Structural and functional characterization of lipid-based mRNA delivery systems
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Abstract
Pharmaceutical research has progressed in rapid manner over the last century. It has seen a shift from small molecule drugs to so called biologicals, which comprise macromolecules (such as proteins or nucleic acids) and have led to significant advantages in some therapeutic fields, such as several forms of cancer, by enabling the possibility of cancer immunotherapy. This therapeutical concept is based on utilizing the body’s own defense mechanisms to combat the mutated cancer cells by training the immune system to recognize tumor antigens, which can for example be achieved through transfection of antigen presenting cells with the tumor antigen by delivering nucleic acids coding for this antigen into these cells. Traditionally, viral vectors have often been used to transfect target cells with genetic information. However, several problems (such as antiviral immune responses against the vector) come with the use of these delivery systems. Therefore, several approaches have been developed to mimic viral vectors while trying to reduce their downsides. One of these approaches is the use of lipid-based nanosized delivery systems, which are called lipoplexes or lipid nanoparticles (LNPs).
Recent years have seen the first approvals of lipid-based delivery systems delivering nucleic acid drug molecules. However, while a lot of effort has been spent on efficacy studies – be it in vitro or in vivo – and on general physicochemical characterization of lipid-based nanomedicines for mRNA delivery in cancer immunotherapy or other applications, a lack of insight into the internal structures, their transformation in relation to environmental changes, and the implications thereof still remains. This thesis therefore gives accurate in situ insights into the structural organization of lipid-based mRNA delivery systems – be it lipoplexes or LNPs – by utilizing potent and seldom applied characterization methods in the form of small angle scattering techniques, as well as traditional nanoparticle and nucleic acid characterization tools such as dynamic light scattering, fluorescence-based pKa determination, microscopy, zeta potential measurements, nucleic acid encapsulation assays, and in vitro transfection efficacy. This enables the confirmation and optimization of previously established models to describe the internal structures and changes thereof more accurately in terms of both formulation and environmental parameters. New models are developed describing the pH-responsiveness of both lipoplexes and lipid nanoparticles, as well as the differences between these kinds of RNA delivery systems. Additionally, a first attempt to draw conclusions about a structure-function relationship is made.
Overall, the results described within this thesis should therefore provide better understanding of the functional and structural coherencies inside lipid-based mRNA delivery systems, which will help in the intelligent design and fine-tuning of the next generation of delivery systems during this just beginning new era of nucleic acid drug products.