Characterization of Nanomaterial Bio-interaction
Project Plan

Goals:
1. Establish in vitro and in vivo nanomaterial NanoEHS testing protocols that are validated to produce the same results in multiple laboratories internationally.
2. Identify nanoEHS in vitro testing protocols that correlate with results in animal test and may be predictive of in vivo effects.
1.0 Project Description
In this project, the physicochemical properties and their nanoEHS interactions of several nanomaterials will be characterized by multiple laboratories in a set of “round robin” experiments supported by an international team of participating experts and organizations. The nanoEHS experiments will be performed using both in vitro and in vivo experimental methods and potential correlations will be assessed between these.

1.1 Project Timeline
The initial experiments are targeted to be completed within one year. At the end of this time, a determination will be made on whether this should be a longer term activity to cover a wider range of materials.

1.2 Funding
At this time, there is no specific funding for the round-robin experiments. Each team will characterize materials, biological interactions and toxicology as part of their normal research activities within existing research projects.

1.3 Deliverables
The primary deliverables for this initial project are validated protocols for
1) Characterizing nanomaterial physicochemical properties
2) Separating, functionalizing and dispersing nanoparticles in biocompatible fluids
3) Generating the biological media and tissue constructs
4) Presentation to cells in for in vitro test
5) Validation of cell uptake rates of nanoparticles
6) Characterization of TiO2, CeO2, ZnO, Ag toxicological interactions with
A. MTT LDH
B. In cell ROS
C. Cytokine Induction
D. Genotoxicity (COMET Test)
7) Animal health and care guideline
8) Nanoparticle Solution Intertracheal Instillation
9) Characterization timeline & testing

1.4: Project Reviews
1.4.1 Face to face project reviews will be held semi-annually
1.4.2 Teleconferences with the Principle Investigators will be held at least once per month to review progress
1.4.3 Teleconferences will be held with the lead and validation teams as required

2.0 Nanomaterials
The initial experiments will be performed on a set of well characterized nanomaterials that have a range of biological interactions.

Nanomaterials to be characterized include TiO2, ZnO, CeO2, Ag, Au, multiwalled carbon nanotubes and polystyrene nanoparticles.

3.0 Round-Robin Experiments
3.1 Physicochemical Characterization: A detailed characterization of the physicochemical properties of test materials will be conducted by the team. Analysis will include size, shape, surface structure, composition, surface composition, zeta potential, redox potential, surface area, radical formation potential, and others as determined appropriate.

3.2 Surface Preparation: The nanoaterials will be surface functionalized with a passivation that will enable stability against agglomeration and reproducible biological interactions.
3.3 In Vitro Test
The initial protocol for evaluating the biointeractions and toxicity will evaluate the interactions of nanoparticles with cells and tissue constructs based on A549, BEAS 2B, and RAW 264.7 cells. The tissue constructs will be generated with cells in a controlled manner to be described in the protocol. The “health” of the cells in the tissue construct will be assessed through monitoring their growth rate and uptake rate of detectable nanoparticles (Au and fluorescent polystyrene) by the cells. Once cell and tissue construct are producing cells with repeatable behavior, nanoparticle interactions with the three cell types will be characterized for MTT LDH, in cell radical oxygen stress (ROS), cytokine induction and genotoxicity.

3.3.1 Uptake of Nanoparticles
The health, age of cells, composition of the fluids and pH can change the uptake and distribution of nanoparticles in different cell types. Prior to testing the bioreaction of unknown nanoparticles, it is important to validate that cells and tissue constructs are absorbing nanoparticles in a reproducible manner. Multiple sizes and concentrations of Au and fluorescent polystyrene will each be introduced in a biocompatible solution to the cells and the number of particles absorbed will be measured with an optical technique for the fluorescent nanoparticles or electron microscope for the Au particles. Once the uptake of the polystyrene and Au nanoparticles has been validated to meet the protocol guidelines and be reproducible, characterization should proceed to the biointeractions with known nanoparticles and then unknown nanoparticles.

3.3.2 Biointeraction & Toxicology : The nanomaterial in the biocompatible fluids will be introduced to A549, BEAS-2B, and RAW264.7 cells and tissue constructs. Characterization will include monitoring cell nanoparticle uptake where possible, MTT LDH, in cell ROS, Cytokine Induction, and Genotoxicity. Common protocols will be developed and refined to ensure consistent experimental procedures and characterization of “end points”. The biological and toxicological responses will be characterized in each lab and the results will be posted on the IANH web site for comparison to other results. The results will be analyzed for consistency at the lab and then against the reference results. If results are outside of the reference results, the validation lab will contact the lead lab and conduct a detailed review of the protocol against the procedure used at the lead lab. Once discrepancies have been resolved, the validation lab will repeat the experiments and the results checked again. The protocol will be updated to clarify any ambiguities or special requirements for reproducible testing.

4.0 In Vivo Characterization
This project will characterize the biokinetics of the nanoparticles and the toxicology in the organs of the animals. Different teams will characterize the toxicology of nanoparticles in 1) “common” rodents and 2) environmentally sensitive aquatic species. The distribution of the nanomaterial will be characterized in the organs of the animals and biological responses characterized. These results will be compared to the in vitro results as a function of exposure dose, exposure surface area and dose rate.

Assessment of Results
The results of these experiments will be compared at multiple points as the protocols are defined and refined. All results will be published in peer reviewed publications highlighting the protocols and consistency of results. Also, critical factors that result in differences in responses will be highlighted. Any correlation between in vitro and in vivo results will be highlighted with recommendations for more detailed study.

Once these protocols are found to be reproducible, they will be presented to standards organizations for consideration.