TRANSPORT PHENOMENA IN ANTI-HIV MICROBICIDE DELIVERY VEHICLES by Anthony R. Geonnotti, III Department of Biomedical Engineering Duke University Date:_______________________ Approved: ___________________________ David F. Katz, Ph.D., Supervisor ___________________________ Patrick F. Kiser, Ph.D. ___________________________ David C. Montefiori, Ph.D. ___________________________ George A. Truskey, Ph.D. ___________________________ Fan Yuan, Ph.D. Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2008 ABSTRACT TRANSPORT PHENOMENA IN ANTI-HIV MICROBICIDE DELIVERY VEHICLES by Anthony R. Geonnotti, III Department of Biomedical Engineering Duke University Date:_______________________ Approved: ___________________________ David F. Katz, Ph.D., Supervisor ___________________________ Patrick F. Kiser, Ph.D. ___________________________ David C. Montefiori, Ph.D. ___________________________ George A. Truskey, Ph.D. ___________________________ Fan Yuan, Ph.D. An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2008 Copyright by Anthony R. Geonnotti, III 2008 Abstract There were 2.5 million people newly infected with HIV in 2007, clearly motivating the need for additional novel prevention methods. In response, topical vaginal antimicrobials, or microbicides, are being developed. These products aim to stop HIV transmission through local, vaginal delivery of antiviral compounds. To succeed, microbicides require a potent active compound within a well-engineered delivery vehicle. A well-engineered delivery vehicle provides an antiviral compound with the greatest opportunity to interact with HIV and/or infected cells, thereby increasing overall microbicide effectiveness. The theoretical and experimental investigations within this dissertation are concerned with the study of HIV and active compound transport within microbicide delivery vehicles and with the mechanisms by which these transport processes can be affected to maximize viral neutralization. To initially investigate the factors contributing to microbicide effectiveness, a combined pharmacokinetic and pharmacodynamic model of HIV transport and neutralization within a microbicide product was created. Model results suggested that thin (~100µm) layers of microbicide product may protect against HIV infection. Model results also indicated that a specific and engineerable property of delivery vehicles – the ability to restrict viral transport – iv may increase the overall effectiveness of a microbicide. Two new experimental assays were developed to test the hypothesis that delivery vehicles can slow viral transport. First, a novel methodology was created to measure particle diffusion over length scales relevant to microbicide delivery (50-500µm). Results showed that current vehicles significantly restrict the transport of small molecules and proteins. The second assay was designed to test HIV transport in a biologically relevant, layered (fluid-microbicide- tissue) configuration of a microbicide product in vivo; infectious HIV was placed above a thin layer of a microbicide delivery vehicle. Assay results showed that HIV transport is significantly slowed by two different placebo gels. This experimental confirmation of viral restriction in hydrogels, combined with the theoretical finding that viral restriction increased microbicide effectiveness, strongly motivates the future development of new delivery vehicles that intentionally slow viral transport. These new experimental methodologies can also be used to screen and compare future delivery vehicles to produce optimal microbicide products. Finally, a two-dimensional, computational finite-element vaginal model was created to evaluate the transport of drugs from an intravaginal ring. This model determined that while IVRs may be effective in the delivery of antiviral compound, their performance is influenced by the flow of vaginal fluid. The analysis also warns about the potential for local toxicity. v Well-engineered delivery vehicles are an essential component to microbicide performance because they maximize the opportunities for active compounds to interact with and neutralize HIV. The studies in this dissertation demonstrate that delivery vehicles have a significant effect on active compound and HIV transport. To create an effective microbicide, vehicle effects on transport processes must be well understood, purposefully engineered, and carefully optimized to ensure maximal interactions between antiviral compounds and virus. Directed engineering of delivery vehicles contribute to the foundation for microbicide success. vi Contents Abstract.........................................................................................................................................iv List of Tables................................................................................................................................xii List of Figures............................................................................................................................xiii Acknowledgements...................................................................................................................xvi 1. Introduction...............................................................................................................................1 1.1 HIV/AIDS..........................................................................................................................1 1.2 Microbicides Overview....................................................................................................3 1.3 Microbicide Development...............................................................................................4 1.3.1 Antiviral Compounds.................................................................................................5 1.3.2 Delivery Vehicles.........................................................................................................7 1.3.3 Engineering Microbicides..........................................................................................9 1.4 Thesis Organization.......................................................................................................12 2. Dynamics of HIV Neutralization by an Entry Inhibitor Microbicide Layer: Biophysical Fundamentals and Transport Theory......................................................................................15 2.1 Chapter Summary..........................................................................................................15 2.2 Introduction.....................................................................................................................16 2.3 Transport Model.............................................................................................................18 2.3.1 Governing Equations of Transport.........................................................................19 2.3.2 Boundary and Initial Conditions.............................................................................21 2.3.3 Viral Neutralization Kinetics...................................................................................22 2.3.4 Complete Non-Dimensionalized Equations..........................................................27 vii 2.3.5 System Parameters....................................................................................................30 2.4 Results..............................................................................................................................34 2.4.1 Reference Conditions................................................................................................34 2.4.2 Less Potent Active Ingredient..................................................................................36 2.4.3 Unrestricted Diffusion..............................................................................................37 2.4.4 Unrestricted Diffusion plus a Thinner Formulation Layer.................................37 2.5 Sensitivity Analysis........................................................................................................38 2.6 Discussion........................................................................................................................40 2.6.1 Implications for Future Work..................................................................................45 3. Measuring Macrodiffusion Coefficients in Microbicide Hydrogels via Postphotoactivation Scanning...................................................................................................47 3.1 Chapter Summary..........................................................................................................47 3.2 Introduction.....................................................................................................................48 3.3 Materials and Methods..................................................................................................52 3.4 Results..............................................................................................................................59 3.5 Discussion........................................................................................................................61 3.6 Conclusions and Future Work......................................................................................64 4. Development of a Novel Bioassay to Measure the Restriction of HIV in Microbicide Hydrogels.....................................................................................................................................67 4.1 Chapter Summary..........................................................................................................67 4.2 Introduction.....................................................................................................................67 4.3 Methods...........................................................................................................................70 4.3.1 Virus Stocks and Concentrations............................................................................70 viii 4.3.2 Media...........................................................................................................................72 4.3.3 Microbicide Vehicles.................................................................................................72 4.3.4 Viral Neutralization/Cytotoxity Assays.................................................................72 4.3.5 Transport Assay.........................................................................................................73 4.3.6 Statistical Analyses....................................................................................................78 4.4 Results..............................................................................................................................79 4.4.1 Assay Performance....................................................................................................79 4.4.2 Neutralization Assay/Cytotoxity Assay.................................................................80 4.4.3 Transport Assay.........................................................................................................80 4.5 Discussion........................................................................................................................85 4.5.2 Clinical Implications of Viral Restriction...............................................................85 4.5.1 Assay Limitations and Future Directions..............................................................87 4.5.3 Estimation of Diffusion Coefficients.......................................................................91 4.6 Conclusion.......................................................................................................................98 5. Creation of an MRI-Derived, Two-Dimensional, Multi-Compartment Model of Vaginal Antiviral Delivery by a Ring.....................................................................................101 5.1 Chapter Summary........................................................................................................101 5.2 Introduction...................................................................................................................101 5.3 Materials and Methods................................................................................................105 5.3.1 Ring Parameters.......................................................................................................105 5.3.2 Fluid Amount and Flow.........................................................................................108 5.3.3 Ring Flux...................................................................................................................110 ix 5.4 Model Implementation................................................................................................111 5.4.1 2-D Model Transport Theory.................................................................................113 Ring Flux.......................................................................................................................113 Dapivirine Transport...................................................................................................115 5.5 Results and Analysis....................................................................................................115 5.6 Discussion......................................................................................................................121 5.6.1 Computational Limitations....................................................................................123 5.6.2 Conclusions..............................................................................................................124 6. Conclusions............................................................................................................................125 Appendix A: Experimental Protocols....................................................................................133 A.1 Protocol to Determine Diffusion Coefficients (Chapter 2)....................................133 A.2 Prepartation of Caged-Fluorescein Labeled Microspheres (Chapter 3)..............136 A.3 Transport Assay Protocol (Chapter 4)......................................................................139 A.3.1 Setup.........................................................................................................................139 A.3.2 Protocol....................................................................................................................140 Appendix B: MATLAB Code...................................................................................................142 B.1 HIV Transport and Neutralization Model................................................................142 B.1.1 Papacode.m..............................................................................................................142 B.1.2 track61constants.m.................................................................................................144 B.1.3 Track61HIV.m.........................................................................................................146 B.1.4 pptplots.m................................................................................................................159 B.2 Code to Analyze Scanned Images of Particle Diffusion.........................................163 x