New clathrin nanotechnology for transport of large protein molecules to the central nervous system

Abstract

Antibodies (Abs) have great promise for detection and treatment of central nervous system (CNS) disorders. However, the blood-brain barrier (BBB) is a major impediment to effective delivery. Only 0.1% of plasma Abs enter the CNS naturally via diffusion through a compromised BBB or via BBB saturation and CNS concentrations may still be insufficient for therapeutic efficacy. Moreover, Abs may take days to diffuse only a few millimeters and intracellular targets are not easily accessible to Abs. Thus, new, efficient, and noninvasive strategies are required for transporting large molecules like Abs into the CNS and inside targeted cells. Here we tested the hypothesis that clathrin, a coat protein naturally used to transport molecules across biological barriers and within cells, could serve as a nanoplatform for high-efficiency delivery of antibodies and imaging agents to the CNS. Clathrin triskelia (17.7 nm in size) were modified to carry 81 gadolinium chelates or 25 fluorescent tags. Nanoplatforms were characterized by size, structure, protein concentration, chelate and gadolinium contents and nanoparticle relaxivity was evaluated at 0.47 T. Clathrin triskelia exhibited ionic relaxivity of 16 mM-1s-1, and molecular relaxivity of 1166 mM-1s-1. A series of studies were conducted to ascertain whether fluorescent-tagged clathrin nanoplatforms could cross the blood brain barrier. Clathrin nanoplatforms were able to cross or bypass the BBB without enhancements following intraperitoneal and intranasal administration in rats. To demonstrate specific targeting clathrin triskelia were modified with dopamine-3- receptor-antibody (D3R-Ab), as there are no small-molecule ligands that bind exclusively to D3 receptors. One molecule of D3R-Ab was attached per clathrin triskelion and antibody remained intact and immunoreactive after the nanoparticle preparation. Low doses (64 μg/ kg) of nanoparticles (42.3±14.8 nm) were delivered intranasally in rats. Three hours later intact D3R-Ab-triskelia nanoparticles were found in D3R-brain regions inside neurons, with the highest concentration detected in islands of Calleja /ventral pallidum (2753 ng/g or 17.2% ID/g) and nucleus accumbens (1028 ng/g). High nanoprobe concentrations (1062 ng/g) were also found in hippocampal cells that have high concentrations of D3- receptors in the cytoplasm, but low expression of D3-receptors on the cell membrane. Low concentrations were detected in the cerebellum (84 ng/g.) Nanoprobes were not detected in regions lacking D3 receptors. D3R-Abs delivered without clathrin intranasally did not enter the brain...