Abstract
BackgroundThe challenge remains to reliably mimic human exposure to high aspect ratio nanoparticles (HARN) via inhalation. Sophisticated, multi-cellular in vitro models are a particular advantageous solution to this issue, especially when considering the need to provide realistic and efficient alternatives to invasive animal experimentation for HARN hazard assessment. By incorporating a systematic test-bed of material characterisation techniques, a specific air-liquid cell exposure system with real-time monitoring of the cell-delivered HARN dose in addition to key biochemical endpoints, here we demonstrate a successful approach towards investigation of the hazard of HARN aerosols in vitro.MethodsCellulose nanocrystals (CNCs) derived from cotton and tunicates, with differing aspect ratios (~9 and ~80), were employed as model HARN samples. Specifically, well-dispersed and characterised CNC suspensions were aerosolised using an “Air Liquid Interface Cell Exposure System” (ALICE) at realistic, cell-delivered concentrations ranging from 0.14 to 1.57 μg/cm2. The biological impact (cytotoxicity, oxidative stress levels and pro-inflammatory effects) of each HARN sample was then assessed using a 3D multi-cellular in vitro model of the human epithelial airway barrier at the air liquid interface (ALI) 24 hours post-exposure. Additionally, the testing strategy was validated using both crystalline quartz (DQ12) as a positive particulate control in the ALICE system and long fibre amosite asbestos (LFA) to confirm the susceptibility of the in vitro model to a fibrous insult.ResultsA rapid (≤4 min), controlled nebulisation of CNC suspensions enabled a dose-controlled and spatially homogeneous CNC deposition onto cells cultured under ALI conditions. Real-time monitoring of the cell-delivered CNC dose with a quartz crystal microbalance was accomplished. Independent of CNC aspect ratio, no significant cytotoxicity (p > 0.05), induction of oxidative stress, or (pro)-inflammatory responses were observed up to the highest concentration of 1.57 μg/cm2. Both DQ12 and LFA elicited a significant (p < 0.05) pro-inflammatory response at sub-lethal concentrations in vitro.ConclusionIn summary, whilst the present study highlights the benign nature of CNCs, it is the advanced technological and mechanistic approach presented that allows for a state of the art testing strategy to realistically and efficiently determine the in vitro hazard concerning inhalation exposure of HARN.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-014-0040-x) contains supplementary material, which is available to authorized users.
Highlights
Commercial production of a diverse range of high aspect ratio nanoparticles (HARN; aspect ratio ≥3) is rapidly increasing, with an inevitability that they will be readily exposed to humans [1,2]
Dispersion by sonication in ultrapure, deionised water led to rod-shaped fibers with a common size distribution, as determined from transmission electron microscopy (TEM), with averages of 170 ± 72 nm in length and 19 ± 7 nm in width for c-Cellulose nanocrystal (CNC) (Figure 1a) and longer fibers of 2.3 ± 1.4 μm in length and 31 ± 7 nm in width for Tunicate cellulose nanocrystals (t-CNCs) (Figure 1b)
Visual examination of the suspension after two weeks (Additional file 1: Figure S1) further supported the stability of the CNC as shown by depolarised dynamic light scattering (DDLS), suggesting that suspensions of this nature can remain stable in this environment for up to several weeks, in the presence of NaCl
Summary
Commercial production of a diverse range of high aspect ratio nanoparticles (HARN; aspect ratio ≥3) is rapidly increasing, with an inevitability that they will be readily exposed to humans [1,2]. Understanding the potential for HARN to cause adverse health effects is limited at this moment in time due to a lack of test systems that can efficiently mimic human exposure to HARN aerosols and provide insight into the mechanistic HARN-cell interaction [3] In this context, as well as the research principles of the 3R (refine, reduce and replace) [7], there is an urgent requirement for realistic and reliable testing strategies that may be used to gain an intrinsic understanding of how HARN could affect the lung at the cellular level [8], as well as provide an alternative to invasive animal experimentation [9]. By incorporating a systematic test-bed of material characterisation techniques, a specific air-liquid cell exposure system with real-time monitoring of the cell-delivered HARN dose in addition to key biochemical endpoints, here we demonstrate a successful approach towards investigation of the hazard of HARN aerosols in vitro
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