Title:

Formation of Biofilm-Like Layer of Probiotic Bacteria in Microgel Using Flagellar Display for Oral Delivery of Cancer Targeting Peptide

Recently engineered bacteria have reemerged as promising avenues for drug delivery and immunotherapy due to the development and advancements of novel peptide-based therapeutics. However, hurdles and critical challenges must be addressed before these modified bacteria can reach clinical setting applications. While non-invasive oral administration is preferred, it presents significant obstacles, including bacterial survival through the harsh gastrointestinal tract environment, prolonged viability during storage, and effective diffusion of therapeutic molecules to targeted cells. Encapsulating bacteria within hydrogel matrices has emerged as a strategy to shield probiotic cells from the acidic and proteolytic conditions of the gastrointestinal tract while providing a suitable microenvironment for cell viability and growth. However, conventional hydrogel encapsulation often leads to a reduced bacterial population due to biomass accumulation at the hydrogel periphery, inhibiting nutrient diffusion to the gel interior and causing extensive cell death. To overcome these limitations, we propose an alternative probiotic encapsulation strategy that achieves spatial control of bacterial colony and growth using a layered microgel formulation. In this innovative system, dextran methacrylate microgel cores are produced independently using a microfluidic device, followed by chemical attachment of engineered probiotic bacteria via a thiol Michael-type addition reaction. Engineered E. coli cells displaying thiol-containing amino acids on modified flagellin proteins will be chemically conjugated onto the surface of dextran methacrylate cores via reaction with available methacrylate residues, followed by an alginate shell encapsulating the bacteria as a 3D monolayer. This design ensures the formation of a single bacterial layer at the interface between the microgel core and alginate shell, facilitating controlled diffusion of molecules and drastically enhancing cell viability and colony clustering by eliminating randomly dispersed bacterial colonies.

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