Autodisplayed LPR1 IV-domain as a tool for identification of drug delivery agents across the blood-brain barrier

Gisbert Fenoy C, Raudszus B, Zlatev I, Nienberg C, Langer K, Jose J

Abstract

LRP1 (Low-density lipoprotein receptor-related protein-1) is a widely expressed cell-surface receptor with diverse biological functions, including ApoE binding and endocytosis. LRP1 is abundantly expressed in neurons and the vascular system, making it an efficient target for delivery of therapeutic substances across the blood-brain barrier (BBB), in case these are part of a system, like nanoparticles, where ApoE is present.1 Previous studies showed the high binding affinity of ApoE to the fourth binding domain of LRP1 (LRP1-IV).2 In this work, LRP1-IV was expressed on the surface of E. coli using Autodisplay technology, to use whole cells for binding studies with ApoE, avoiding cumbersome protein purification. For this purpose an artificial gene for a fusion protein was constructed. The fusion protein consisted of an N-terminal signal peptide that directs the protein across the inner membrane of E. coli, the LRP1-IV domain, and the C-terminal autotransporter (AT). The AT facilitates the transport of the LRP1-IV to the surface of the cell.3 Surface display of LRP1-IV was verified by western blot of outer membrane preparations and flow cytometry of whole cells with a specific LRP1 antibody. Flow cytometric analysis also indicated that cells displaying LRP1-IV bind purified ApoE3 and ApoE3 labeled with PromoFluor-NHS 633. The assay as established enables to investigate the binding of ApoE3 variants to LRP1-IV or variants thereof and will allow determination of binding affinities. Our next step will be to test the binding of ApoE3 modified nanoparticles to surface displayed LRP1-IV. These nanoparticles could be used as carriers across the BBB for drugs which normally are not able to cross this barrier.4,5