Engineering PEG-based living hydrogels for controlled transport and bacterial microenvironments

Abstract

Engineered living hydrogels (ELHs) are an emerging class of biomaterials that integrate living microorganisms within hydrogel matrices to create dynamic materials capable of sensing, responding to, and modifying their surrounding environment. Bacteria are attractive as living components because of their genetic programmability, metabolic diversity, rapid growth, and ability to produce a wide range of functional biomolecules. Hydrogels are three-dimensional, water-swollen, crosslinked polymer networks that provide a hydrated environment for cell encapsulation while allowing molecular transport throughout the material, making them well suited as the structural component of engineered living hydrogels. ELHs have been investigated for applications including therapeutic delivery, biosensing, bioelectronics, and environmental remediation. However, as this novel field has emerged, quantitative relationships between hydrogel architecture, molecular transport, and bacterial function remain poorly understood, limiting the rational design of these systems. This dissertation addresses these challenges through the development of experimental platforms and poly (ethylene glycol) (PEG)-based ELHs. Collectively, these studies establish quantitative design principles for engineering bacterial behavior and the local microenvironment within engineered living hydrogels. The first portion of this dissertation focuses on establishing quantitative relationships between hydrogel network architecture and molecular transport in PEG-based hydrogels. Because molecular transport depends on both hydrogel properties and the physicochemical characteristics of diffusing molecules, quantitative characterization is necessary to predict solute transport and engineer hydrogel function. Experimental methods were developed to quantify diffusion coefficients, partition coefficients, and hydrogel mesh size, providing complementary descriptions of transport behavior and network architecture. Together, these measurements establish a quantitative framework for characterizing molecular transport and hydrogel network architecture in PEG-based hydrogels that are used throughout the remainder of this dissertation. The second portion of this dissertation applies the quantitative transport framework established in the first portion to develop increasingly sophisticated PEG-based ELHs. A photodegradable PEG hydrogel platform integrated within a microfluidic device was first developed to investigate how controlled hydrogel degradation influences bacterial migration, chemotaxis, and cellular delivery. Controlled degradation regulated bacterial speed and directionality, enabling selective delivery of motile bacteria to a defined location while excluding nonmotile particles. PEG molecular weight was then systematically varied to engineer hydrogels with distinct mesh sizes, transport properties, and mechanical characteristics. Hydrogel network design and the resulting nanoscale confinement regulated bacterial growth and biomolecule production, while encapsulated endospores created a resilient material that maintained bacterial function following dehydration and temperature stress. These hydrogel design principles were then integrated with enzyme immobilization to engineer the local biochemical microenvironment surrounding encapsulated bacteria. A reaction-diffusion model predicted hydrogen peroxide profiles guided the selection of catalase loadings, which maintained bacterial viability, recovery, and function following oxidative stress. The final portion of this dissertation discusses several remaining challenges that limit broader implementation of engineered living hydrogels. These include integrating multiple biochemical functionalities, developing increasingly sophisticated hydrogel architectures, extending predictive transport models to dynamic environments, and validating these materials under application-relevant conditions. Addressing these will support the continued development of engineered living hydrogels for biomedical, environmental, and industrial applications.