Constructing artificial systems that can effectively replace or supplement natural biological machinery within cells is one of the fundamental challenges underpinning bioengineering. At the sub-cellular scale, artificial organelles (AOs) have significant potential as long-acting biomedical implants, by mimicking the action of native organelles in conducting intracellularly compartmentalized enzymatic reactions. We have become interested in enhancing the potency of these AO approaches by judiciously combining AOs with advances in genetic bioengineering to produce highly tailorable biohybrid cellular systems that can be manipulated with non-natural stimuli. We have recently developed a microliter scale (10 µL) polymersome (PSome) synthesis based on polymerization-induced self-assembly (PISA) that produces AOs at a much more cost-efficient scale compared to traditional self-assembly methods (typically milliliter scale) allowing for the encapsulation of otherwise cost-prohibitive enzymes. These enzyme-loaded PSomes present ideal features to be employed as AOs such as their enhanced enzymatic resistance to thermal, proteolytic and importantly, long term intracellular stresses. To demonstrate an example of their biomodulation potential, we have encapsulated <i>Gaussia</i> luciferase (GLuc), as a model luminescent enzyme, within PSomes and coupled their luminescence output to stimulate optogenetically engineered cardiomyocytes. This process generates a non-natural pathway for the stimulation of cardiac beating through treatment with coelenterazine (CTZ) as the substrate for GLuc. This process is highly robust with the long-term intracellular stability of these luminescent AOs allowing for this cardiostimulation to be reinitiated with fresh CTZ even after 7 days in culture. This synergistic combination of organelle mimicking synthetic materials with genetic engineering is therefore envisioned as a highly universal strategy for the generation of new biohybrid cellular systems displaying unique triggerable properties.

Microliter scale synthesis of luciferase-encapsulated polymersomes as artificial organelles for optogenetic modulation of cardiomyocyte beating

ACS Spring 2022

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