In vitro and in vivo investigation of dendronized streptavidin and fluorescent nanodiamonds, two flexible nanosystems efficiently crossing the blood-brain barrier to improve nanotheranostics in neurological disease treatment
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Abstract
In treatment of central nervous system (CNS) diseases, the blood-brain barrier (BBB) is the main obstacle preventing drug molecule penetration from the bloodstream into the brain. In this thesis dendronized streptavidin (DSA) and fluorescent nanodiamonds (fNDs) encapsulated by a human serum albumin (HSA)-based biopolymer (dcHSA-fNDs) are presented as promising platforms for treatment of neurological disorders via crossing an intact BBB. DSA provides a flexible bio-click system composed of an adapter of tetrameric streptavidin linked to biotinylated PAMAM dendrimers, holding the potential to apply synergistically intrinsic therapeutic function of PAMAM dendrimers combined with any biotinylated drug activity on one platform. dcHSA-fNDs represent a very peculiar nanotheranostics tool characterized by the unique optical and magnetic properties of fNDs. Moreover, the HSA-based coating provides colloidal stability and improved functionalization capability. Thus, dcHSA-fNDs exhibit the potential of real time monitoring of drug delivery.
In this thesis it is successfully demonstrated that both nanocompounds (1) are able to penetrate neurovascular unit (NVU) cells as shown by confocal imaging, immunocytochemical and biochemical analysis; (2) are transported from one side to the other of in vitro BBB models employing porcine and murine mono-cultures or murine triple co-cultures and, (3) the transport occurs via intracellular trafficking without induction of autophagy demonstrated by confocal analysis of colocalization with specific intracellular markers; (4) do not affect NVU cells viability and BBB integrity according to transendothelial electrical resistance monitoring, (5) are able to reach the brain and target NVU cells in vivo after intravenous injection as observable from screening of brain slices combined with immunohistochemistry. Specifically, DSA undergo lysosomal escape preserving cargo-carrier intracellular integrity and do not induce BBB disruption also in vivo validated by Evans Blue assay; dcHSA-fNDs show direct cell-cell migration in NVU cells moving along tunneling nanotubes; the mechanism occurs also among different cell types as observable combining live cell imaging and immunocytochemical analysis. For fNDs, this doctoral thesis provides for the first time reliable data on in vivo brain delivery and mechanisms of transport across the BBB.
In summary, in this thesis two nanoplatforms targeting the brain using highly biocompatible non-invasive strategies to develop various therapeutic approaches are investigated in detail.