THE NEED FOR NANOMATERIAL EVALUATION IN A PHYSIOLOGICALLY

THE NEED FOR NANOMATERIAL EVALUATION IN A PHYSIOLOGICALLY RELEVANT MODEL: CONNECTING ENVIRONMENTAL VARIABLES AND NM BEHAVIOR TO TOXICOLOGICAL RESPONSES Kristen K. Comfort Department of Chemical and Materials Engineering University of Dayton

DEFINING THE NANOBIO INTERFACE Nano-Bio interface = dynamic physicochemical interactions, kinetics, and thermodynamic exchanges between nanomaterials (NM) surfaces and biological components Influenced By: Determines:

MOTIVATION • Tremendous advances have been made in NM characterization, synthesis, and dosimetry • Parallel progress in biological models needs to be developed and implemented • Long term goals: • Generate in vitro models that mimic an in vivo system • Improve predictive modeling of NM-behavior and bioresponses • Design accurate, high-throughput in vitro systems

IN VITRO VS IN VIVO SYSTEMS in vitro in vivo Advantages: • Simplified model • Lower cost • Rapid assessment/ High-throughput capabilities • Can explore mechanistic response • Cell line specificity • Complete physiological response Inclusion of immune/inflammatory systems • Disadvantages: • Difficult to extrapolate to human system • Applicability is dependent on design Ethical concerns – Europe is phasing out • High cost • Time requirements • Dosimetry and distribution concerns • Difficult to puzzle out NM mechanisms

IN VITRO VS IN VIVO SYSTEMS • in vitro • in vivo Current Limitation: Poor correlation Need to improve in vitro models to bridge this gap

LET’S EXAMINE A TISSUE/ORGAN SYSTEM • What are its unique characteristics? 1) 3 -Dimensional

LET’S EXAMINE A TISSUE/ORGAN SYSTEM • What are its unique characteristics? 1) 3 -Dimensional 2) Comprised of multiple cell types • (hepatocytes, endothelial, Kupffer)

LET’S EXAMINE A TISSUE/ORGAN SYSTEM • What are its unique characteristics? 1) 3 -Dimensional 2) Comprised of multiple cell types • (hepatocytes, endothelial, Kupffer) 3) Physiological fluid • Interstitial fluid or secreted bile

LET’S EXAMINE A TISSUE/ORGAN SYSTEM • What are its unique characteristics? 1) 3 -Dimensional 2) Comprised of multiple cell types • (hepatocytes, endothelial, Kupffer) 3) Physiological fluid • Interstitial fluid or secreted bile 4) Dynamic environment • Connected to the CVS

PRIMARY GOALS… (1) To transform this: Into something that is more representative of: (2) Which will lead to augmented in vitro applicability: Increased Correlation & Predictive Modeling

EXPERIMENTAL RESULTS

STUDY APPROACH • Target system: Alveolar region • Model contains: • Human alveolar epithelial cells • Artificial alveolar fluid (AAF) • Dynamic movement • 60 nm tannic acid gold nanoparticles (Au. NPs) • Characterize • Evaluate nano-bio interface

DYNAMIC FLOW • Introduced to the cell culture system through use of a peristaltic pump • Tubing was inserted into lid of 24 well plate • Each well was singularly connected, producing unilateral flow across the surface • Target volumetric flow rate was selected: • Velocity in tubing = 0. 2 cm/s (capillary rate) • Velocity across cells = 0. 003 cm/s (diffusion-based rate)

ENVIRONMENTAL INFLUENCE ON CELL MORPHOLOGY A 549 cells cultured with: (A) Media, static (B) AAF, static (C) Media, dynamic (D) AAF, dynamic Conclusions: • Dynamic flow induced elongation • AAF causes curvature • BOTH are seen in vivo

AUNP CHARACTERIZATION Primary size (nm) 65. 1 ± 5. 3 Agglomerate size (nm) 74. 8 ± 4. 6 Zeta potential (m. V) -31. 8 ± 0. 9 Ionic dissolution (%) 0. 8 ± 0. 5

AUNP CHARACTERIZATION Conclusions: Exposure to AAF significantly altered Au. NP properties and behavior.

AUNP DEPOSITION • Deposition = percentage of administered NPs that are bound to the cell surface or internalized • The deposited dose has been strongly correlated to cytotoxicity Conclusions: In media: dynamic flow reduces deposition In AAF: deposition is unchanged due to sedimentation of large agglomerates

AUNP INTERNALIZATION • TEM images of (A) Media, static (B) AAF, static (C) Media, dynamic (D) AAF, dynamic Conclusions: • Increased Au. NP number with AAF • AAF/dynamic – no internalization

NANO-BIO INTERFACE Conclusions: • Cells maintained altered morphology • Increased Au. NP number with AAF

TAKE AWAY MESSAGE • It is possible to modify traditional in vitro systems to more closely mimic in vivo models • We introduced dynamic flow and biological fluids • NP characteristics and behavior are strongly dependent upon the surrounding environment • This has been linked to bioresponses • Therefore, modified in vitro systems allow for identification of novel responses previously unobtainable. • Bridging the in vitro – in vivo gap

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