Bridging between bioactive and biomimicking materials : cascade reactions in catalytic compartments

Based on the theme of this thesis, Chapter 1 introduces the concept cells as the paramount example of compartmentalization in nature and the use of polymeric assemblies encapsulating enzymes as mimics. It then proceeds to discuss the principles behind self-assembly of polymers and applications of such systems.
Building on that, Chapter 2 states the aim of the thesis, delineating its background and the vision that lead to a coherent research process. For this thesis, vesicular polymeric compartments composed of the triblock copolymer PMOXA-b-PDMS-b-PMOXA were produced, harbouring various proteins in their lumen and membranes, for catalysis and membrane permeabilization.
In a first step, I contributed to the development of multicompartment cell mimics, micrometer-sized polymeric vesicles that behave like cells in their internal organization and segregation, triggered environmental responses and architectural plasticity. In Chapter 3, such assemblies are able to sense the redox potential of the exterior and, with a cascade resembling receptor-mediated pathways in cells, activate responses ranging from enzymatic activity to selective permeability and cytoskeleton reorganization.
In Chapter 4 and 5, the polymeric vesicles were “shrunk” to diameters of 200 nm and less, to work on biological settings, using sizes smaller than cells for future biomedical applications, with binary mixture of vesicles encapsulating a single type of enzyme. They lost their internal compartmentalization but gained a more intimate relationship with living matter, acting first as cell models, then as symbionts to detoxify the cell medium from uric acid (Chapter4.1) and finally as artificial organelles to study the effect of the overproduction of the signaling molecule cGMP through an already-present cascade (5.1). These two studies shed light not only on the general behavior of binary cascades at the nanoscale, but also on technological limitations of such system, that is the difficult transmembrane diffusion through the porin OmpF, and the effect of distance.
To solve the first matter, we studied melittin as a replacement for OmpF. The pore-forming peptide was studied in its interaction with PMOXA-b-PDMS-b-PMOXA membranes (Chapter 6), and we determine the parameters governing their interaction, both from the polymer (stiffness, length, chain dispersity, roughness), from the geometry of the assembly (curvature) and its stability when it interacts with the peptide. A kind of catalytically active polymeric vesicles was produced to prove melittin’s functionality.
To solve the problem of substrate diffusion, we designed clusters of catalytic vesicles, tethered via complementary DNA strands, and permeabilized by melittin. Enzymes part of the same cascade were in close proximity, below 20 nm, leading to a net gain in reaction efficiency when compared to the same unclustered conditions. Additionally, the DNA clusters adhered to the surface of lung cells, suggesting a future as targeted delivery. The conclusions of Chapter 8 summarize the results of this work and suggest the future outlook for research in this field, whereas Chapter 9 lists all the materials and methods used.