Mechanisms of Nanoparticle Toxicity in Cancer and Normal Cells

Nanotechnology offers vital tools for cancer diagnosis, early detection and novel treatments. Gold nanoparticles (AuNPs) are at the forefront of biological and biomedical research and show an increased promise in targeted cancer therapy due to their unique physicochemical properties. They offer the advantage of biomolecular interaction both at the cell surface level and inside the cell. In this thesis, 2nm AuNPs surface functionalised with a 50:50 ratio of a thiolated α-Galactose derivative and a thiol-PEGamine were examined for their toxicity and uptake in normal and cancer cell lines in vitro. Using this simple ligand structure, a selective cancer toxicity was observed, which was highly dependent upon the nanoparticles’ synthesis duration. This observation was correlated to ligand density, which decreased with shorter synthesis time, making particles less toxic. Detailed insight into the mechanism of toxicity and uptake was gained by energy-dependent experiments and cell death assays, revealing a significant adhesion of these particles to filopodia. The proposed mechanism of cell death in HSC oral squamous carcinoma cells exposed to AuNPs was identified to be the extrinsic apoptotic pathway, as toxicity was abrogated by inhibition of either caspase 3/7 or caspase 8, but not by inhibition of caspase 9. Future directions include batch standardisation of the optimum nanoparticle synthesis conditions and elucidation of the mechanisms of cell uptake and toxicity in a variety of cancer and normal cell lines

Galactose:PEGamine coated gold nanoparticles adhere to filopodia and cause extrinsic apoptosis

Ultra-small gold nanoparticles, surface functionalised with a 50:50 ratio of a thiolated α-Galactose derivative and a thiolated hexaethylene glycol amine, are toxic to HSC-3 oral squamous carcinoma cells. Differences in nanoparticle toxicity were found to be related to synthesis duration, with 1 h reaction nanoparticles being less toxic than 5 h reaction nanoparticles. Ligand density decreased with longer reaction time, although size, charge and ligand ratio remained similar. The concentration of sodium borohydride in the reaction decreased logarithmically over 5 h but remained within a concentration range sufficient to desorb weakly-bound ligands, possibly explaining the observed gradual decrease in ligand density. Nanoparticle toxicity was abrogated by inhibition of either caspase 3/7 or caspase 8, but not by inhibition of caspase 9, consistent with extrinsic apoptosis. Electron microscopic analysis of cellular uptake demonstrated predominantly cytoplasmic localization. However, when energy-dependent transport was inhibited, by lowering the temperature to 4ºC, a remarkable adhesion of nanoparticles to filopodia was observed. Inhibition of filopodial assembly with a fascin inhibitor prevented nanoparticle adhesion to HSC-3 cells at 4ºC, while fascin inhibition at 37ºC resulted in less cytoplasmic uptake. More adhesion to HSC-3 filopodia was seen with the higher toxicity 5 h reaction time nanoparticles than with the 1 h nanoparticles. By including a further two cell types (HaCaT keratinocytes and hCMEC/D3 endothelial cells), a pattern of increasing toxicity with filopodial binding of 5 h reaction nanoparticles became apparent

Cancer-selective, single agent chemoradiosensitising gold nanoparticles

Two nanometre gold nanoparticles (AuNPs), bearing sugar moieties and/or thiol-polyethylene glycol-amine (PEG-amine), were synthesised and evaluated for their in vitro toxicity and ability to radiosensitise cells with 220 kV and 6 MV X-rays, using four cell lines representing normal and cancerous skin and breast tissues. Acute 3 h exposure of cells to AuNPs, bearing PEG-amine only or a 50:50 ratio of alpha-galactose derivative and PEG-amine resulted in selective uptake and toxicity towards cancer cells at unprecedentedly low nanomolar concentrations. Chemotoxicity was prevented by co-administration of N-acetyl cysteine antioxidant, or partially prevented by the caspase inhibitor Z-VAD-FMK. In addition to their intrinsic cancer-selective chemotoxicity, these AuNPs acted as radiosensitisers in combination with 220 kV or 6 MV X-rays. The ability of AuNPs bearing simple ligands to act as cancer-selective chemoradiosensitisers at low concentrations is a novel discovery that holds great promise in developing low-cost cancer nanotherapeutics

High expression of BCL-2 predicts favorable outcome in non-small cell lung cancer patients with non squamous histology

Abstract Background Bcl-2 promotes cell survival by inhibiting adapters needed for the activation and cleavage of caspases thus blocking the proteolytic cascade that ultimately dismantles the cell. Bcl-2 has been investigated as a prognostic factor in non small cell lung cancer (NSCLC) patients with conflicting results. Methods Here, we quantitatively assessed Bcl-2 expression in two large and independent cohorts to investigate the impact of Bcl-2 on survival. AQUA®, a fluorescent-based method for analysis of in situ protein expression, was used to measure Bcl-2 protein levels and classify tumors by Bcl-2 expression in a cohort of 180 NSCLC patients. An independent cohort of 354 NSCLC patients was used to validate Bcl-2 classification and evaluate outcome. Results Fifty % and 52% of the cases were classified as high expressers in training and validation cohorts respectively. Squamous cell carcinomas were more likely to be high expressers compared to adenocarcinomas (63% vs. 45%, p = 0.002); Bcl-2 was not associated with other clinical or pathological characteristics. Survival analysis showed that patients with high BCL-2 expression had a longer median survival compared to low expressers (22 vs. 17.5 months, log rank p = 0.014) especially in the subset of non-squamous tumors (25 vs. 13.8 months, log rank p = 0.04). Multivariate analysis revealed an independent lower risk for all patients with Bcl-2 expressing tumors (HR = 0.53, 95% CI 0.37-0.75, p = 0.0003) and for patients with non-squamous tumors (HR = 0.5, 95% CI 0.31-0.81, p = 0.005). Conclusions Bcl-2 expression defines a subgroup of patients with a favorable outcome and may be useful for prognostic stratification of NSCLC patients.</p

Clonogenic assay of HSC-3 cells, demonstrating a partial rescue of 50:50 αGal:PEG-amine AuNP-induced cell death by 50 μM Z-VAD-FMK caspase inhibitor.

<p>10 μM Antimycin A was used as an apoptosis positive control. For each condition, n = 3 and data are presented ±SEM. * Denotes a significant difference (P<0.05 ANOVA, Tukey multiple comparisons post-test).</p

AuNP physical characteristics.

<p>AuNP physical characteristics.</p

Clonogenic assay dose-response curves for three different 50:50 sugar:PEG-amine AuNPs on adherent HSC-3 and HaCaT cells.

<p>Cells were loaded with a range of AuNP concentrations for: A) 1 h, B) 3 h, C) 6 h and D) 24 h. The graphs represent the percentage of cell colonies compared to the no-nanoparticle control for each sugar:PEG-amine AuNP ±SEM.</p

Clonogenic assay dose-response of adherent MCF-7 and MCF-10 cells exposed for 3 h to different concentrations of 50:50 or 0:100 αGal:PEG-amine AuNPs.

<p>The graphs represent the percentage of cell colonies compared to the no-nanoparticle control ±SEM.</p