DESCRIPTION: Engineered Intestinal Microenvironments as Preclinical Drug Screening Platforms Getting a drug to market is an intensive process costing $800 million and taking 12 years. Therefore, preclinical screening is heavily relied upon to identify drug candidates with a high probability of market translation. Orally administered drugs, which treat myriad conditions ranging from heart disease and diabetes to chronic pain and infection, must first be absorbed by the body to be physiologically effective, regardless of their anatomical location of action. Currently, the most widely used in vitro absorption model is the Caco-2 monolayer assay. A key limitation of this assay is a negligible level of paracellular transport through tight junctions between Caco-2 cells compared to healthy small intestinal tissue. This inaccuracy results in erroneous abandonment of promising drug molecules due to the false prediction of poor pharmacokinetic parameters. We propose development of an engineered extracellular matrix (eECM) to replace the collagen type I matrix typically used in the Caco-2 assay. We hypothesize that engineering of the matrix biochemistry (Aim 1) and biomechanics (Aim 2) will reproducibly control focal adhesion formation and cytoskeletal organization, leading to the formation of tight junctions that are more physiologically relevant and capable of modeling paracellular transport. While others have tried to address the limitations of the Caco-2 assay, they have typically relied on use of chemical agents, cellular co-culture systems, or primary cells. While scientifically interesting, unfortunately these strategies are technically cumbersome and therefore not readily translatable to high-throughput industrial laboratory settings. There has yet to be a focus on utilizing biomaterials engineering strategies to guide Caco-2 cellular behavior along a more physiologically relevant pathway. Using recombinant techniques, we synthesize modular eECM materials containing elastin-like structural domains and cell-binding sites derived from native ECM proteins. This strategy enables decoupled control and investigation of matrix biochemistry and biomechanics. In Aim 1, cell-binding site identity and concentration are systematically altered to affect Caco-2 monolayer maturation and permeability, as quantified via integrin engagement studies, cell proliferation rate, number and size of focal adhesions, cellular density, expression and organization of tight junction proteins and epithelial markers, and paracellular transport measurements of model drugs. In Aim 2, matrix biomechanics is altered independently of matrix biochemistry to regulate cell-matrix traction forces (as measured by traction force microscopy) and hence focal adhesion and tight junction formation and Caco-2 monolayer permeability (quantitatively measured as in Aim 1). In both aims, cell density, expression of epithelial markers, expression and organization of tight junction proteins, and paracellular transport rates will be compared to values for human small intestinal tissue. This work will result in the development of an in vitro preclinical absorption model with improved physiological accuracy within a protocol format that can be easily adopted by industrial laboratories through the simple replacement of collagen with a novel eECM material.