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Controlled Delivery of Bioactive Factors for Tissue Regeneration

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Controlled Delivery of Bioactive Factors for Tissue Regeneration CONTROLLED DELIVERY OF BIOACTIVE FACTORS FOR TISSUE REGENERATION by Noah Ray Johnson Bachelor of Science, The Pennsylvania State University, 2010 Submitted to the Graduate Faculty of Swanson School of Engineering in partial fulfillment of the requirements for the degree of Ph.D. in Bioengineering University of Pittsburgh 2014 UNIVERSITY OF PITTSBURGH SWANSON SCHOOL OF ENGINEERING This dissertation was presented by Noah Ray Johnson It was defended on November 25, 2014 and approved by William J. Federspiel, Ph.D., Professor, Department of Bioengineering Patricia A. Hebda, Ph.D., Professor, Departments of Otolaryngology and Pathology Alan Wells, M.D., DMSc, Professor, Department of Pathology Dissertation Director: Yadong Wang, Ph.D., Professor, Department of Bioengineering ii Copyright © by Noah Ray Johnson 2014 iii CONTROLLED DELIVERY OF BIOACTIVE FACTORS FOR TISSUE REGENERATION Noah Ray Johnson, Ph.D. University of Pittsburgh, 2014 Growth factors have enormous clinical potential as they orchestrate all repair and regenerative processes in the body. However, their half-lives in vivo when applied alone are very short, on the order of minutes to hours. Therefore, large doses and multiple applications are necessary which is expensive and raises safety concerns considering their potency. To address this issue we developed a coacervate delivery system which protects growth factors from degradation and sustains and localizes their release at the site of injection. By imitating the native signaling environment involving ligands, proteoglycans, and cell receptors our delivery vehicle also enhances growth factor bioactivity, enabling the use of minute and clinically-safe dosages. This dissertation describes the translational potential of this growth factor therapy to address three significant clinical needs: 1) Diabetic wound healing, 2) Cardiac repair following myocardial infarction, 3) Bone regeneration for congenital defects and trauma. iv TABLE OF CONTENTS 1.0 INTRODUCTION ............................................................................................................... 1 1.1 REGENERATIVE THERAPIES: INSIGHTS AND CHALLENGES ................. 1 1.2 CONTROLLED PROTEIN DELIVERY ................................................................ 3 1.3 DRUG DELIVERY SYSTEMS FOR WOUND HEALING .................................. 4 1.3.1 Hydrogels ........................................................................................................ 6 1.3.2 Scaffolds .......................................................................................................... 9 1.3.3 Particles ......................................................................................................... 12 1.3.4 Complexes and Conjugates ......................................................................... 16 1.3.5 Coacervates ................................................................................................... 18 1.3.6 Future goals .................................................................................................. 19 1.4 THE POTENTIAL OF COACERVATE SYSTEMS IN DRUG DELIVERY ... 20 1.4.1 Elastin-like peptides ..................................................................................... 21 1.4.2 Carboxymethyl chitosan-based coacervates .............................................. 22 1.4.3 Mussel adhesive proteins ............................................................................. 23 1.4.4 The future of coacervate delivery vehicles ................................................. 23 1.5 A POLYCATION:HEPARIN COACERVATE DELIVERY VEHICLE .......... 24 1.6 SPECIFIC AIMS...................................................................................................... 25 2.0 DEVELOPMENT OF A POLYCATION:HEPARIN VEHICLE WITH TUNABLE RELEASE KINETICS ...................................................................................................... 28 v 2.1 INTRODUCTION .................................................................................................... 28 2.2 MATERIALS AND METHODS ............................................................................ 31 2.2.1 Materials ....................................................................................................... 31 2.2.2 Synthesis of PELD ........................................................................................ 31 2.2.3 Characterization of PELD ........................................................................... 32 2.2.4 Preparation of polycation:heparin coacervates ......................................... 33 2.2.5 Zeta potential and dynamic light scattering (DLS) measurements ......... 33 2.2.6 Fluorescent imaging of PELD coacervates ................................................ 33 2.2.7 Cytotoxicity assay ......................................................................................... 34 2.2.8 BSA release assay ......................................................................................... 34 2.2.9 Coacervate adsorption to polymer scaffolds .............................................. 35 2.2.10 Scanning electron microscopy ..................................................................... 35 2.2.11 Statistical analysis ........................................................................................ 36 2.3 RESULTS AND DISCUSSION .............................................................................. 36 2.3.1 PELD synthesis and characterization ........................................................ 36 2.3.2 Characterization of PELD:heparin coacervates ....................................... 39 2.3.3 PELD cytocompatibility .............................................................................. 42 2.3.4 Controlled release from PELD coacervates ............................................... 44 2.3.5 Coacervate coating of polymeric scaffolds ................................................. 46 2.4 CONCLUSIONS ...................................................................................................... 48 3.0 CONTROLLED DELIVERY OF HB-EGF TO ACCELERATE DERMAL WOUND HEALING .......................................................................................................................... 50 3.1 INTRODUCTION – NORMAL WOUND HEALING ......................................... 50 3.2 MATERIALS AND METHODS – NORMAL WOUND HEALING ................. 52 3.2.1 HB-EGF coacervate preparation ................................................................ 52 3.2.2 Fluorescent imaging of the coacervate ....................................................... 52 vi 3.2.3 HB-EGF release profile ............................................................................... 53 3.2.4 In vitro scratch wound assay with primary human keratinocytes ........... 53 3.2.5 In vitro proliferation of primary human keratinocytes ............................ 54 3.2.6 Murine wound model and wound closure analysis ................................... 54 3.2.7 Histological analysis ..................................................................................... 55 3.2.8 Immunohistochemical analysis ................................................................... 55 3.2.9 Statistical analysis ........................................................................................ 56 3.3 RESULTS – NORMAL WOUND HEALING....................................................... 56 3.3.1 Characterization of HB-EGF coacervate ................................................... 56 3.3.2 HB-EGF stimulates keratinocyte migration .............................................. 57 3.3.3 Controlled release of HB-EGF from the coacervate does not inhibit keratinocyte proliferation ............................................................................ 59 3.3.4 HB-EGF coacervate accelerates mouse wound closure ............................ 59 3.3.5 HB-EGF coacervate increases keratinocyte proliferative and migratory potential in vivo ............................................................................................. 62 3.3.6 Angiogenesis accompanies faster healing ................................................... 64 3.4 DISCUSSION – NORMAL WOUND HEALING ................................................ 64 3.5 INTRODUCTION – DIABETIC WOUND HEALING ....................................... 68 3.6 MATERIALS AND METHODS – DIABETIC WOUND HEALING ................ 70 3.6.1 HB-EGF coacervate preparation ................................................................ 70 3.6.2 Diabetic mouse wound healing model ........................................................ 71 3.6.3 Histological analysis ..................................................................................... 71 3.6.4 Wound healing assessments ........................................................................ 72 3.6.5 Keratinocyte scratch migration assay ........................................................ 73 3.6.6 Statistical analysis ........................................................................................ 73 vii 3.7 RESULTS – DIABETIC WOUND HEALING ..................................................... 74 3.7.1 HB-EGF coacervate accelerates re-epithelialization of diabetic wounds 74 3.7.2 HB-EGF coacervate stimulates diabetic keratinocyte proliferation ....... 75 3.7.3 HB-EGF coacervate increases collagen deposition in diabetic wounds .. 78 3.7.4 HB-EGF coacervate enhances diabetic wound contraction ..................... 79 3.7.5 HB-EGF coacervate encourages diabetic
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