Subcellular localization and therapeutic efficacy of polymeric micellar nanoparticles encapsulating bedaquiline for tuberculosis treatment in zebrafish

The combination drug regimens that have long been used to treat tuberculosis (TB), caused by Mycobacterium tuberculosis, are fraught with problems such as frequent administration, long duration of treatment, and harsh adverse effects, leading to the emergence of multidrug resistance. Moreover, there is no effective preventive vaccine against TB infection. In this context, nanoparticles (NPs) have emerged as a potential alternative method for drug delivery. Encapsulating antibiotics in biodegradable NPs has been shown to provide effective therapy and reduced toxicity against M. tuberculosis in different mammalian models, when compared to conventional free drug administration. Here, we evaluate the localization, therapeutic efficacy and toxic effects of polymeric micellar NPs encapsulating a promising but highly hydrophobic and toxic antitubercular drug bedaquiline (BQ) in zebrafish embryos infected with Mycobacterium marinum. Our study shows that the NP formulation of BQ improves survival and reduces bacterial burden in the infected embryos after treatment when compared to its free form. The intravenously injected BQ NPs have short circulation times due to their rapid and efficient uptake into the endothelial cells, as observed by correlative light and electron microscopy (CLEM)

Nanoparticle-mediated drug therapy against tuberculosis in the zebrafish embryo model

Mycobacterium tuberculosis (M.tb), the primary agent of human tuberculosis (TB), is a global problem of pandemic dimensions, despite the introduction of antibiotics against the disease happened more than 70 years ago. Today TB is the largest cause of death by a single infectious agent. Harsh and inadequate treatment often associated with lack of patient compliance, has in the past two decades led to the emergence of drug-resistant TB, which is hard to treat successfully. However, this negative development has in turn attracted significant attention in the field and consequently resulted in an increase in funding and research of new antibiotics against TB. One of the new drugs developed is pretomanid, a hydrophobic compound formerly known as PA 824. The drug is currently in phase III clinical trials and have displayed great promise. An important property of pretomanid is its ability to kill replicating, as well as non-replicating bacteria. The latter, being associated with persister bacilli, the main reason for why it is so difficult to eradicate TB completely. With the aim of improving the effectiveness of pretomanid against TB, in the last decade, approximately one thousand second-generation analogues have been synthesized and evaluated. The work described in this thesis investigates the therapeutic effect of a selection of these analogues, and more specifically the potential benefit of nanoparticle (NP) encapsulation. Although NP-based delivery of drugs is a relatively new field, there are several studies reporting on improved therapeutic outcome and lowering of toxicity when compared to free drug delivery. The NP-based approach is especially interesting with respect to hydrophobic compounds with poor oral bioavailability. Our work is performed using the embryonic zebrafish model for fish-TB (caused by Mycobacterium marinum, M.m) as an initial platform for rapid screening of the efficacy and general safety of the compounds. Based on these results, testing is then moved on to preclinical model, the mouse. In the zebrafish embryo, we observed a significant therapeutic effect when the compounds were in NP formulation. The free form of the hydrophobic drugs was extremely difficult to administer, and therefore only one of the compounds could be thoroughly examined as a free drug. Nevertheless, for this compound, the NP formulation was superior in fighting against the infection. Finally, this thesis describes the establishment of a new model for studying the fitness of M.m during TB infection by using the zebrafish embryo in combination with M.m expressing two fluorescent proteins

The zebrafish embryo as an in vivo model for screening nanoparticle-formulated lipophilic anti-tuberculosis compounds

International audienceWith the increasing emergence of drug-resistant Mycobacterium tuberculosis strains, new and effective antibiotics against tuberculosis (TB) are urgently needed. However, the high frequency of poorly water-soluble compounds among hits in high-throughput drug screening campaigns is a major obstacle in drug discovery. Moreover, in vivo testing using conventional animal TB models, such as mice, is time consuming and costly, and represents a major bottleneck in lead compound discovery and development. Here, we report the use of the zebrafish embryo TB model for evaluating the in vivo toxicity and efficacy of five poorly water-soluble nitronaphthofuran derivatives, which were recently identified as possessing anti-TB activity in vitro. To aid solubilization, compounds were formulated in biocompatible polymeric micelles (PMs). Three of the five PM-formulated nitronaphthofuran derivatives showed low toxicity in vivo, significantly reduced bacterial burden and improved survival in infected zebrafish embryos. We propose the zebrafish embryo TB-model as a quick and sensitive tool for evaluating the in vivo toxicity and efficacy of new anti-TB compounds during early stages of drug development. Thus, this model is well suited for pinpointing promising compounds for further development

Real-time imaging of polymersome nanoparticles in zebrafish embryos engrafted with melanoma cancer cells: Localization, toxicity and treatment analysis

Background The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. Methods Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. Findings Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. Interpretation The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells