Safety/Toxicity Assessment of Ceria (A Model engineered NP) to the Brain: Scope for Grulke

Objectives: The long-term objectives are to determine the physico-chemical properties of engineered nanomaterials (ENM) that influence their distribution into the cells comprising the blood-brain barrier and the brain and to characterize their beneficial and/or hazardous effects on the brain. The work will be conducted with cerium oxide (Ce02) as a model insoluble, stable metal oxide tracer. Initial studies will utilize systemic ENM administration in the rat for hazard identification to define the NOAEL and LOAEL and a dose-response assessment. The Specific Aims will test the following 4 null hypotheses. 1) That the size (- 10, 30 and 100 nm diameter spherical Ce02 ENMs) and shape (- 30 nm spherical, disk and rod) Ce02 ENMs do not influence their distribution into, or effects on, the rat brain. 2) That the surface properties (hydrophilicity/hydrophobicity. surface charge, and steric inhibition) do not influence ENM distribution into, or effects on, the rat brain. The functionalized Ce02 ENMs to be given to rats will be based on an extensive in vitro comparison of the physico-chemical properties of silanes and polymers bound to the surface of Ce02 ENMs. 3) That the surface properties of ENMs do not affect their protein opsonization and that the opsonizing protein nature does not influence ENM distribution into, or effects on, the rat brain. 4) That, over time, there are no changes in the physico-chemical properties of ENMs after they enter the brain and that there is unaltered biopersistence. Additionally, this work will 5) determine the rate and mechanism(s) of brain uptake of ENMs that have the greatest potential for toxicity to the BBB and the brain and the target cells of the BSS and brain for ENM-induced toxicity and 6) the mechanism(s) mediating the toxicity. Experimental Approaches: Particle size determination will be conducted by light scattering for spherical ENMs and TEM. ENM morphology will be characterized by TEM. ENM surface chemistry will be characterized by zeta potential (charge), TEM/EELS and molecular modeling of ENM surfaces and x-ray photoelectronl Auger spectroscopy (surface functionalization), critical surface tension (hydrophilicity/hydrophobicity), 2-propanol oxidation kinetics (chemical reactivity) and the use of Accelrys® software to predict interaction between ENM surface groups and proteins. Aggregation will be assessed by light-scattering analysis. Surface chemistry will be modified by functionalizations using various coupling agents, such as silane, and coupling agents attached to polymers. Rats will be given Lv. ceria ENMs to enable characterization of their beneficial and adverse effects once they reach systemic circulation, as would occur after their oral, inhalation or dermal absorption. Multiple endpoints will be studied related to blood-brain barrier (BBB) and brain function as well as ENM localization and biotransformation, such as opsinization. Endpoints will include BBB integrity using various substances that are unable to cross the intact BBB; ENM localization, aggregation, redistribution and clearance from the brain, using high resolution and scanning TEM and energy dispersive X-ray analysis; and pro- and antioxidative stress responses, assessed by multiple endpoints. Ceria ENMs that distribute into the BBB and brain cells in significant amounts will be subsequently studied using the in situ brain perfusion method to determine the rate of their brain entry. The capillary depletion method will be employed for ENMs that significantly accumulate in the BBB cells. Based on the above results, studies will be conducted using BBB and/or brain cells in culture to identify the target cells of hazardous effects and determine the mechanism(s) mediating the effects. The choice of cell type will be influenced by our prior results of cell types showing the greatest ENM accumulation and/or considered most likely to be mediating observed effects. The studies will be designed so that the results can be interpreted in the framework of risk assessment; to define the NOAEL and LOAEL, the dose-response relationship, and the characteristics of the toxic effect(s). Results: The results will indicate the influence of the size, shape and various surface chemistry properties of ENMs on their entrance into BBB cells and the brain, compared to selected peripheral organs, the effects they produce in the brain, their biopersistence and biotransformation in the brain. The results should also define the rate of brain entry of those ENMs that most rapidly enter the brain and the cells most susceptible to adverse effects of ENMs. These studies address the following suggested research foci of the solicitation: systemic distribution of nanoscale materials, identification of critical physic-chemical parameters of nanomaterials that correlate with biological responses, biotransformation and bioaccumulation of nanomaterials, toxicological responses following nanoscale material exposure, and the determination of the influence of physico-chemical properties of nanoscale materials on biological compatibility or toxicity. The studies will be designed to maximize the application of the results to riskassessment. Supplemental Keywords: animal, biodistribution, biotransformation, brain exposure, health effects, heavy metals, oxidative stress