PROJECT SUMMARY Traditional drug delivery platforms are limited by the amount of drug that can be loaded into the delivery system. While drug loading capacity can reach 50% by weight for some polymeric systems, the time period over which the delivery of the therapeutic can be sustained is often limited. In addition, the therapeutic efficacy of a local drug delivery system is related to the local bioavailability of the active agent. Devices that bio-catalytically produce the therapeutic in situ could thus provide more effective local delivery of the active drug and improve therapeutic efficacy. The long-term vision for this program is to create the next-generation 3D printed in situ drug production and delivery devices, including drug-eluting stents, microneedles, and patches, based on engineered living materials (ELMs) for localized and sustained therapeutic delivery. ELMs are composites of microorganisms incorporated within a polymeric matrix, wherein the cells maintain their viability and can be metabolically engineered to produce a therapeutic compound. Despite the immense potential of ELMs for drug delivery applications, the primary challenges to the deployment of ELMs as drug-eluting stents or patches to treat intestinal diseases are that ELMs must (i) be processable into precise form factors (e.g., patient-specific stents) with the requisite mechanical properties, (ii) biodegrade into non-cytotoxic components at predetermined rates, and (iii) bio-catalytically produce and elute the therapeutic agent in situ for the lifetime of the device. The objective of this proposal is to develop 3D printable resins that afford biodegradable hydrogel constructs with a tunable stiffness, and to demonstrate the fabrication of a prototype ELM device for sustained delivery of a model compound. In Aim 1, we will create aqueous resins with non-pathogenic E. coli Nissle 1917 (EcN) that can be 3D printed into hydrogel constructs using a commercially available 3D printer. We will formulate aqueous resins comprised of soluble globular protein derivatives that can be co-polymerized with water-soluble acrylate monomers upon photo-initiated polymerization. The mechanical properties of the ELM hydrogels (stiffness, strength, and toughness) and rates of enzymatic degradation will be quantified for each resin formulation. In Aim 2, we will metabolically engineer non-pathogenic EcN to produce berberine as a model therapeutic compound. We will further evaluate the cytotoxicity and epithelial integrity of Caco-2 cells in the presence of 3D printed ELMs. As validation of these ELMs we will use an in vitro model to confirm the production and elution of berberine from the 3D printed ELM by evaluating the Caco-2 response to proinflammatory stimulation. We envision these 3D printed ELMs to be used as devices for local drug delivery in the treatment of malignancies or inflammatory diseases affecting the intestines. While our ELM platform is compatible with a broad array of micro...