1. 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

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

3. 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

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

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

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

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

8. 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

9. 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

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

11. EXPERIMENTAL RESULTS

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

13. 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)

14. ENVIRONMENTAL INFLUENCE ON CELL MORPHOLOGY A549 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 Conclusions: Dynamic flow induced elongation AAF causes curvature BOTH are seen in vivo

15. AUNP CHARACTERIZATION Primary size (nm)65.1 ± 5.3 Agglomerate size (nm)74.8 ± 4.6 Zeta potential (mV)-31.8 ± 0.9 Ionic dissolution (%)0.8 ± 0.5

16. AUNP CHARACTERIZATION Conclusions: Exposure to AAF significantly altered AuNP properties and behavior. Conclusions: Exposure to AAF significantly altered AuNP properties and behavior.

17. 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 Conclusions: In media: dynamic flow reduces deposition In AAF: deposition is unchanged due to sedimentation of large agglomerates

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

19. NANO-BIO INTERFACE Conclusions: Cells maintained altered morphology Increased AuNP number with AAF Conclusions: Cells maintained altered morphology Increased AuNP number with AAF

20. 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

21. THANK YOU