Magnetite nanoparticles as a vehicle to transport recombinant hexosaminidase A and B through an in vitro model of the bloodbrain barrier

Nanoparticles are efficient vehicles to pharmacological delivery to different target tissues. Currently, magnetite nanoparticles (MNP) conjugated to recombinant enzymes, have been proposed as an alternative carrier to allow the enzymes penetration through the blood brain barrier (BBB) during the intravenous enzyme replacement therapy (ERT) administration in lysosomal disorders with central nervous system involvement. This novel strategy would avoid the use of invasive approaches such as intrathecal or intracerebroventricular injections to reach the cell target. Previously, we have observed that MNP/HexA-conjugates are uptaked and sorted to the lysosomes reducing the stored lipids after 24 and 48 h of incubation with a single administration on GM2-gangliosidoses fibroblast models. In this work, a BBB in vitro model were used to evaluate the ability of MNP/HexA and MNP/HexB nanobioconjugates to cross throughout. MNP were synthesized and conjugated with recombinant HexA and HexB, and characterized to obtain adequate size nanobioconjugates. The BBB in vitro model was generated using the endothelial cells in upper chamber and Tay Sachs and Sandhoff fibroblasts in the bottom chamber of transwell plates. Cytotoxicity assays were performed on endothelial cells using MTT approach. Trans-endothelial resistance (TEER) was measured daily with and without nanobioconjugates and recombinant proteins administration. To assess passage through the endothelial layer, labeled nanoparticles were used, and fluorescence were measured in the bottom medium. Enzyme activity was evaluated on culture medium and fibroblasts lysate. Endothelial cells maintain a stable TEER during 7 days without treatment. Upon nanoparticles administration a TEER variation was observed on the third day of treatment. Fluorescence increases in bottom chamber after one day of incubation with labeled-nanoparticles. Likewise, changes in the fibroblast's activity levels were observed in the first two days of treatment, where TEER were not altered. Nanobioconjugates are promissory carriers in the development of ERT to patients with LD involving CNS.