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    Transferrin receptor-mediated gene delivery using functionalised gold nanoparticles.

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    Padayachee_Jananee _2018.pdf (4.827Mb)
    Date
    2018
    Author
    Padayachee, Jananee.
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    Abstract
    Gene therapy strategies have shown their potential in treating numerous central nervous system (CNS) disorders, including highly aggressive brain cancers. Gold nanoparticles (AuNPs) are popular vectors for gene delivery, due to their low toxicity, and ease of synthesis and functionalisation. However, the in vivo efficacy of these vectors is dependent on their ability to cross the blood-brain barrier (BBB), a specialised capillary network preventing the movement of compounds into the CNS. Passage across the BBB is often facilitated through targeting of the transferrin (Tf) receptor, leading to uptake by receptor-mediated transcytosis. This study aimed to develop untargeted and Tf-targeted functionalised AuNP (FAuNP) vectors and assess their potential as gene delivery vectors. AuNPs were prepared through citrate reduction and functionalised with chitosan (CS) and poly(ethylene) glycol 2000 (PEG2000) in two weight ratios [2% and 5% (ww⁄)] to produce untargeted FAuNPs. The holo-transferrin protein was conjugated to both PEGylated and unPEGylated FAuNPS to produce the Tf-targeted FAuNPs (TfAuNPs). The physicochemical characteristics of FAuNPs were evaluated using UV spectroscopy, Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). TEM revealed AuNP to be spherical and relatively monodisperse. FAuNPs displayed hydrodynamic diameters ranging from 94.7 – 196.4 nm with good colloidal stability, as evidenced by NTA. Binding studies viz. band shift and ethidium bromide intercalation assays showed that all FAuNPs were able to fully complex and efficiently condense pCMV-luc plasmid DNA, with PEGylated and targeted FAuNPs being capable of partially protecting DNA from nuclease degradation, as determined in nuclease protection assays. In vitro studies were conducted in the HEK293, Caco-2, and the Tf receptor-positive HeLa cell lines. Cytotoxicity was assessed using the MTT cytotoxicity assay, which revealed FAuNPs to be relatively non-toxic to HeLa and HEK293 cells. Notably, TfAuNPs displayed low cytotoxicities, and generally exhibited increased cell viabilities compared to the untargeted FAuNPs. The luciferase gene reporter assay was conducted to assess the transfection efficiency of the FAuNPs. Transfection levels were highest in Caco-2 cells, with PEGylated FAuNPs observed to produce reduced transfection compared to the unPEGylated FAuNPs. TfAuNPs displayed favourable transfection in HeLa cells; with the competition binding assays confirming receptor-mediated uptake for AuCSTf and AuCSTf-5% PEG FAuNPs only, suggesting that a grafting density of the 2% (ww⁄) PEG interfered with receptor binding. These Tf-targeted FAuNPs show the potential to be utilised as vectors for brain delivery; however further optimisation and investigations in an in vivo system are required.
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    https://researchspace.ukzn.ac.za/handle/10413/18378
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