An investigation into the uptake of gold nanoparticles by isolated cells and whole vessels and their influence on function

Mohamed, Teba (2014) An investigation into the uptake of gold nanoparticles by isolated cells and whole vessels and their influence on function. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Background: Nanoparticles (NPs) of different material composition (silica, gold) have gained increasing attention in drug therapeutics and diagnosis of disease. In particular, Gold nanoparticles (AuNPs) demonstrate potential for cell tracking and imaging diagnostics, however, their impact on cellular and vascular function remains uncertain. The AuNPs emission characteristics can vary when they are suspended in different physiological fluids due to their aggregation; hence, to overcome this effect, organic polymer composite coatings have been applied. The aim of the present study is to investigate the effects AuNPs and their surface modifiers on endothelial cell and murine aortic vascular function, ex vivo. Methods: A number of nanoparticles were fabricated (silica, gold), but AuNPs were selected because they were simple to synthesise and characterise. AuNPs (12±3nm) were synthesised according to the Turkervich method. They were then surface modified with the polymers polyvinylpyrrolidone (PVP) and mercapto-polyethylene glycol - (mPEG) and characterised by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDAX) and UV-Vis spectroscopy. Silica NPs (SiNPs, 100 and 200 nm) were synthesised using a modification of the Stöber method. They were characterised using TEM and the Malvern Zeta Sizer. Cellular uptake of AuNPs by cultured bovine aortic endothelial cells (BAECs) was visualized using transmission electron microscopy (TEM). The effects of AuNPs on BAEC proliferation, cell viability and apoptosis were determined using the automated cell counter and flow cytometry for cell number counting, exclusion dye propidium iodide (PI) and Annexin V/PI detection, respectively. Aortic vessel rings from male Wistar rats were mounted between two fine steel wires in an organ bath system and constantly superfused in oxygenated physiological salt solution (PSS) at 37oC. Cumulative doses of the endothelial dependent agonist acetylcholine (ACh; 0.01-100μM) and endothelial independent sodium nitroprusside (SNP; 0.1nm-10μM) were added to KCl preconstricted vessels, before and 30 minutes after incubation with modified and non-modified AuNPs. Results: Maximum cellular uptake of AuNPs was observed 24 hours and 48 hours, after incubation in non-modified and modified AuNPs, respectively. Both non-modified and modified AuNPs significantly decreased cell viability and proliferation and increased apoptosis up to 24 hours after incubation, whereas no inhibitory effect was observed after 48 hours of incubation. Both modified and non-modified AuNPs influenced cellular signalling and reduced Erk and Akt phosphorylation. Non-modified AuNPs had no overall effect on ACh responses, but significantly reduced responses to SNP. PVP modified AuNPs (AuPVP), but not mPEG modified AuNPs (AumPEG) lead to a significant attenuation in ACh responses (ACh concentration 0.01-100μM; p<0.05). AumPEG at 2.9μg/mL had no overall significant effect on either ACh or SNP responses; however, at higher concentration (5.8μg/mL) AumPEG NPs led to a significant reduction in ACh dilator response at most ACh concentrations. PVP alone, at the minimal concentration required to stabilise the AuNPs, reduced ACh dilator responses. Conclusions: We demonstrate that surface modification of AuNPs using polymers enhances their stability in physiological solutions and culture media, and also reduces cellular uptake by BAECs. Furthermore, we show that cellular and vascular effects of AuNPs depends on the type and concentration of polymer modification used. mPEG modified AuNPs show greater biocompatibility and are less detrimental to vasodilator function, than PVP modified AuNPs, thus showing greater potential use as agents for diagnostic imaging and therapeutics.