Gold nanocarriers for transport of oligonucleotides across brain endothelial cells

Abstract

Treatment of diseases that affect the CNS by gene therapy requires delivery of oligonucleotides to target cells within the brain. As the blood brain barrier prevents movement of large biomolecules, current approaches involve direct injection of the oligonucleotides, which is invasive and may have only a localised effect. The aim of this study was to investigate the potential of 2 nm galactose-coated gold nanoparticles (NP-Gal) as a delivery system of oligonucleotides across brain endothelium. DNA oligonucleotides of different types were attached to NP-Gal by the place exchange reaction and were characterised by EMSA (electrophoretic mobility shift assay). Several nanoparticle formulations were created, with singleor double-stranded (20nt or 40nt) DNA oligonucleotides, or with different amounts of DNA attached to the carriers. These nanocarriers were applied to transwell cultures of human brain endothelium in vitro (hCMEC/D3 cell-line) or to a 3D-hydrogel model of the blood-brain barrier including astrocytes. Transfer rates were measured by quantitative electron microscopy for the nanoparticles and qPCR for DNA. Despite the increase in nanoparticle size caused by attachment of oligonucleotides to the NP-Gal carrier, the rates of endocytosis and transcytosis of nanoparticles were both considerably increased when they carried an oligonucleotide cargo. Carriers with 40nt dsDNA were most efficient, accumulating in vesicles, in the cytosol and beneath the basal membrane of the endothelium. The oligonucleotide cargo remained attached to the nanocarriers during transcytosis and the transport rate across the endothelial cells was increased at least 50fold compared with free DNA. The nanoparticles entered the extracellular matrix and were taken up by the astrocytes in biologically functional amounts. Attachment of DNA confers a strong negative charge to the nanoparticles which may explain the enhanced binding to the endothelium and transcytosis by both vesicular transport and the transmembrane/cytosol pathway. These gold nanoparticles have the potential to transport therapeutic amounts of nucleic acids into the CNS.

Tissue Distribution and Cellular Localization of Gold Nanocarriers with Bound Oligonucleotides

“…The size of the gold core of the DNA-conjugated NP-Gal was determined by TEM (Supplementary Figures 3 & 4) and the hydrodynamic diameter by FPLC gel filtration. These data have been reported previously [13] and are summarized in Supplementary Figure 1.…”

“…As determined by molecular mass of the gold core (∼20,000) and physical size, these nanoparticles have approximately 100 gold atoms in the core and 40 covalently bound C2-galactose molecules on the outside. The characteristics of these nanoparticles were reported previously [13] and are summarized in Supplementary Figure 1. These glyconanoparticles are designated 'NP-Gal'.…”

“…The method for production and characterization of the gold glyconanoparticles with bound oligonucleotides has been described previously [13]. It involves a chemical exchange reaction in which the ligand with a free -SH group exchanges with the C2-galactose which is bound to the gold core via a sulfur atom.…”

“…Attachment of cargo molecules to the glyconanoparticles can radically alter their physicochemical characteristics and their interaction with endothelial cells. For example, coupling 40-nt double-stranded DNA oligonucleotides to the outside of the nanoparticles makes them negatively charged, increases their hydrodynamic diameter to approximately 7 nm and increases the rate of transcytosis of the carriers up to tenfold and cargo 50-fold [13].…”

“…The aim of this study was to determine whether attachment of an oligonucleotide cargo to the gold nanocarriers affects their tissue distribution and cellular localization in vivo. Two formulations of nanoparticle were used, one with 1-3 bound oligonucleotides per nanoparticle (NP-DNA-40 LO ) and the other with 3-6 oligonucleotides per nanoparticle (NP-DNA-40 HI ) [13]. They were compared with the unconjugated glyconanoparticle (NP-Gal).…”