Gold Nanocarriers To Deliver Oligonucleotides Into The CNS

Fatima, Nayab (2020). Gold Nanocarriers To Deliver Oligonucleotides Into The CNS. PhD thesis The Open University.

Abstract

Delivery of therapeutical oligonucleotides to the central nervous system is challenging due to their inability to cross the blood-brain barrier. Current approaches to overcome this problem disturb the blood-brain barrier, administer too low dose for therapeutic purposes or have serious side effects. Therefore, nanocarriers, that are able to penetrate the blood-brain barrier, can be used to deliver them. Hence, 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 into the brain.

DNA oligonucleotides were attached to the NP-Gal via place exchange reaction. Several nanoparticle formulations were created, such as 20nt single-stranded versus double stranded versions of DNA-coated gold nanoparticles (NP-DNA-20ss, NP-DNA-20ds) and 40nt long double stranded versions of DNA-coated gold nanoparticles (NP-DNA-40ds). These were analysed by (1) electrophoresis mobility shift assay (for detection of nanoparticles with different numbers of DNA molecules), and (2) fast protein liquid chromatography (for isolation of NP-DNA-40ds) forming two fractionated formulations - low-density 40nt DNA nanoparticle formulation (NP-DNA-40LO) and high-density 40nt DNA nanoparticle formulation (NP-DNA-40HI). The rate and route of transport across brain endothelial cells (hCMEC/D3) was assessed by transmission electron microscopy. A 3D co-culture model of the blood brain barrier was used to detect nanoparticle uptake into astrocytes once they passed through brain endothelial cells. Next, we performed in vivo examination of tissue distribution of the nanoparticles following intracarotid and intravascular infusion (using ICP mass spectroscopy along with light and electron microscopy). Lastly, qPCR was used to quantify transport rate and efficiency of DNA cargo across brain endothelial cells.

DNA oligonucleotides attached to NP-Gal by place exchange reaction produced six different nanoparticle formulations. The cell uptake efficiency of NP-DNA-20ss, NP-DNA-20ds and NP-DNA-40ds was compared; the more negatively-charged DNA bound nanoparticles had increased cellular uptake in human brain endothelial cells compared with the NP-Gal. This investigation pointed out the possibility that attaching more DNA onto each nanoparticle may increase the uptake efficiency of NP-Gal by brain endothelium. The transport rate of DNA nanoparticles free of unreacted DNA and nanoparticles - NP-DNA-40LOco-culture model of blood-brain barrier. Both the NP-DNA-40LO and NP-DNA-40HI were more effective in crossing the brain endothelial cells and entering astrocytes compared to NP-Gal. Moreover, we found our nanoparticles to be not cytotoxic for hCMEC/D3 at the dose used in our studies (8µg/mL). Next, we investigated tissue localization of NP-Gal, NP-DNA-40LO and NP-DNA-40HI 10 minutes after intravascular infusion in rats. All three nanoparticles formulations localised in the brain endothelial cells and glial cells of the cortex. While NP-Gal were mainly found in the kidney, DNA-coated nanoparticles were found in the cytoplasm of liver hepatocytes and macrophages of the red pulp in the spleen. Lastly, we found that DNA cargo canLO and NP-DNA-40HI carried over half of the DNA applied (apical side) across the brain endothelium. In conclusion, our study demonstrated the possibility to deliver 40nt DNA oligonucleotide across the blood-brain barrier using a 2nm gold nanocarrier. There was improvement in the bioavailability of oligonucleotides across the brain endothelial cells carried by NP-DNA-40LO and NP-DNA-40HI compared to naked DNA in vitro. In vivo, we found similar brain penetration of NP-Gal, NP-DNA-40LO and NP-DNA-40HI so further studies may be needed.

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