Robert Tshikhudo

Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines.

BackgroundReliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles. In this study, the cytotoxicity and uptake of 14 nm and 20 nm citrate stabilised gold nanoparticles (AuNPs) in the bronchial epithelial cell line BEAS-2B, the Chinese hamster ovary cell line CHO, and the human embryonic kidney cell line HEK 293 were investigated.MethodsCytotoxicity of the AuNPs was assessed via traditional XTT-, LDH-, and ATP-based assays, followed by cell impedance studies. Dark-field imaging and hyperspectral imaging were used to confirm the uptake of AuNPs into the cells.ResultsInterference of the AuNPs with the XTT- and ATP-based assays was overcome through the use of cell impedance technology. AuNPs were shown to be relatively non-toxic using this methodology; nevertheless CHO cells were the most sensitive cell type with 20 nm AuNPs having the highest toxicity. Uptake of both 14 nm and 20 nm AuNPs was observed in all cell lines in a time- and cell type-dependent manner.ConclusionsUsing the cell impedance and dark-field hyperspectral imaging technologies, it was possible to study the toxicity of AuNPs in different cell lines and show that these cells could internalize AuNPs with their subsequent intracellular aggregation. It was also possible to show that this toxicity would not correlate with the level of uptake but it would correlate with cell-type and the size of the AuNPs. Therefore, these two label-free methodologies used in this study are suitable for in vitro studies on the effects of AuNPs, and could present themselves as appropriate and valuable methodologies for future nanoparticle toxicity and uptake studies.

Monolayer-protected clusters of gold nanoparticles: impacts of stabilizing ligands on the heterogeneous electron transfer dynamics and voltammetric detection.

Surface electrochemistry of novel monolayer-protected gold nanoparticles (MPCAuNPs) is described. Protecting ligands, (1-sulfanylundec-11-yl)tetraethylene glycol (PEG-OH) and (1-sulfanylundec-11-yl)poly(ethylene glycol)ic acid (PEG-COOH), of three different percent ratios (PEG-COOH:PEG-OH), 1:99 (MPCAuNP-COOH(1%)), 50:50 (MPCAuNP-COOH(50%)), and 99:1 (MPCAuNP-COOH(99%)), were studied. The electron transfer rate constants (k(et)/s(-1)) in organic medium decreased as the concentration of the surface-exposed -COOH group in the protecting monolayer ligand is increased: MPCAuNP-COOH(1%) (approximately 5 s(-1)) > MPCAuNP-COOH(50%) (approximately 4 s(-1)) > MPCAuNP-COOH(99%) (approximately 0.5 s(-1)). In aqueous medium, the trend is reversed. The surface pK(a) was estimated as approximately 8.2 for the MPCAuNP-COOH(1%), while both MPCAuNP-COOH(50%) and MPCAuNP-COOH(99%) showed two pK(a) values of about 5.0 and approximately 8.0. These results have been interpreted in terms of the quasi-solidity and quasi-liquidity of the terminal -OH and -COOH head groups, respectively. MPCAuNP-COOH(99%) excellently suppressed the voltammetric response of the ascorbic acid but enhanced the electrocatalytic detection of epinephrine compared to the other MPCAuNPs studied. This study reveals important factors that should be considered when designing electrode devices that employ monolayer-protected gold nanoparticles and possibly for some other redox-active metal nanoparticles.

Gold nanoparticle based Tuberculosis immunochromatographic assay: The quantitative ESE Quanti analysis of the intensity of test and control lines

A rapid dual channel lateral flow assay for the detection of Mycobacterium Tuberculosis antibodies (MTB 38 kDa monoclonal antibody) in human blood was developed. The MTB 6-14-38 kDa fusion antigen and anti-Protein A were used as the capture proteins for test and control lines respectively. Protein A labeled 40 nm gold nanoparticles were used as the detection conjugate. Whole blood and serum were spiked with MTB 38 kDa monoclonal antibody to make a positive sample model. The developed lateral flow was used to test MTB 38 kDa monoclonal antibody, and a detection limit of 5 ng/ml was used as a cut-off concentration of the analytes. The effect of the analyte concentration on the MTB lateral flow assay was studied using the variation of the intensity obtained from a ESE Quanti reader. There was a direct correlation between the analyte (MTB 38 kDa monoclonal antibody) concentration and the intensity of the test line. The intensity increased with an increase in the concentration of MTB 38 kDa monoclonal antibody, while in contrast, an increase in analyte concentration decreased the intensity of the control line.

Facile Attachment of TAT Peptide on Gold Monolayer Protected Clusters: Synthesis and Characterization

High affinity thiolate-based polymeric capping ligands are known to impart stability onto nanosized gold nanoparticles. Due to the stable gold-sulfur bond, the ligand forms a protective layer around the gold core and subsequently controls the physicochemical properties of the resultant nanogold mononuclear protected clusters (AuMPCs). The choice of ligands to use as surfactants for AuMPCs largely depends on the desired degree of hydrophilicity and biocompatibility of the MPCs, normally dictated by the intended application. Subsequent surface modification of AuMPCs allows further conjugation of additional biomolecules yielding bilayer or multilayered clusters suitable for bioanalytical applications ranging from targeted drug delivery to diagnostics. In this study, we discuss our recent laboratory findings on a simple route for the introduction of Trans-Activator of Transcription (TAT) peptide onto the surface of biotin-derivatised gold MPCs via the biotin-strepavidin interaction. By changing the surface loading of biotin, controlled amounts of TAT could be attached. This bioconjugate system is very attractive as a carrier in intercellular delivery of various delivery cargoes such as antibodies, proteins and oligonucleotides.

Polyanilino-Carbon Nanotubes Derivatised Cytochrome P450 2E1 Nanobiosensor for the Determination of Pyrazinamide Anti-Tuberculosis Drugs

Pyrazinamine (PZA) is one of the most commonly prescribed anti-tuberculosis (anti-TB) drug due to its ability to significantly shorten the TB treatment period. However, excess PZA in the body causes hepatotoxicity and liver damage. This, therefore, calls for new methods for ensuring reliable dosing of the drug, which will differ from person to person due to interindividual differences in drug metabolism. A novel biosensor system for monitoring the metabolism of PZA was prepared with nanocomposite of multi-walled carbon nanotubes (MWCNTs), polyaniline (PANI) and cytochrome P450 3A4 (CYP3A4) electrochemically deposited on a glassy carbon electrode (GCE). The nanocomposite biosensor system exhibited enhanced electroactivity that is attributable to the catalytic effect of the incorporated MWCNTs. The biosensor had a sensitivity of 7.80 μA/μg mL-1 PZA and a dynamic linear range of 4.92 160 ng/mL PZA.

Stable, Hydrophilic Nitrilotriacetic Acid-Capped Gold Monolayer Protected Clusters

A one step synthesis and functionalization of robust, hydrophilic monolayer protected clusters of gold (Au-MPCs) containing a nitrilotriacetic acid (NTA) is described. The 14 nm Au-MPCs were prepared by the attachment of two bifunctional thioalkylated-poly(ethylene glycol) ligands on the nanoparticles, one containing a nitrilotriacetic acid (NTA) terminal group, while the second hydroxyl (OH) terminated ligand was used as a co-stabilizer to promote the stability of the MPCs. The resulting PEGylated NTA functionalized Au MPCs, which are characterized by TEM, UV-vis and agarose gel electrophoresis are attractive probes for many target species e.g. hexahistidine-tagged proteins. Importantly, the NTA functionality on MPCs ligand shell can be varied.

Investigating the Stabilisation Effect of Carboxylic vs. Hydroxyl Groups on Fe3O4 and Fe3O4@Au Nanoparticles

The high-temperature solution phase reaction of iron(III) acetylacetonate, Fe(acac)3 and 1,2-hexadecanediol was used to synthesise iron oxide and gold-coated iron oxide nanoparticles. Different surface functionalities, such as sebacic acid (SA) and 1, 10–Decanediol (DD), were introduced on the surface of the particles to investigate the stabilising effect of carboxylic groups (SA) in comparison to the hydroxyl groups (DD). Nanoparticle thermal stability, composition, state of aggregation, size and morphology were investigated and the results from techniques such as Fourier Transform-Infra Red spectroscopy (FT-IR), Ultraviolet visible spectroscopy (UV-vis), Transmission Electron Microscopy (TEM) and thermal analysis are discussed.