INTRODUCTION

Engineered living materials have driven a major shift in the paradigm of materials research in the recent years. One of the recent advances in the development of drug encapsulation and delivery systems has been the emergence of living materials, that contain genetically modified living organisms performing enhanced functions. Such living therapeutic materials include genetically programmed living organisms for performing enhanced functions like productive, adaptable, on-demand drug delivery with replenishable repositories.¹ However, much is yet to be understood in the effects of encapsulation on bacterial performance. To address this issue, we used a bacterially orthogonal hydrogel system based on Pluronic F127, in which network stabilization could be established with both non-covalent and covalent interactions that could be proportionally tuned.² The interplay between the dynamic viscoelastic properties of the pluronics and the functional performance of the encapsulated bacteria was studied. The effect of the dynamic mechanical properties of the surrounding biocompatible and scale-up friendly hydrogel matrix on the growth behaviour, microcolony growth kinetics, viability and functionality of the encapsulated microbial cells was studied providing essential cues for living materials applications. We demonstrate the development of this tunable hydrogel system to encapsulate genetically engineered E. coli bacterial strain, in core-shell 3D bioprinted scaffolds, leading to a light-regulated, localized, tunable and prolonged drug/protein release.

METHODS

Pluronic F127 polymer was chosen as a bioink. Pluronic hydrogel forms physically assembled gel networks at room temperature and it diacrylated derivative, pluronic diacrylate, can be chemically crosslinked upon UV light exposure using a free-radical photoinitiator. Hydrogel matrices with a range of viscoelastic properties were obtained by mixing pluronic and pluronic diacrylate in varying proportions. The dynamic mechanical properties were studied using rheology. A light-regulated dVio drug-producing E. coli strain was engineered by incorporating the genes related to the metabolic synthesis of the drug into a light responsive optogenetic plasmid.2 Bacteria was encapsulated in 3D-bioprinted core-shell constructs and the effect of change in the crosslinking density and the resulting mechanical properties on the bacterial growth rate, drug/protein production and viability was studied using epifluorescence and confocal laser scanning microscopy.

RESULTS

Bacteria in physically crosslinked Pluronic hydrogels grew unidimensionally, as a chain with cells predominantly arranged along their longitudinal axis (Figure 1a). In contrast, in covalently crosslinked Pluronic Diacrylate hydrogel, the growing chain buckled already in the 2nd or 3rd division cycle, and the dividing bacterial population formed rounded colonies (Figure 1b). These results evidence that the nature of the crosslinks of the hydrogel network influences the growth of the encapsulated microorganisms. The compressive forces imposed by the network as the bacterial colony grows act as physical modulators of bacteria proliferation in the confined state. The 3D printed core-shell fibres with violet colored dVio drug were fabricated (Figure 2). The bacteria were viable in the 3D scaffolds stored at various temperature conditions for over 14 months.