Mechanisms facilitating uptake of carboxyl-polyethylene glycol-functionalised gold nanoparticles into multicellular spheroids

Abstract

The drug discovery pipeline is hindered by confounding and non-representative in vitro cellular models. Traditionally, monolayer cell cultures have been used to evaluate drug toxicity and efficacy; however, are not sufficiently representative of the in vivo milieu. More advanced culture methods, including three-dimensional (3D) multicellular tumour spheroids, offer a solution by presenting a more appropriate physiological state for the cellular system. Equally damaging to the drug discovery pipeline, however, are candidate drug compounds with little potential. Nanomedicines have shown promise in drug delivery and various other theragnostic applications, and increasingly continue to do so. Generally, medicines are required to permeate structures, especially solid tumours, into the intracellular environment to exert activity. As such, there is a need for both uptake mechanisms and intracellular trafficking pathways to be well characterised. Further to the potential for cancer research, the National Institute for Occupational Health (South Africa) researches the Health, Safety, and Environment (HSE) of engineered nanomaterials. This study aimed to establish an A549 alveolar carcinoma spheroidal drug discovery and toxicity testing platform for the elucidation of uptake mechanisms employed for the pilot nanoparticles (NPs) in this study: 14 nm carboxyl-polyethylene glycol-functionalised gold nanoparticles (PCOOH-AuNPs). The PCOOH-AuNPs were manufactured and characterised in terms of size, surface charge, spectral activity, and concentration by Mintek (South Africa). The A549 alveolar carcinoma cell line was used in conjunction with the liquid overlay technique, allowing for efficient and reproducible spheroid formation. Phase contrast microscopy was used alongside ImageJ analysis to monitor morphological aspects of spheroid growth, as well as cell lysis and lactate dehydrogenase (LDH) release for relative enumeration of cells. Live/dead staining was used to visualise areas of metabolic activity (viability) and compromised membranes (cell death) within a spheroid. Cytotoxicities of the PCOOH-AuNPs and pharmacological uptake inhibitors were assessed by monitoring LDH release from spheroids after exposure. Uptake mechanisms were assessed via CytoViva® hyperspectral imaging of 5 µm cryotomed spheroid sections after exposure to the pharmacological uptake inhibitors; sodium azide, dynasore, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), genistein, and chlorpromazine. Cells formed compact and reproducible spheroids within seven days after seeding, showing an average diameter and circularity index of 702.11 μm and 0.80, respectively. Zones of rapid metabolism and viability were evident from live/dead staining towards the superficial layers of a spheroid, as were zones of cell death towards the core of the spheroid. The Day 7 spheroids exhibited no greater LDH release than negative controls when exposed to either the AuNPs (2 and 24 h exposures) or pharmacological inhibitors (3 h exposures). Internalised AuNPs were counted in the presence or absence of uptake inhibitors to deduce employment of endocytic mechanisms. Counts were obtained and the following proportions of uptake mechanisms were calculated to have been employed for PCOOH-AuNP uptake: 9.2% passive diffusion, 6.6% macropinocytosis, 17.1% clathrin- and caveolae-independent pathways, 33.5% to 54.8% clathrin-mediated endocytosis, and 3.1% to 24.4% caveolae-mediated endocytosis. Penetration of AuNPs into spheroids was, on average, 4.5 μm, which is low, and indicates low levels of transcytosis, as well as intracellular retention of the PCOOH-AuNPs. Uptake mechanisms employed by A549 spheroids for PCOOH-AuNPs were found to be diverse; however, primarily occurred via clathrin-mediated endocytosis. This means that PCOOH-AuNPs are likely to be trafficked towards a degradative fate in the lysosome. Such a destination largely invalidates their use for intracellular drug delivery, unless drugs or NPs are to induce lysosomal membrane permeabilization, exerting action in that way. Findings made in this study promise to inform intelligent design of future AuNP renditions having the goal of greater safety, efficacy, and achievement of desired theragnostic purpose. Further, the lack of cytotoxicity of both the PCOOH-AuNPs and all the uptake inhibitors, validates the methods in this study as reliable. A reproducible, representative 3D in vitro NP testing model has been established, using the A549 alveolar carcinoma cell line, which can be used to assess uptake strategies employed by NPs.