Development and Evaluation Of HIV gp120 Responsive Microbicide Formulation for the Prevention of HIV Sexual Transmission
No Thumbnail Available
Authors
Meeting name
Sponsors
Date
Journal Title
Format
Thesis
Subject
Abstract
Sexual transmission of HIV remains the primary route (75 to 85%) of HIV infection among all new infection cases. Furthermore, women represent the most vulnerable population and are more susceptible to HIV infections than their male counterpart. Thus, there is an urgent need to develop topical (vaginal/rectal) microbicide formulations capable of preventing HIV sexual transmission. The objective of this dissertation is to develop a mannose specific, lectin-based topical microbicide formulation capable of targeting HIV gp120 for the prevention of HIV sexual transmission. In Chapters 1 and 2, the general hypothesis, aims and scope of this work are introduced. Chapter 3 covers the literature review of anti-HIV lectins and current delivery approaches. In Chapter 4, the binding interactions between the mannose specific lectin Concanavalin A (ConA) and glycogen from Oster, as well as mannan from Saccharomyces cerevisiae, were studied using a quartz crystal microbalance (QCM). The equilibrium dissociation constant describing the interaction between Con A and glycogen (KD = 0.25 ฮผM) was 12 fold lower than the equilibrium dissociation constant describing the binding between Con A and mannan (KD = 2.89 ฮผM). That is, Con A was found to have a higher affinity for the glucose-base polysaccharide, than for the mannose-based. This observation was mainly attributed to steric effects, the difference in molecular weight and branching pattern of both polysaccharides. The knowledge gained in Chapter 4 was applied in Chapter 5 for the development of HIV 1 gp120 and mannose responsive particle (MRP) formulations. Thus, core dissolved MRP (๐ถโ๐๐
๐) and core containing MRP (๐ถ+๐๐
๐) were prepared through the layer-by-layer coating of calcium carbonate (CaCO3) with the mannose specific lectin (Con A) and a polysaccharide cross-linker (Glycogen). Particles were characterized and tested in vitro on Lactobacillus crispatus, Human vaginal keratinocytes (VK2/E6E7) and murine macrophage [RAW 264.7 (TIB 71)] cell lines. ๐ถ+๐๐
๐ average size and ฮถ-potential were 1130ยฑ15.72 nm [PDI = 0.153] and 15.1ยฑ0.55 mV, (n=3). Similarly, ๐ถโ๐๐
๐ average size and ฮถ-potential were 1089ยฑ23.33 nm (n=3) and -14.2ยฑ0.25 mV (n=3). Tenofovir (TFV) encapsulation efficiency in CaCO3 was 74.4% with drug loading of 16.3% w/w and 6.0% w/w in ๐ถ+๐๐
๐ and ๐ถโ๐๐
๐, respectively. Both ๐ถโ๐๐
๐ and ๐ถ+๐๐
๐ were nontoxic to L. crispatus and did not induce any significant pro-inflammatory nitric oxide release in VK2 and RAW 264.7 cell culture. However, ๐ถโ๐๐
๐ was found to significantly affect VK2 and RAW 264.7 cells viability at concentrations โฅ 100 ยตg/ml. Similarly, ๐ถโ๐๐
๐ significantly increased pro-inflammatory cytokines (IL1ฮฑ, IL1ฮฒ, IL6, IL7, MKC and TNFฮฑ) release at concentrations โฅ 100 ยตg/ml. Conversely, ๐ถ+๐๐
๐ did not induce any significant changes in VK2 and RAW 264.7 cells viability nor in pro-inflammatory cytokinesโ levels, in the concentration range tested (โค 1000 ยตg/ml), for 24 h. ๐ถ+๐๐
๐ was then selected for further in vitro drug release studies as well as ex vivo vaginal mucoadhesion studies. HIV gp120 triggered TFV release from ๐ถ+๐๐
๐ in a concentration dependent manner, and following HixsonโCrowell and Hopfenberg kinetic models, consistent with drug release from diminishing surface or matrix eroding drug particles. ๐ถ+๐๐
๐ was further optimized by varying the number of Con A layer in the formulation, and in order to achieve lower HIV gp120 sensitivity (โค 100 ยตg/ml). Furthermore, bioadhesion studies, performed ex vivo on porcine vaginal tissue, demonstrated that FITCโ๐ถ+๐๐
๐ adheres to vaginal tissue at levels varying between 10% ยฑ 1 and 20% ยฑ 2, depending on the number of Con A layers in the formulation. In chapter 6, ๐ถ+๐๐
๐ preclinical safety was evaluated in 8-12 weeks old female C57BL/6 mice model. First, mice were treated with Depo-Proveraยฎ to maintain them in a diestrus-like state. Then the microbicide formulation was delivered vaginally at a dose of 100 mg/kg in PBS. Vaginal histology, immunohistochemistry evaluations, as well as pro-inflammatory cytokines release (vaginal lavage and tissue extract) were investigated after 24 h. The vaginal retention of FITCโ๐ถ+๐๐
๐ was also evaluated, up to 24 h. Vaginal and major reproductive organsโ histology did not show major damage of the epithelial layer. This result was also consistent with immunohistochemistry evaluation of CD45+ cells infiltration in the vaginal epithelial layer, unlike the positive control treated groups (BZK and N-9). Furthermore ๐ถ+๐๐
๐ did not induce any significant changes in pro-inflammatory cytokines IL1ฮฑ, Ilฮฒ, IL7, IP10 and TNFฮฑ. In addition, it was also observed that FITC labeled ๐ถ+๐๐
๐ does not have a long-term retention in mice vaginal tract. This result suggested that a precoital or multiple vaginal application (i.e., BID) approaches of ๐ถ+๐๐
๐ should be investigated. Overall, this study demonstrates the feasibility of lectin-based microbicide formulations to target HIV gp120 for the prevention of HIV sexual transmission.