The zebrafish: a preclinical screening model for the optimization of nanomedicine formulations

Abstract

Nanomedicines are a valuable option to achieve drug accumulation specifically in diseased cells or tissues and therefore reduce side effects. Since the introduction of the revolutionary concept called the magic bullet for such sophisticated treatments more than 100 years ago, a lot of hope and expectations were placed into the field of nanoparticulate drug delivery. Initial forms of nanoparticles such as liposomes were described and extensively characterized which finally resulted in the FDA approval of the first cancer nanomedicine, namely Doxil, in 1996. This early success fueled the already gold-rush like atmosphere and resulted in a huge amount of time and money invested in nanomedicine research and development. As it is often the case for such a much-noticed field of medicinal research, the number of approved and clinically applied nanomedicines was not able to keep up with the unrealistic expectations resulting from the exponential increase of nanomedicine related publications. This triggered a lot of criticism questioning basic principles such as the enhanced permeability and retention effect or even the general use of nanomedicines. Despite the fact that the raised points are legitimate to a certain degree, the field of nanomedicine is far away from suffering from a general crisis, underlined by the steadily (but slowly) increasing number of approved formulations. Nevertheless, it cannot be denied that nanomedicine development is a cumbersome process suffering from a lot of drop-outs during very early phases of clinical trials. Among other things, this is due to the fact that formulation design and optimization is mainly based on in vitro studies, which are not able to fully mimic complex biological conditions. Moreover, only a selected number of formulations can subsequently be assessed in rodent in vivo experiments, since such studies are expensive, time consuming and suffer from ethical concerns. Obviously, there is a huge gap between in vitro cell culture and rodent in vivo studies, which makes the selection of potentially successful nanomedicine formulations extremely difficult. In addition, this situation does not allow a thorough formulation design and optimization under complex biological conditions and hampers a detailed understanding of basic nanomedicine interactions with biological environments at a macromolecular level. Therefore, this PhD thesis aimed to introduce the zebrafish as a complementary and easy accessible in vivo model in order to bridge the gap between in vitro and rodent in vivo studies during nanomedicine development. In the first part (Chapters I-I to III-I), the current nanomedicine development process prior to rodent in vivo studies was reviewed and the zebrafish model was set-up, validated, and further characterized. Briefly, already described formulation effects on nanomedicine pharmacokinetics were reproduced and the predictive power of the zebrafish model system was verified. Thereby, a special focus was put on two main nanomedicine clearance mechanisms, namely phagocytosis by macrophages as a part of the mononuclear phagocytic system and scavenger receptors expressed on cells, which belong to the reticuloendothelial system. Based on the successful completion of the first part, the zebrafish model was used for the development of sophisticated nanoparticulate delivery systems (Chapter IV). For example, the optimal ligand density for an actively targeted nanoparticle was established in the zebrafish model and verified in a subsequent rodent biodistribution experiment. In addition, two different nanoparticle-enzyme systems were tested regarding their stability, biocompatibility, and functionality in this living biological system, i.e. zebrafish. During this thesis, general advantages of the zebrafish model such as large clutch size, optical transparency, availability of many transgenic lines, the possibility to screen a large number of formulations, and relatively low regulatory requirements became evident. All parameters were adapted to the purpose of nanomedicine formulation design and optimization. The promising findings will be further pursued in detailed follow-up studies regarding the development of an accurate and quantitative pharmacokinetic model, the elucidation of exact formulation dependent nanomedicine cell uptake and trafficking mechanisms under in vivo conditions, or to support the formulation design and optimization of nanomedicines for infectious diseases. Altogether, the presented zebrafish model showed to be a valuable and promising tool for several applications in the field of nanomedicine development and will hopefully foster the successful translation of further nanomedicines from bench to bedside.