Formulation of Hyaluronidase Enzyme Sensitive Topical Nanomicrobicides for HIV Virus Transmission Prevention
The objective of this dissertation is to design and optimize a nanoformulations (nanoparticle and nanofiber) delivery system loaded with anti-HIV topical microbicides for HIV prevention in women. In chapters 1 and 2, the overview of the problem, research objectives as well as the literature review of the technical and scientific background of this dissertation are introduced. In chapter 3, a study was designed to test the hypothesis that a triggered release of a topical anti-HIV microbicide (tenofovir: TFV) from hyaluronic acid based nanoparticles (HA NPs) can be achieved under the influence of hyaluronidase (HAase) enzyme. The Fractional Factorial Experimental Design (FFED) was employed to examine the formulation variables such as: molar concentrations of adipic acid dihydrazide (X₁) and 1-Ethyl-3-[3 dimethylaminopropyl]carbodiimide hydrochloride (X₂), volume of acetone (X₃) and reaction time (X₄), and their influence on the responses such as Y₁; particle mean diameter: PMD (nm), Y₂; polydispersity index: PDI and Y₃; zeta (ζ) potential: (mV). The cross-linking efficiency of NPs was characterized by Fourier Transform Infra-Red (FT-IR), and ¹³C-nuclear magnetic iv resonance (NMR) analyses. When formulated with X₁; 2.49 mM, X₂; 9.96 mM, X₃; 60 mL, X₄; 6 h, the HA-NPs exhibited a spherical shape with PMD, PDI, ζ potential, and drug loading of 70.6 ± 4.1 nm, 0.07 ± 0.02, -38.2 ± 2.8 mV, and 26.1 ± 1.2% w/w, respectively, (n = 3). Unlike for HA based gel, HAase notably triggered the drug release and HA degradation from the NPs after 24 h (~90% w/w and 65% w/w, respectively); whereas, in its absence, these values were ~39% w/w and 26% w/w, respectively. The NPs were non-cytotoxic to human vaginal VK2/E6E7 and End1/E6E7 cells and had no effect on Lactobacillus viability. These data suggested the possibility of using HA-NPs as a delivery system for intravaginal delivery of topical microbicides for the prevention of HIV transmission. In chapter 4, a study was designed to test the hypothesis that a stimuli-sensitive, safe and mucoadhesive thiolated hyaluronic acid (HA) based nanofibers (NFs) loaded with a topical vaginal microbicide (TFV) can be used for the prevention of HIV virus vaginal transmission in women. To test this, a novel thiolated sulfhydryl (-SH) group modified HA (HA-SH) was synthesized to fabricate the TFV loaded HA-SH-NFs (mean diameter ~75 nm) using electrospinning method. Sulfhydryl (-SH) group modified HA (HA-SH) were characterized for their size distribution, surface morphology, surface chemistry, crystallinity, mucoadhesion property, and in vitro drug release profile using size exclusion chromatography, powder X-ray diffraction, FT-IR, and ¹H-NMR analyses. Mucin interaction and ellipsometer measurements confirmed that mucoadhesion of HA-SH-NFs was increased compared to that of native HA polymer based on an increase in the size (~4 fold), thickness (~3 fold) and adsorbed mucin amount (~2 fold) after 3 h incubation of HA-SH-NFs with mucin. A triggered drug release (~87% w/w) from NFs (drug loading ~17% w/w) occurred after 1 h in the presence of seminal hyaluronidase enzyme. It was observed that in the absence of HAase, the drug release from NFs followed the Peppas kinetic model whereas, in the presence of HAase, NFs followed Weibull model. The HA-SH-NFs were non-cytotoxic to vaginal VK2/E6E7 and End1/E6E7 v cells and L. crispatus bacteria for 48 h. The results suggested that TFV loaded HA-SH-NFs templates developed in this study have the potential of vaginal delivery of topical microbicides for the prevention of HIV transmission. In chapter 5, in vivo evaluations of the developed HA-NPs and HA-SH-NFs were performed in female C57BL/6 mice. The histological analysis on the mice genital tract and other organs did not show any signs of damage upon once-daily administration of HA-NPs or HA-SH-NFs up to 7 days. Following 24 h exposure, HA-NPs or HA-SH-NFs did not show any significant immune (CD45) cell infiltration in mice vaginal tissues. The cytokines ((IL-1α, IL 1β, IL-6, IP-10, IL-7, MKC, TNF-α) levels (pg/mL) in cervicovaginal lavage and cervicovaginal tissues were not significantly changed compared to control mice data analyzed after 24 h. The cytokine results confirmed the non-immunogenicity of developed nanoformulations. The in vitro anti-HIV activity of HA-NPs and HA-SH-NFs was analyzed at the MOI of 10,000, 5,000, and 1,000 using a luciferase assay. The pseudotyped HIV virus particles were generated using lipofectamine plasmid transfection method. The size distribution, mean diameter (~128 nm), and titer (~3.07×10¹⁰) of pseudotyped virus particles was analyzed using nanoparticle tracking analysis measurements. The in vitro anti-HIV activity data showed that the TFV loaded HA-NPs and HA-SH-NFs were able to inhibit the pseudotyped HIV virus replication. Moreover, the results also confirmed that the structural integrity and anti-HIV activity of TFV was preserved after the nanofabrication processes. The in vivo results illustrated that these nanoformulations (HA-NPs and HA-SH-NFs) are promising delivery systems and offered a safe delivery of anti-HIV microbicide candidates. Overall, the data presented here highlight the applicability and potential of TFV loaded HA-NPs and HA-SH-NFs templates for the topical vaginal delivery of anti-HIV/AIDS microbicide candidates.
Table of Contents
Introduction -- Overview and pandemic of HIV infections -- Development of hyaluronidase sensitive tenofovir loaded hyaluronic acid based nanoparticles -- Fabrication of hyaluronidase sensitive tenofovir loaded hyaluronic acid based mucoadhesive nanofibers -- Preclinical safety and in vitro HIV efficacy evaluaitons of hyaluronidase sensitive tenofovir loaded hyaluronic acid based nanoformulations -- Summary, conclusion and future directions -- Appendix