Design and optimization of nanoformulation loaded with HIV microbicides
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The objective of this dissertation is to design and optimize nanoformulation for microbicides intended for the prevention of HIV/AIDS transmission. In chapters 1 and 2, the aims and the scope of this work are introduced, as well as the literature review of its technical background. In chapter 3, the formulation and process variables involved in the production of spray-dried oily-core nanocapsules (NC) are investigated using Box-Behnken Design (BBD) and Fractional Factorial Experimental Design (FFED). Indomethacin (IND) is chosen as a model drug for lipophilic HIV microbicides. Three formulation variables (the amount of polymer, oil, and surfactant) and four process variables (inlet temperature, feed flow rate, atomizing air flow, and aspiration rate) are optimized for NC with smaller mean diameter, higher encapsulation efficiency (EE%) and higher process yield. The spray-dried NC are also characterized for their morphology, density, structure, in vitro drug release, and powder flowability. The optimized production process successfully resulted in NC with mean diameter less than 200 nm, with a drug EE% of 95% and an overall yield of 30.8%. Morphological analysis and density-gradient centrifugation confirmed the existence of an oily core and spherical nanostructure with no detectable drug crystals. The NC have longer sustained drug release profile than nanosphere (NS) control, with a good fit to the Ritger-Peppas model of drug release (R2 > 0.930). In chapter 4, we test the hypothesis that actual anti-HIV microbicides such as tenofovir (TNF) or tenofovir disoproxil fumarate (TDF) loaded nanoparticles (NP) prepared with a blend of poly (lactic-co-glycolic acid) (PLGA) and methacrylic acid copolymer (Eudragit® S-100, or S-100) are noncytotoxic and exhibit pH-responsive release of anti-HIV microbicides in the presence of human semen fluid simulant (SFS). After the preparation by emulsification diffusion process, their size, EE%, drug release profile, morphology, and cytotoxicity are characterized by dynamic light scattering, spectrophotometry, transmission electron microscopy, and cellular viability assay/transepithelial electrical resistance measurement, respectively. Cellular uptake is elucidated by fluorescence spectroscopy and confocal microscopy. The NP have an average mean diameter of 250 nm, maximal EE% of 16.1% and 37.2% for TNF and TDF, respectively. There is a 4-fold increase in drug release rate from the 75% S-100 NP in the presence of SFS over 72 h. At a concentration up to 10 mg/ml, the PLGA/S-100 NP are noncytotoxic for 48 h to vaginal endocervical/epithelial cells and Lactobacillus crispatus. The particle uptake (∼ 50% in 24h) by these vaginal cell lines mostly occurred through caveolin-mediated pathway. In chapter 5, we modify the production process, and develop a dual-functional mucoadesive and pH-responsive microparticles (MS) formulation for TNF. The formulation variables and process parameters are screened and optimized using a 24-1 FFED. The MS are characterized for their mean diameter, zeta potential, yield, EE%, Carr's index, drug loading, in vitro release, cytotoxicity, inflammatory responses and mucoadhesion. The optimal MS formulation has an average diameter of 4.73 μm, zeta potential of -26.3mV, 68.9% yield, EE% of 88.7%, Carr's index of 28.3 and drug loading of 2% (w/w). The MS formulation release 91.7% of its payload in the presence of simulated human semen over 4 hours. At a concentration of 1 mg/ml, the MS are noncytotoxic to vaginal/endocervical epithelial cells and Lactobacillus crispatus when compared to control media. There is also no statistically significant level of inflammatory cytokine (IL1-α, IL-1β, IL-6, IL-8, and IP-10) release triggered by these MS. Their percent mucoadhesion is 2-fold higher than that of 1% HEC gel formulation. These MS are found to be 3.8-5.8 fold more potent compared to native drug in HIV-1 inhibition, as a result of CD4-dependent CCR5 tropic cell-free HIV-1 entry essay. Overall, the present work demonstrates two nanoformulations that could be used. The oily core NC (300 mg of PLA, 0.56 ml of oil, and 239.57 mg of Pluronic F127) which is suitable for lipophilic microbicides, and a microparticle formulation (mean diameter of 4.73 μm with drug loading of 2% w/w) for the more water-soluble TNF. These formulations are promising alternative delivery systems for the purpose of AIDS prevention, offering safe and controlled intravaginal delivery of HIV microbicides.
Table of Contents
Introduction -- Current state of the art in HIV microbicides delivery -- Design and optimization of spray dried oily core nanocapsule for encapsulation of lipophilic anti-HIV microbicides -- pH-responsive nanoparticles releasing tenofovir for the prevention of HIV transmission -- Spray drying tenofovir loaded mucoadhesive and pH-sensitive microshperes intended for HIV prevention -- Summary and conclusions -- Appendix