Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2015 Design of polyanhydride-based targeted nanovaccines against HIV Julia Eulalia Vela Ramirez Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biomedical Commons, and the Chemical Engineering Commons Recommended Citation Vela Ramirez, Julia Eulalia, "Design of polyanhydride-based targeted nanovaccines against HIV" (2015). Graduate Theses and Dissertations. 14476. https://lib.dr.iastate.edu/etd/14476 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Design of polyanhydride-based targeted nanovaccines against HIV by Julia Eulalia Vela Ramirez A dissertation submitted to the graduate committee In partial fulfillment of requirements for the degree of DOCTOR OF PHILOSOPHY Major: Chemical Engineering Program of Study Committee: Balaji Narasimhan, Major Professor Michael J Wannemuehler Nicola L. B. Pohl Ian C. Schneider Jennifer Heinen Iowa State University Ames, Iowa 2015 Copyright © Julia Eulalia Vela Ramirez, 2015. All rights reserved. ii TABLE OF CONTENTS LIST OF FIGURES...…….…………...……………………………………………….viii LIST OF TABLES…….…………………………………………………………………xi ACKNOWLEDGEMENTS……………………………………………………………..xii ABSTRACT…..………………………………………………………………………..xvii CHAPTER 1 ......................................................................................................... 1 1.1. Introduction .................................................................................................... 1 1.2. References .................................................................................................... 6 CHAPTER 2 ....................................................................................................... 10 2.1 Summary ...................................................................................................... 10 2.2 Biodegradable polymers for drug and vaccine delivery ................................ 11 2.2.1 Natural polymers .................................................................................... 11 2.2.2. Polyesters ............................................................................................. 16 2.2.3. Polyurethanes ....................................................................................... 19 2.2.4. Vinyl polymers ....................................................................................... 20 2.2.5. Poly(alkyl cyanoacrylates) ..................................................................... 22 2.2.6. Polyethers ............................................................................................. 23 2.2.7. Polyanhydrides ...................................................................................... 25 2.3. Nanoparticle characteristics as adjuvants and/or delivery vehicles ............. 29 2.3.1. Particle size ........................................................................................... 29 2.3.2. Shape .................................................................................................... 34 2.3.3. Hydrophobicity and surface charge ....................................................... 36 2.4. Targeting mechanisms for drug and vaccine delivery .................................. 38 2.4.1 Introduction ............................................................................................ 38 2.4.2. Desired immune system components ................................................... 39 iii 2.4.2.1. Tumor cells ..................................................................................... 39 2.4.2.2. Antigen presenting cells .................................................................. 41 2.4.2.3. Targeting approaches ..................................................................... 45 2.4.2.4. Antigen specificity ........................................................................... 46 2.4.2.5. Environmentally responsive delivery vehicles ................................. 47 2.4.2.6. Channeling molecules ..................................................................... 48 2.4.3. Nanoparticle targeting strategies ........................................................... 48 2.4.3.1. Passive targeting ............................................................................. 49 2.4.3.2. Active targeting ............................................................................... 49 2.5. HIV: Overview and current challenges ........................................................ 52 2.5.1. Overview of HIV infection ...................................................................... 53 2.5.2. Vaccine strategies ................................................................................. 55 2.5.3 Current therapies and challenges........................................................... 57 2.6. Summary ..................................................................................................... 58 2.7. References ............................................................................................... 60 CHAPTER 3 ....................................................................................................... 78 CHAPTER 4 ....................................................................................................... 80 4.1. Abstract ....................................................................................................... 81 4.2. Introduction .................................................................................................. 82 4.3. Materials and Methods ................................................................................ 84 4.3.1. Materials ................................................................................................ 84 4.3.2. Construction of pET-gp41-54Q-GHC .................................................... 85 4.3.3. Expression and purification of gp41-54Q-GHC ..................................... 86 4.3.4. Monomer and polymer synthesis ........................................................... 87 4.3.5. Nanoparticle synthesis .......................................................................... 88 4.3.6. Nanoparticle functionalization................................................................ 89 4.3.6.1. Synthesis of carboxylated di-mannose ............................................ 89 4.3.6.2. Surface functionalization ................................................................. 89 4.3.7. Nanoparticle characterization ................................................................ 90 4.3.8. Antigen release kinetics ........................................................................ 91 iv 4.3.9. Gel electrophoresis ............................................................................... 91 4.3.10. Antigenic analysis of released immunogen ......................................... 92 4.3.11. In vitro DC uptake and activation ......................................................... 93 4.3.11.1 Mice ............................................................................................... 93 4.3.11.2. DC culture and stimulation ............................................................ 93 4.3.11.3 Particle internalization .................................................................... 94 4.3.11.4 Flow cytometric analysis of cell surface markers and CLRs .......... 94 4.3.12. Cytokine secretion ............................................................................... 95 4.3.13. Statistical analysis ............................................................................... 95 4.4. Results......................................................................................................... 95 4.4.1 Chemistry-dependent gp41-54Q-GHC kinetics and stabilization upon release from polyanhydride nanoparticles ....................................................... 95 4.4.2. Functionalization and characterization of carbohydrate-modified nanoparticles ................................................................................................... 97 4.4.3. gp41-54Q-GHC release kinetics from functionalized nanoparticles ...... 99 4.4.4. gp41-54Q-GHC antigenicity was preserved upon release from functionalized nanoparticles .......................................................................... 100 4.4.5. Internalization by DCs was enhanced by functionalization .................. 101 4.4.6. Functionalized nanoparticles enhanced DC expression of CD40 and CD206 .................................................................................................... 102 4.4.7. Functionalized nanoparticles modulated DC secretion of pro- inflammatory cytokines .................................................................................. 104 4.5. Discussion ................................................................................................. 105 4.6. Conclusions ............................................................................................... 112 4.7. Acknowledgments ..................................................................................... 113 4.8. References ...............................................................................................