Surface engineering of T cells with polymer nanoparticles to enhance drug delivery to the brain

Firstly, this thesis presents the in vivo evaluation of a previously developed model system that utilizes CD4+ TE/M cells decorated with up to 100 polystyrene based nanoparticles per cell by maleimide thiol conjugation. These nanoparticle T-cell conjugates were previously shown to successfully deliver nanoparticles across a monolayer of brain endothelial cells in different in vitro models of the BBB. To evaluate their particle delivery potential to the brain parenchyma in vivo, cells were labeled with different cytosol stains, conjugated to the nanoparticles and injected into the carotid artery of healthy wildtype mice. To distinguish cells that still resided in the brain endothelium and not the brain parenchyma, brains were sectioned into 16µm slices, stained for the parenchymal basement membrane marker laminine and imaged by fluorescence widefield microscopy. As commercially available cytosol stains either drastically reduce cell viability of the T cells or interfere with the nanoparticle conjugation process, intrinsically fluorescent T cells expressing tdtomato were used in further studies to visualize the antigen independent delivery of polystyrene beads to the brain parenchyma. Secondly, this thesis explores different conjugation strategies for the immobilization of biodegradable polymersomes onto the surface of T cells. This work presents the technology transfer of the previously studied model system which used polystyrene beads to the pharmaceutically relevant carrier system using biodegradable polymersomes. It was shown that the anti-biofouling surface properties of the polymersomes prevented their covalent immobilization on the surface of T cells. However, by surface coating of the particles with NeutrAvidin the surface fouling properties could be improved drastically, enabling the NeutrAvidin-biotin based conjugation of polymersomes to the surface of the T cells in therapeutically relevant amounts. The conjugation of polymersomes to T cells was shown to be stable over 24h without impairing cellular key functions like cell viability or T cell binding to the endothelial inflammatory marker ICAM-1 which mediates the T cell extravasation at the level of the brain endothelium. Lastly, this thesis compares different NeutrAvidin based conjugation strategies of polymersomes to T cells and aims to identify the membrane proteins that are utilized throughout the nanoparticle conjugation process to anchor the nanoparticle surface immobilization. Therefore, polystyrene based nanoparticles were conjugated to T cells using NeutrAvidin-biotin based conjugation strategies. The linker between the nanoparticles and the surface proteins was engineered to be selectively cleavable while tagging the proteins for identification by LC-MSMS.