Design and applications of enzyme powered motile hybrid organic/inorganic-microcapsules

Protocells have been established as an important platform in the understanding of contemporary naturally occurring, cellular phenomena or in the study of the origin of life. By designing protocells from the bottom up, the cell complexity can be reduced to the bare minimum, which allows the research of very specific functionalities, which often mimic naturally occurring properties of cells or microcompartments. The previously published work by Kumar et al. from the group of Prof. Stephen Mann found inspiration in gas vesicles of Halobacterium salinarium, which allowed the organism to move vertically via the manipulation of the buoyant force the organism experiences. In a similar manner, Kumar et al. designed giant buoyant microcapsules which maintained their vertical motility through enzymatic control of an O₂-microbubble inside the capsule. The first experimental chapter of this thesis presents a novel protocell-system in protamine/DNA-microcapsules and compares it to the previously presented AMP/DNA-microcapsules from Kumar et al. The cells are demonstrated as very robust and easy to fabricate with high stability in various chemical environments and under physical stress. What makes the capsules so valuable for the further experiments of this thesis though is their capability of entrapping various functional components like enzymes, or micro- or nano particles within them. The second half of this chapter then investigates the entrapment capabilities, by assessing the capsule's membrane thickness, permeability and its structure microscopically to establish a thorough hypothesis on the localisation of the entrapped enzymes, their stability, how they are entrapped and what the capsule-membrane looks like. The following chapter then establishes the core functions of microcapsule motility. Entrapment of catalase and glucose oxidase enables the nucleation and consumption of an O₂-microbubble which in return causes the capsule to ascend or descend, facilitated by buoyancy. The first half of this chapter focuses on showcasing both the catalase mediated ascent and the glucose oxidase-mediated descent before combining both concepts into an oscillatory motion. Furthermore, the effect of the growing O₂-bubble on the capsule and its structural integrity is studied, which finally establishes protamine/DNA-microcapsules as superior to their predecessors. The second half of this chapter then focuses on the concept of microcapsule oscillations. Due to the complexity of the experimental design and since diffusion of spatially separated substrates plays a big role in them, the experimental devices will be explained and assessed both through computational simulation and practical diffusion studies with dyes. Stable oscillations and the novel concept of damped oscillations are presented in context to their experimental setups and are analysed through computer-assisted tracking of the microcapsule. Since the understanding of the growth- and depletion rate of the O₂-microbubble are so important, the last part of this chapter focuses on analysing the microbubble dynamics in relation to the substrate concentrations. Furthermore, enzyme leakage appeared as a reoccurring phenomenon throughout this thesis, which will be discussed regarding microcapsule oscillations and whether it causes any issues for the general legitimacy of the concept. Lastly, the third experimental chapter utilises the previously established tools to exploit microcapsule oscillations to perform a rudimentary uptake, transport and release of functional cargo. This work introduces polyoxometalate coacervate vesicles (PCVs) as secondary cargo containers, which can retain molecular cargo via sequestration, and which are subsequently loaded onto protamine/DNA-microcapsule through electrostatic- and other non-defined surface-surface interactions. Next, the uptake conditions for protamine/DNA-capsules and PCVs and the release of the cargo, which is conceptualised around disintegration of the PCVs in response to a rising pH to ~9 will be discussed. To initiate the PCV-disintegration internally, urease was entrapped and used as the trigger for PCV-disintegration through the reaction with urea. Finally, multiple full and successive uptake, transport and release cycles are presented and discussed with several different cargo species.