University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 8-2016 Multipurpose tenofovir disoproxil fumarate electrospun fibers for the prevention of HIV-1 and HSV-2 infections. Kevin Tyo Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Other Chemicals and Drugs Commons, Polymer Chemistry Commons, Preventive Medicine Commons, and the Virus Diseases Commons Recommended Citation Tyo, Kevin, "Multipurpose tenofovir disoproxil fumarate electrospun fibers for the prevention of HIV-1 and HSV-2 infections." (2016). Electronic Theses and Dissertations. Paper 2486. https://doi.org/10.18297/etd/2486 This Master's Thesis is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. MULTIPURPOSE TENOFOVIR DISOPROXIL FUMARATE ELECTROSPUN FIBERS FOR THE PREVENTION OF HIV-1 AND HSV-2 INFECTIONS By Kevin Tyo B.S. Virginia Tech, 2010 A Thesis Submitted to the Faculty of the School of Medicine of the University of Louisville In Partial Fulfillment of the Requirement for the Degree of Master of Science In Pharmacology and Toxicology Department of Pharmacology and Toxicology School of Medicine University of Louisville Louisville, KY August, 2016 MULTIPURPOSE TENOFOVIR DISOPROXIL FUMARATE ELECTROSPUN FIBERS FOR THE PREVENTION OF HIV-1 AND HSV-2 INFECTIONS By Kevin Tyo B.S. Virginia Tech, 2010 Thesis approved on By the following Thesis Committee: Dr. Jill Steinbach-Rankins Dr. Gavin Arteel Dr. Hermann Frieboes Dr. Nobuyuki Matoba Dr. Kenneth Palmer ii DEDICATION To my Grandmother Teruko Reynolds iii ACKNOWLEDGEMENTS I would like to acknowledge and give thanks to my mentor Dr. Jill Steinbach-Rankins. She has continuously offered support and guidance to both me and my scientific research. Despite my occasional failures, she has never wavered in her support. Additionally, I would like to acknowledge the additional support given from my fellow lab members as well as colleagues in the Department of Pharmacology and Toxicology. iv ABSTRACT MULTIPURPOSE TENOFOVIR DISOPROXIL FUMARATE ELECTROSPUN FIBERS FOR THE PREVENTION OF HIV-1 AND HSV-2 INFECTIONS Kevin Tyo Sexually transmitted infections affect hundreds of millions of worldwide. Both human immunodeficiency virus (HIV-1 and -2) and herpes simplex virus-2 (HSV- 2) remain incurable, urging the development of new prevention strategies. While current prophylactic technologies are dependent on strict user adherence to achieve efficacy, there is a dearth of delivery vehicles that provide discreet and convenient administration, combined with prolonged-delivery of active agents. To address these needs, we created electrospun fibers (EFs) comprised of FDA- approved polymers, poly(lactic-co-glycolic acid) (PLGA) and poly(DL-lactide-co- ε-caprolactone) (PLCL), to provide sustained-release and in vitro protection against HIV-1 and HSV-2. PLGA and PLCL EFs, incorporating the antiretroviral, tenofovir disoproxil fumarate (TDF), exhibited sustained-release for up to 4 weeks, and provided complete in vitro protection against HSV-2 and HIV-1 for 24 hr and 2 wk, respectively. In vitro tests confirmed the safety of these fibers in vaginal and cervical cells, highlighting the potential of polymeric EFs as multipurpose next-generation drug delivery vehicles. v TABLE OF CONTENTS PAGE DEDICATION ....................................................................................................... iii ACKNOWLEDGEMENTS ..................................................................................... iv ABSTRACT ......................................................................................................... v LIST OF TABLES ............................................................................................... vii LIST OF FIGURES ............................................................................................ viii BACKGROUND AND INTRODUCTION .............................................................. 1 CURRENT HIV AND HSV-2 THERAPEUTICS ................................................... 6 ORAL AND TOPICAL PrEP ................................................................................. 9 ELECTROSPUN FIBERS .................................................................................. 13 METHODS AND MATERIALS ........................................................................... 19 RESULTS .......................................................................................................... 27 DISCUSSION .................................................................................................... 56 SUMMARY AND CONCLUSIONS .................................................................... 67 REFERENCES .................................................................................................. 69 CURRICULUM VITAE ....................................................................................... 74 vi LIST OF TABLES PAGE 1. Quantification of TDF fiber loading and encapsulation efficiency ................... 35 2. PLGA and PLCL fiber IC50s (against HIV-1) after administration of the 1 and 24 hr eluates ...................................................................................................... 44 3. PLGA and PLCL fiber IC50s (against HIV-1) after administration of the 1, and 2 wk eluates ..................................................................................................... 47 4. PLGA and PLCL fiber IC50s (against HSV-2) after administration of the 1 and 24 hr eluates ...................................................................................................... 52 vii LIST OF FIGURES PAGE 1. A heat map depicting the prevalence of HIV infection ..................................... 3 2. Chemical structure of PLGA and PCL ............................................................ 15 3. Structure of Tenofovir Disoproxil Fumarate (TDF) ......................................... 18 4. Scanning electron microscopy images (SEM) of blank PLGA and PLCL fibers electrospun using different solvents .................................................................. 29 5. SEM images of PLGA and PLCL fibers prepared with increasing concentrations of TDF (1%, 10%, 20% w/w) using HFIP as solvent .................. 30 6. Average diameters of electrospun fibers measured from SEM images using Image J .............................................................................................................. 31 7. TDF standard curve for loading and encapsulation efficiency.........................36 8. TDF standard curve used to assess sustained release...................................37 9. Sustained-release profile of TDF fibers in SVF .............................................. 38 10. Results from in vitro HIV-1 infection assays ................................................ 42 11. Results from in vitro HSV-2 infection assays ............................................... 50 12. IC50 curve of HSV-2 plaque assay inhibition ................................................ 51 viii 13. Representative pictures from HSV-2 plaque assays using undiluted fiber eluates ............................................................................................................... 53 14. Cytotoxicity assessed via MTT assay ........................................................... 55 iii BACKGROUND AND INTRODUCTION Sexually transmitted infections (STIs) are a global health challenge, with over one million new cases of STIs reported daily. Over 530 and 36 million people globally are infected by herpes simplex virus type-2 (HSV-2) and human immunodeficiency virus (HIV), respectively [1]. Compounding these statistics, HSV-2 infection has been shown to significantly enhance HIV infection by as much as 2 to 7-fold [2, 3]. Furthermore, co-infected individuals harbor higher concentrations of both viruses relative to individuals with only one infection [3]. Despite the existence of multiple strategies to prevent STIs, no cure exists for these diseases, and rates of infection among particular demographics remain high [4, 5]. The first known incidence of HIV infection originated in the late 1950s in what is today known as the Democratic Republic of the Congo [6]. The general consensus of scientists is that HIV originated from cross-species contamination of simian immunodeficiency viruses (SIVs), which normally infect primates. The first known cases of the current HIV pandemic in the United States date back to 1981, where previously healthy homosexual men began suffering and dying from opportunistic infections [7]. At first this emerging disease, designated Acquired Immune Deficiency Syndrome (AIDS) was widely believed to only affect homosexuals. However, in 1984 it was discovered that AIDs is caused by the HIV, which is transmitted by hetero- and homosexual intercourse, blood-to-blood 1 transmission, or mucosal exposure [8]. Today, over 36 million people worldwide carry the virus, with women in the developing world bearing the highest burden of infection (Figure 1). Currently, there two types of HIV known: HIV-1 and HIV-2, with HIV-1 being more prevalent due to higher virulence