PEG-Based Living Hydrogels Engineered for Tunable Production of Bioactive Lipopeptides

Encapsulation of microbial cells within nanoporous hydrogels creates dynamic and responsive living materials well-suited for biotherapeutic applications. Bacillus subtilis is a promising microbe in this application as it is generally regarded as safe, can be sporulated for long-term stability and resistance to nonideal environments, and can produce antimicrobial and anticancer molecules such as the cyclic lipopeptide surfactin. Here, we examine the growth of B. subtilis cells and corresponding production of surfactin after encapsulation within poly-(ethylene glycol) (PEG) hydrogels at varied levels of nanoconfinement. Encapsulation was achieved through Michael-type addition reactions between PEG diacrylate and PEG tetrathiol macromers, where macromer molecular weight was systematically varied to generate hydrogels across a range of average mesh sizes (9-19 nm). Hydrogels had varied Young's modulus (7.3 ± 1.7 kPa to 16.4 ± 0.7 kPa) and provided a 7-fold range in small molecule diffusivity. In-situ cellular growth monitoring and surfactin quantification revealed that all hydrogels stimulated the production of surfactin with verified antibacterial activity and in a manner tunable with mesh size. Smallest mesh sizes drove highest surfactin production, a ∼5-fold increase relative to equivalent cultures of unconfined cells. Cell loading was then varied in 9 nm mesh size hydrogels to reveal that low cell loading (0.1-1 × 10⁴ cells per μL hydrogel) promoted sustained growth and surfactin production proportional to the number of cells loaded. Conversely, hydrogels loaded with excessive cells (2.5-7.5 × 10⁴ cells per μL hydrogel) resulted in unsustained growth and diminished surfactin production. Finally, to develop a more robust material, B. subtilis endospores were encapsulated into hydrogels at optimized conditions. Spore-laden hydrogels retained the capability to produce surfactin after exposure to dehydration and temperature stress. These results indicate that hydrogel encapsulation stimulates B. subtilis surfactin production according to the level of nanoconfinement to achieve a tunable engineered living material for production of bioactive molecules.