microcatheter and a suitable gelation rate and gel strength for stable embolization. The drug release properties of the polymer matrix were determined for small molecule chemotherapeutics such as doxorubicin and anti-angiogenic sorafenib. Preliminary in vivo performance of the novel system for TACE was evaluated using a rodent model. Future directions include expansion of in vivo studies, particularly in an animal model for HCC and TACE to study therapeutic efficacy and long­ term biocompatibility. iv I dedicate this thesis to my grandmothers who started addressing me as “doctor” from the advent of my dream of higher education dating to before the new millennium. TABLE OF CONTENTS ABSTRACT............................................................................................................................iii LIST OF FIGURES............................................................................................................. viii LIST OF TABLES....................................................................................................................x ABBREVIATIONS............................................................................................................... xi ACKNOWLEDGMENTS .....................................................................................................xv Chapters 1. INTRODUCTION............................................................................................................. 1 1.1 Introduction............................................................................................................ 1 1.2 Silk-elastinlike protein polymers for localized tumor therapy.......................... 3 1.3 Aims and scope of this dissertation..................................................................... 4 1.4 References............................................................................................................ 10 2. LITERATURE BACKGROUND...................................................................................13 2.1 Introduction.......................................................................................................... 13 2.2 Commercially available materials...................................................................... 21 2.3 Research and development of advanced materials...........................................45 2.4 Conclusions.......................................................................................................... 63 2.5 References............................................................................................................ 72 3. IN SITU GELLING SILK-ELASTINLIKE PROTEIN POLYMER FOR TRANSARTERIAL CHEMOEMBOLIZATION........................................................87 3.1 Introduction.......................................................................................................... 87 3.2 Materials and methods........................................................................................ 90 3.3 Results...................................................................................................................95 3.4 Discussion.......................................................................................................... 109 3.5 Conclusion......................................................................................................... 115 3.6 References.......................................................................................................... 116 4. SILK-ELASTINLIKE PROTEIN POLYMER LIQUID CHEMOEMBOLIC FOR LOCALIZED RELEASE OF DOXORUBICIN AND SORAFENIB......................119 4.1 Introduction........................................................................................................119 4.2 Experimental section......................................................................................... 124 4.3 Results................................................................................................................ 131 4.4 Discussion.......................................................................................................... 149 4.5 Conclusion......................................................................................................... 157 4.6 References.......................................................................................................... 157 5. CONCLUSIONS AND FUTURE DIRECTIONS......................................................162 5.1 Conclusions........................................................................................................162 5.2 Challenges and future directions...................................................................... 168 5.3 References.......................................................................................................... 172 Appendices A. PRELIMINARY IN VIVO EVALUATION OF SELP CHEMOEMBOLIC IN A RAT HEPATOMA MODEL....................................................................................... 175 B. ATR-FTIR EVALUATION OF DRUG LOADED SELP-815K GELS.......................................................................................................193 vii LIST OF FIGURES 1.1 Diagram of SELP-47K and SELP-815K protein polymers......................................... 5 1.2 Diagrammatic representation of TACE using DEBs to treat HCC as compared to a SELP liquid embolic that is capable of co-delivery of single to multiple drugs........7 2.1 Schematic comparing physical properties of four commercially available embolic microspheres used in bland embolization procedures.................................................35 2.2 Schematics demonstrating loading mechanisms of two commonly used drug eluting microspheres__________________________________________________ 39 2.3 Mechanisms of in situ gelation for liquid embolics__________________________ 57 2.4 General biosynthetic strategy of recombinant polymers.............................................69 3.1 Three-dimensional model of the microfluidics device designed and fabricated to represent the vasculature of a hepatic tumor_______________________________ 96 3.2 Viscosity traces of candidate polymer compositions................................................. 99 3.3 Rheological characterization of candidate polymer compositions......................... 100 3.4 Comparison of the rheological characteristics between candidates____________102 3.5 Schematic of the in vitro test setup.............................................................................103 3.6 12% w/w sheared SELP-815K tested in vivo in male New Zealand

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