An in vitro testing strategy towards mimicking the inhalation of high aspect ratio nanoparticles

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.

Human Asthmatic Bronchial Cells Are More Susceptible to Subchronic Repeated Exposures of Aerosolized Carbon Nanotubes At Occupationally Relevant Doses Than Healthy Cells

“…The ALICE system is a well-established aerosolization system for spherical NPs 21,24 and was recently demonstrated to be efficient for the aerosolization of fiber-shaped nanomaterials, such as cellulose nanocrystals and MWCNTs for single and short-term repeated exposures. 25,26 For the current study a stable, well-characterized, and well-dispersed MWCNT 27 suspension was aerosolized using the ALICE system. For the current work Pluronic F127 has been used as a dispersant since it prevents MWCNT aggregation during the experimental work procedure and results in a well-dispersed suspension, allowing an efficient aerosolization.…”

Repeated exposure to carbon nanotube-based aerosols does not affect the functional properties of a 3D human epithelial airway model

“…The testing strategy for single inhalation exposures was previously validated by Endes et al (2014), in terms of biological sensitivity by applying DQ12 as a positive particulate aerosol control for (pro-)inflammatory responses. DQ12 demonstrated the applicability of ALICE nebulisations by causing significant (pro-)inflammatory reactions when exposed to the triple cell co-culture at the air-liquid interface.…”

“…Nebulisation of MWCNTs was performed using the ALICE as described by Lenz et al (2009) and Endes et al (2013Endes et al ( , 2014. Briefly, the ALICE consists of a nebuliser (droplet generator), an exposure chamber, an incubation chamber (which is connected to a flow system providing humidity and temperature conditions suitable for cell cultivation) as well as a quartz crystal microbalance (QCM; detection limit 0.09 mg/cm 2 , 5 MHz, Stanford Research Systems, GMP SA, Renens, Switzerland) for real-time determination of the deposited material.…”

Elucidating the Potential Biological Impact of Cellulose Nanocrystals

“…Nonetheless, the US Occupational Safety and Health Administration had previously set a specific permissible exposure limit (PEL) of 200-750 µg/m 3 over an eight hour timed weighted average (TWA) for cotton dust. Irrespective of the issues surrounding both exposure limits, they do provide a significant basis for research to dictate that investigations undertake exposures at 'realistic' concentrations/doses so that extrapolation towards human exposure can be made [47]. Furthermore, such exposure limit values provide a valuable 'stop-gap' until regulatory bodies are able to provide direction towards the use and exposure of nanomaterials [48].…”

“…Whilst these models show a different biochemical/biomolecular response to monoculture systems (whether it be a similar trend, but different concentration-based effects, or a completely different biological effect) [100], there is still much debate and unknown as to how they correlate to the in vivo scenario, albeit efforts are underway to address this knowledge gap [101]. Such systems have recently been shown to be advantageous in determining the hazard posed by nanocellulose [47], as well as its interaction with cellular systems [102]. Table 2.…”