Abstract:
Nanoparticles are attractive candidates for drug development given their variable size, functionalisation, and reactivity with molecules. Gold nanoparticles possess the potential to elicit anticancer effects, though often these are determined in two-dimensional cell models that fail to represent the in vivo environment appropriately. Three-dimensional cultures, such as multicellular spheroids, offer greater representation, particularly due to their heterogenous phenotype and complex molecular architecture. This study aimed to develop an A549 alveolar carcinoma spheroid model to use as a mechanistic cytotoxicity testing platform for functionalised gold nanoparticles.
A549 multicellular spheroids were generated using an agarose micro-mould, with characterisation (morphology, acid phosphatase activity, protein content) over 21 days of growth. Cytotoxicity of 14 nm carboxyl-polyethylene glycol- and 20 nm amine-coated gold nanoparticles in spheroids were assessed on the seventh day post-seeding by determining the effect on morphology, acid phosphatase activity, protein content, caspase-3/7 activity and cell cycle alterations.
A549 spheroids were maintained over 21 days of growth, however they started to lose structural integrity on day 14. The 14 nm carboxyl-polyethylene glycol-liganded gold nanoparticles were not cytotoxic at a maximum concentration of 1.2 x 1012 nanoparticles/mL. The 20 nm amine-coated gold nanoparticles yielded dose- and time-dependent cytotoxicity, where decreased spheroid compactness was observed over a three-day exposure period at 4.5 x 1012 nanoparticles/mL. Over the same exposure time, no effect on the protein content was observed. Increased debris formation and deoxyribonucleic acid (DNA) fragmentation was observed through flow cytometric analysis, as well as a 75.00% decrease (significant, p ≤ 0.001 in acid phosphatase activity, though no increase in caspase-3/7 activity occurred.
Spheroids were successfully formed and remained stable over the experimental period. Furthermore, differentiation between cytotoxic and non-cytotoxic gold nanoparticles, and further elucidation of its potential cytotoxic mechanism, was accomplished. Successful implementation of this method has therefore afforded greater representation of in vitro findings to the in vivo environment.
