Design and Evaluation of HIV Microbicides Loaded mucoadhesive Nanoformulation

Abstract

HIV/AIDS had caused more than 25 million deaths since it was first recognized. Women are more susceptible to HIV infection in comparison with men due to human physiology, social, economic disadvantages. To protect women from HIV transmission, there is an urgent need to develop a formulation with proper vaginal retention time for the topical application of anti-HIV microbicides. The aim of this dissertation is to test the hypothesis that a mucoadhesive polymer based nanomedicine can prolong the contact time with the vaginal tissue, provide the controlled release of the drug, and is safe in vivo. Various formulation and are investigated, their physicochemical properties, mucoadhesive properties, and safety are evaluated. In Chapter 3, a chitosan (CS) based nanoparticle (NP) delivery system suitable for the encapsulation of tenofovir (TFV) is designed and optimized. The physicochemical characteristics of the NPs including encapsulation efficiency (EE%), diameter, morphology, in vitro drug release as well as in vitro cytotoxicity are evaluated. the size of CS NPs ranged from 168 nm to 277 nm. It is shown that NPs are safe to both vaginal epithelial cell line and Lactobacillus over 48 h. The mucoadhesion is about 12%. The CS NPs are mucoadhesive and safe as a microbicide carrier, the drawback of the CS NPs is the low EE%. In Chapter 4, the hypothesis that TFV loaded thiolated chitosan (TCS) NPs exhibit superior biophysical properties for mucoadhesion compared to those of native chitosan NPs is tested. The NPs are prepared by ionic gelation. The particle mean diameter, EE%, release profile, in vitro cytotoxicity, cellular uptake, uptake mechanism, and percent mucoadhesion are assessed. The particles are spherical with diameters ranged from 148 nm to 255 nm. The EE% and drug loading is 25% and 1.62% (w/w), respectively. The NPs provide a controlled release over of the drug following Higuchi model. The TCS NPs are not cytotoxic to both vaginal epithelial cell line and Lactobacillus over 48 h. The cellular uptake is time dependent. It is mainly occurred via caveolin mediated endocytosis. The mucoadhesive properties of TCS NPs is 5-fold higher than that of CS NPs. Compare to the CS NPs, the TCS NPs exhibit relatively higher EE%, drug loading, and mucoadhesion. In Chapter 5, we develop a TCS coated multilayer microparticles (MPs). Sodium alginate MPs are prepared by spray drying. The multilayer MPs are developed by coating the optimal alginate MPs with the TCS solution using a layer-by-layer method. The morphological analysis, drug loading, in vitro drug release, cytotoxicity, mucoadhesion and in vivo toxicity are evaluated. The MPs diameter ranges from 2 μm to 3 μm with a drug loading of 7-12% (w/w). The MPs show a controlled drug release. The MPs are found to have a high mucoadhesion (~50 folds at a higher ratio, and ~20 folds at a lower ratio of mucin: MPs) compare to non-layered sodium alginate MPs in both vaginal fluid and semen fluid simulant buffers. The multilayer MPs are non-cytotoxic to vaginal and endocervical epithelial cells. Histological analysis of the female C57BL/6 mice genital tract and other organs shows no damage upon once-daily administration of MPs up to 24 h and 7 days. The drug loading of the TCS MPs is significantly enhanced (from 1.62% to 12.73%) compare to that of the TCS NPs. However, the mucoadhesion of the MPs is slightly lower than that of the NPs due to the larger particle size. In chapter 6, the TCS core/shell nanofibers (NFs) are fabricated by a coaxial electrospinning technique. The drug loading is13%-25% (w/w), the EE% is about 100% because no loss of material during the electrospinning process. The NFs exhibit smooth surface with average diameters in the range of 50 to 100 nm. The NFs are non-cytotoxic at the concentration of 1 mg/ml. The core-shell NFs exhibit a release kinetic following Weibull model, and are 40-60 fold more bioadhesive than NFs made solely with PEO. H&E and immunohistochemical (CD45) staining analysis of genital tract indicates non-toxicity and non-inflammatory effects of the NFs daily treatment for up to 7 days. The TCS NFs exhibit both high drug loading and high mucoadhesion; these data highlight the potential of TCS NFs templates for the topical vaginal delivery of anti-HIV/AIDS microbicides. Overall, the present work demonstrates TFV loaded NF can be considered as a good candidate for the delivery of water-soluble small-molecule drugs, and a promising vaginal delivery system for the prevention of HIV transmission.