Abstract
At the moment, one-third of the world’s population is estimated to be infected with Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB) in humans. Our treatment strategies have been greatly challenged due to the lack of an effective vaccine and the increasing emergence of drug-resistant strains. Following the lead of Gopal Khuller, we set out to elucidate a novel drug delivery system based on biodegradable nanoparticles (NPs). NPs loaded with anti-mycobacterial drugs, such as rifampicin (RIF), can be used to more effectively deliver drugs to the site of infection. In addition, these NPs facilitate sustained release and therefore, drastically decrease the number of doses needed during a typical treatment regimen. In this thesis, work towards the improvement of TB therapy was executed in two separate ways. First, using the Mycobacterium marinum (M.marinum)-zebrafish model system established by the group of Lalita Ramakrishnan, we evaluated the effects of the antibiotic, RIF, and the anti-mycobacterial compound that blocks bacterial efflux pumps, thioridazine (TZ), free in solution. This was accomplished with the use of drug baths; water supplemented with the bactericidal agent. With this method the morphological effects could readily be observed, and due to the transparency of zebrafish embryos, the fluorescent M.marinum bacilli could be visualized in vivo. Second, we were interested in using antimicrobial peptides to treat mycobacterial infections, as an alternative to antibiotics. Due to the already successful production of NPs loaded with RIF in our group, we sought to encapsulate plasmid DNA (pDNA) encoding these antimicrobial peptides using the nanoprecipitation method. The effects of different concentrations of RIF and TZ were observed in infected and uninfected zebrafish embryos. The infecting bacteria readily acquired tolerance towards RIF, as has been shown by others, but higher concentrations were found to show higher bactericidal activity and no observable toxicity. TZ is believed to increase the effects of antibiotics, such as RIF, and therefore eliminate the transient phenomenon of tolerance, but these effects were not observed under the conditions of the preliminary experiment performed here. Although pDNA encapsulation was not successful, NPs were fabricated in a narrow size range and protocols for preparation of NPs and for the analyses of pDNA integrity and loading were established. These protocols provide the foundation for others in our group to continue the further development of this promising NP-based therapy.