Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713920
Title: Gold nanoparticles as a delivery system of oligonucleotides into the brain
Author: Gromnicova, Radka
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2017
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Abstract:
The treatment of brain disease is challenging due to the blood-brain barrier, a physiological structure that prevents the majority of potential therapeutic agents from entering the brain. One approach to overcome this problem is the use of nanoparticles as a delivery system. Several types of nanoparticle have shown promise as drug carriers, including gold nanoparticles. They exhibit relatively low cytotoxicity and can enter cells by both active and passive uptake mechanisms and can cross the blood-brain barrier in vivo and in vitro. The aim of this study was to investigate the potential of 5nm gold glyconanoparticles as a delivery system for oligonucleotides into the brain. Three ligand formulations of gold glyconanoparticles were investigated, covalently coated with glucose, OEG-amine/galactose or OEG-amine/galactose/insulin. The two formulations with OEG-amine showed higher uptake efficiency into both human brain endothelial cells (hCMEC/D3) and primary astrocytes, as determined by electron microscopy. Nanoparticles located in subcellular compartments of endothelium were quantitated. Inhibition studies demonstrated that both active and passive transport mechanisms were involved in the uptake of these nanoparticles. However, knockdown of the insulin-receptor on the endothelium did not reduce transport of insulin-coated nanoparticles. It appeared that the OEG-amine coating alone induced much higher levels of vesicular transport, relative to cytosolic uptake. The uptake efficiency of OEG-amine/galactose nanoparticles into different endothelial cells (kidney (ciGENC), bone marrow (BMEC) and lung primary (HMVEC-L)) was compared. Kidney endothelium had higher nanoparticle uptake than brain endothelium. Cellular properties that might influence this cell-selective uptake were investigated; the high level of nanoparticle uptake by kidney endothelium was correlated with a higher level of endocytosis and a different glycocalyx composition on these cells. The transport characteristics of the three formulations of glyconanoparticles were investigated in vivo. The nanoparticles were injected intracarotid into rats and left to circulate for 10 min, in order to capture the first contact of glyconanoparticles with the brain, as detected by electron microscopy. The nanoparticles were observed in brain parenchyma of the cortex, striatum, hippocampus, median eminence and choroid plexus. However, a biodistribution study of the gold, by ICP-mass spectrometry showed that the great majority of the injected nanoparticles were present in the kidney. Finally, a cargo molecule of DNA oligonucleotide was attached to the gold glyconanoparticles (galactose-coated) by the place-exchange reaction, forming ssDNA/galactose nanoparticles. Nanoparticles with different amounts of bound DNA were fractionated by FPLC and analysed by a novel electrophoretic mobility shift assay (EMSA). When comparing the uptake efficiency of dsDNA/galactose nanoparticles to galactose nanoparticles there was no reduction in uptake efficiency, despite addition of the highly negatively charged cargo. To conclude, gold glyconanoparticles can cross the blood-brain barrier and enter cells of the brain in vivo and in vitro. Addition of a DNA oligonucleotide cargo does not alter their ability to cross endothelium and hence < 5 nm gold glyconanoparticles may be a useful carrier of oligonucleotides into the brain.
Supervisor: Not available Sponsor: Basil Sharrack ; OU
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.713920  DOI: Not available
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