Tissue Engineered Platforms for Nanoparticle Screening

Nanotechnology innovations are expected to be a major driver of the world economy in the next decade with market size estimates ranging from conservative ($5B) to astronomical ($3T). Nanoparticles display unique physical properties due to their size; they may also possess unique biological properties.

Products utilizing nanoparticles are already on the market despite limited toxicology information and despite public concern being high. Media coverage of a 2008 Nature Nanotechnology research article included headlines such as “Are nanotubes the next asbestos?” and “Cancer risk seen in nanotechnology.” Unfortunately high-throughput measurement tools to screen nanoparticle toxicity do not exist. 

Cell culture assays do not mimic the essential components of a biological system; whole animal studies are expensive, slow, and increasingly unpopular. Adding to the challenge are the hundreds of existing and emerging nanoparticles which exhibit different sizes, shapes, aspect ratios, and surface chemistries; the physico-chemical properties relevant to nanotoxicity have not been identified. Furthermore nanoparticle properties are expected to change in complex biological environments.

We are designing tissue-engineered platforms to characterize the impact of nanoparticles on cells in a developing neurological environment and the reliability of nanoparticles in biological environments. Engineered tissues are functional equivalents which can mimic key biological responses; another promising application is individual patient therapies (theragnostics).

Neural Cell Differentiation in Hydrogels. Current investigations utilize a model neural cell (PC12) derived from rat pheochromocytoma. These cells respond to nerve growth factor (a neurotrophin) by terminal differentiation, that is, they cease proliferating and extend neurites. Collagen I forms compliant gels known to support PC12 differentiation. Layered cell cultures have been created to permit rapid quantification of neurite extension. Cells are seeded onto a gelled collagen matrix containing nanoparticles, allowed to attach, then surrounded by additional matrix. Although cells are suspended with the gel and can migrate or extend neurites in all directions, they exist within a thin layer, facilitating analysis.

Encapsulation of Neural Progenitor Cells. Tumor-derived cells are not true precursors, nor do they form true neurons upon differntiation. Rat-derived adult hippocampal progenitor cells (AHPC) have the capacity to grow (divide) and differentiate into neuronal and glial populations -- an ideal system once the scaffold's chemical and mechanical cues are optimized. Hydrogels for tissue-engineered platforms must be biocompatible, but do not have the stringent requirements of implantable materials. We are investigating PEG hydrogels formed from dimethacrylate macromers, photoinitiator, and uv light.

Dispersal of Nanoparticles. Quantum dots (QDs) have been dispersed in Collagen I hydrogels and are easily visualized via their fluorescence at 565 nm. QDs are typically surrounded by a biocompatible surface coating, since core materials are known to be toxic, making them easily dispersed in aqueous solutions. In contrast, dispersing agents are essential for suspending carbon nanotubes (CNTs) in cell culture medium. CNTs dispersed by ultrasonic treatment alone begin to re-aggregate within a few hours. Although we anticipate that dispersion within a hydrogel will slow re-aggregation, cell-tolerated surfactants will be critical for long-term experiments.

Differentiating PC12 cells were exposed to surfactants and cell proliferation was quantified via metabolic activity i.e. the ability of the cell population to reduce resazurin to resorufin. Several surfactants proved toxic at useful concentrations (~ 1 mg/mL) however Poloxamer 188 is cytocompatible over a range of concentrations. Comparison with non-neuronal (smooth muscle) cells suggests this response is general.