title: An in vitro testing strategy towards mimicking the inhalation of high aspect ratio nanoparticles.
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.
  
  
  CONCLUSIONS
  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.
authors.forename: Carola
authors.initials: C
authors.lastname: Endes
authors.forename: Otmar
authors.initials: O
authors.lastname: Schmid
authors.forename: Calum
authors.initials: C
authors.lastname: Kinnear
authors.forename: Silvana
authors.initials: S
authors.lastname: Mueller
authors.forename: Sandra
authors.initials: S
authors.lastname: Camarero-Espinosa
authors.forename: Dimitri
authors.initials: D
authors.lastname: Vanhecke
authors.forename: E Johan
authors.initials: EJ
authors.lastname: Foster
authors.forename: Alke
authors.initials: A
authors.lastname: Petri-Fink
authors.forename: Barbara
authors.initials: B
authors.lastname: Rothen-Rutishauser
authors.forename: Christoph
authors.initials: C
authors.lastname: Weder
authors.forename: Martin J D
authors.initials: MJ
authors.lastname: Clift
mesh_terms: D000336:Aerosols; D017639:Asbestos, Amosite / Q000633:toxicity; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002482:Cellulose / Q000633:toxicity*; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D019570:Inhalation Exposure / Q000009:adverse effects*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D057139:Nanofibers; D053758:Nanoparticles / Q000633:toxicity*; D009330:Nebulizers and Vaporizers; D018384:Oxidative Stress / Q000187:drug effects; D011791:Quartz / Q000633:toxicity; D058525:Quartz Crystal Microbalance Techniques; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D018570:Risk Assessment; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D000336:Aerosols; D018836:Inflammation Mediators; D017639:Asbestos, Amosite; D011791:Quartz; D002482:Cellulose