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

Our plain-English summary of An in vitro testing strategy towards mimicking the inhalation of high aspect ratio nanoparticles, by C. Endes, O. Schmid, C. Kinnear, et al (Particle and Fibre Toxicology, 2014).

The study

• Authors: C. Endes, O. Schmid, C. Kinnear, S. Mueller, S. Camarero-Espinosa, D. Vanhecke, E.J. Foster, A. Petri-Fink, B. Rothen-Rutishauser, C. Weder, M.J. Clift
• Journal: Particle and Fibre Toxicology
• Published: 2014;11:40
• Cited by: 63 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: The 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.

Methods: Cellulose 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.

Results: A 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.

Conclusion: In 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.

In plain English

This laboratory study tested whether an air-liquid interface exposure system could be used to deliver high aspect ratio nanoparticles to lung cells in vitro. Cellulose nanocrystals (CNCs) from cotton (c-CNC, aspect ratio about 9) and from tunicates (t-CNC, aspect ratio about 80) were suspended, characterised and nebulised with the "Air Liquid Interface Cell Exposure System" (ALICE) onto a three-dimensional triple cell co-culture model of the human epithelial airway barrier, with deposited doses of 0.14, 0.81 and 1.57 micrograms per square centimetre measured by quartz crystal microbalance and an anthrone assay. Crystalline quartz (DQ12, deposited dose 0.23 micrograms per square centimetre) served as a particulate positive control and long fibre amosite asbestos (LFA, 100 microlitres of 0.05 mg/mL applied to the apical side) as a fibrous positive control. Neither CNC type caused significant cytotoxicity, oxidative stress or (pro-)inflammatory response (p greater than 0.05) at 24 hours; DQ12 and LFA caused significant (p less than 0.05) TNF-alpha and IL-8 release. The authors concluded the approach provides a testing strategy for inhalation hazard of such particles and highlights the benign nature of CNCs.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: In vitro laboratory study: aerosol exposure of a 3D human lung cell co-culture model, with material characterisation
• Who was studied: A three-dimensional triple cell co-culture model of the human epithelial airway barrier based on A549 alveolar epithelial cells, with monocyte-derived macrophages apically and dendritic cells baso-laterally; three independent experiments per material
• Exposure: Nebulised cellulose nanocrystals from cotton (c-CNC) and tunicates (t-CNC) at deposited doses 0.14 to 1.57 micrograms per square centimetre; nebulised crystalline quartz DQ12 as particulate control; long fibre amosite asbestos applied by pseudo-ALI as fibrous control
• Main results:
  • Deposited CNCs measured by TEM: c-CNCs 161 ± 61 nm long and 16 ± 5 nm wide; t-CNCs 2.1 ± 1.0 micrometres long and 20 ± 5 nm wide; aspect ratios 11 ± 5 and 108 ± 57
  • Average deposited doses achieved for both CNC materials were 0.14 ± 0.04, 0.81 ± 0.03 and 1.57 ± 0.03 micrograms per square centimetre; DQ12 deposited dose was 0.23 ± 0.08 micrograms per square centimetre
  • No significant cytotoxicity (lactate dehydrogenase release) was seen for either CNC type at any tested concentration, nor for nebulised DQ12 or for LFA
  • For both CNC types, no significant change (p greater than 0.05) in oxidative stress status or (pro-)inflammatory response; in contrast DQ12 and LFA gave statistically significant (p less than 0.05) TNF-alpha and IL-8 release at 24 hours
  • Long fibre amosite did not produce an oxidative stress response in the triple cell co-culture at the pseudo-ALI
• The authors' conclusion: The authors concluded that the combined approach forms a testing strategy for realistic and efficient in vitro hazard assessment of inhaled high aspect ratio nanoparticles, and that the low-toxicity potential of CNCs, independent of length, was demonstrated alongside inflammogenic particulate and fibrous positive controls
• Limitations the authors state:
  • The authors state that the lack of biochemical effects after acute CNC exposure does not necessarily mean no effects would occur in a chronic study, and that pro-fibrogenic effects need consideration over longer exposure
  • The authors note all exposure concentrations used can be defined as overload situations
  • Long fibre amosite could not be nebulised because its suspensions are highly unstable, so it was applied as a pseudo-ALI exposure instead
  • The authors state further studies applying realistic doses chronically to a relevant target tissue are needed to clarify effects on oxidative state

Topics

The National Library of Medicine indexes this study under subjects our site covers: Amosite.

Medical terms in this study, explained in our Lexicon: Aspect Ratio (Length-to-diameter ratio), Oxidative Stress, Crystalline, Amosite, Deposition, State-of-the-Art Defense.

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