Abstract
Probiotics offer therapeutic benefits by modulating the local microbiome, the host immune response, and the proliferation of pathogens. Probiotics have the potential to treat complex diseases, but their persistence or colonization is required at the target site for effective treatment. Although probiotic persistence can be achieved by repeated delivery, no biomaterial that releases clinically relevant doses of metabolically active probiotics in a sustained manner has been previously described. Here, we encapsulate stiff probiotic microorganisms within relatively less stiff hydrogels and show a generic mechanism where these microorganisms proliferate and induce hydrogel fracture, resulting in microbial release. Importantly, this fracture-based mechanism leads to microorganism release with zero-order release kinetics. Using this mechanism, small (∼1 μL) engineered living materials (ELMs) release >10 (8) colony-forming-units (CFUs) of E. coli in 2 h. This release is sustained for at least 10 days. Cell release can be varied by more than three orders of magnitude by varying initial cell loading and modulating the mechanical properties of encapsulating matrix. As the governing mechanism of microbial release is entirely mechanical, we demonstrate controlled release of model Gram-negative, Gram-positive, and fungal probiotics from multiple hydrogel matrices. SIGNIFICANCE: Probiotics offer therapeutic benefits and have the potential to treat complex diseases, but their persistence at the target site is often required for effective treatment. Although probiotic persistence can be achieved by repeated delivery, no biomaterial that releases metabolically active probiotics in a sustained manner has been developed yet. This work demonstrates a generic mechanism where stiff probiotics encapsulated within relatively less stiff hydrogels proliferate and induce hydrogel fracture. This allows a zero-order release of probiotics which can be easily controlled by adjusting the properties of the encapsulating matrices. This generic mechanism is applicable for a wide range of probiotics with different synthetic matrices and has the potential to be used in the treatment of a broad range of diseases.