Development of nanoparticle drug carriers against tuberculosis in zebrafish via control of the vasculature.

Our group has focused on developing nanoparticles as novel drug delivery vehicles against tuberculosis and cancer and towards this goal in 2010 we introduced the transparent zebrafish embryo into the nanoparticle (NP) field. The standard practice now is to characterize NP in vitro and in cell culture, this allows promising NP to be tested in mice, the most popular preclinical testing systems. However, mice, being opaque do not allow fluorescent NP to be seen in detail in the live animals. In contrast zebrafish embryos allow not only NP but all the cells of this vertebrate to be seen at high resolution; this is emerging as an ideal system to screen and characterize different NP in detail in order to select only the most promising for final testing in mouse, still the most popular preclinical system prior to clinical trials. We have established two different disease models in the zebrafish embryo. In the first we inject fish tuberculosis (TB) bacteria (fluorescent Mycobacterium marinum) which are taken up by macrophages and assemble into a cell aggregate, the granuloma, where in the fish, as in humans with TB the vast majority of the pathogenic bacteria accumulate. In the second system we inject fluorescent mouse melanoma cancer cells into the fish embryo; these multiple and develop a tumor. The TB granuloma and the tumours are remarkably similar structures and this agrees with other publications arguing that they are also functionally similar. What is strikingly evident in the zebrafish models is that both structures stimulate the growth of new blood vessels that grow into them. , this is easily seen in fish expressing fluorescent blood vessels. This similarity was never realized in 70 years of studies of TB and cancer in mouse models. In principle this opens up many generations of research into cancer as being potentially relevant for the development and treatment of TB, and vice versa. We are now actively following the crucial open question in both diseases: when NP carrying drugs against TB or cancer are injected into the blood system how can these NP leave the blood vessels. The dominant hypothesis, from the cancer field since 1986 is that the particles take advantage of the fact that next to tumors the endothelial cells that line the blood vessels fail to make a proper seal and that the NP diffuse through these newly opened spaces; this scheme is called the enhanced permeability and retention (EPR) model. In earlier papers we reported that a mechanism like this operates in both the cancer and the TB systems, still more evidence of the similarity between the two diseases. The availability of superb state of the art spinning disc microscopes at our IBV facility has revealed that what we thought was a trickling out of NP from the blood vessels to the disease structures is in fact an explosion of fluorescence, in dramatic events we refer to as ?flashes?. A closer inspection has revealed that two types of blood cells macrophages and neutrophils pass through the endothelial cells together with the nanoparticle flashes. We are investigating this surprising result now by electron microscopy.

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Nanoparticle (NP) drug therapy is a highly promising therapeutic approach for diseases such as cancer, and tuberculosis (TB), that is caused by Mycobacterium tuberculosis. Using NP the drug packet can be targeted to the disease site and released locally in a sustained manner, thereby reducing adverse effects associated with free drug systemic therapy. Development of NP is the focus of hundreds of research groups worldwide. The routine strategy is to fabricate NP-enclosing drugs or fluorescent dyes and to screen these in cultured cells before testing them in mouse models. A serious limitation of the latter is the difficulty in imaging the NP at high resolution in live animals. For this reason, we developed zebrafish embryos as models for visualising NP in the context of TB, and human cancer models. The transparency of this system allows high resolution in vivo imaging, not only of fluorescent NP and bacteria (TB), or cancer cells, but also of selectively labelled host cells, such as macrophages, erythrocytes or endothelial cells. We have provided proof of principle that NP-encapsulated rifampicin is very effective against TB mycobacteria in the zebrafish. There, as in humans, both TB (granulomas) and cancer (tumors) share an organised, multi-cellular organisation of disease tissues that are nurtured by a dynamic network of blood vessels. Evidence argues that this vasculature in and around tumours and granulomas can be manipulated by drugs that block blood vessel development (angiogenesis) in order to both improve therapy directly and to enhance NP access. The goal of this proposal is: 1. To collaborate with four chemists, one making novel drugs against TB and three other experts in NP fabrication to develop new generation anti TB-NP. 2. To manipulate the zebrafish vasculature to enhance access of NP to the TB granulomas. This part will be facilitated by collaboration with computational experts who will model blood flow and NP dynamics.