Biomimetic engineering of colloidal nanoarchitectures with "in vitro" and "in vivo" functionality

Biomimetic engineering opens unprecedented possibilities of combining biomolecules (i.e. proteins, DNA, polysaccharides) with synthetic materials (i.e. synthetic polymers). This combination results in unique hybrid systems with functionalities that mimic processes in living organisms. While the translational value of functional biomimetically engineered structures is of exceptional importance in fields such as technology, engineering, chemistry, biology and medicine, due to the properties the structures inherit from both the synthetic and bio-materials, the understanding of how biomimetically engineered systems self-assemble and function is equally important, as it gives insight in how non-living systems progressed to living organisms. Some of the most prominent examples of functional biomimics include polymersome based catalytic nanocompartments, multicompartment systems that mimic cellular organization and artificial organelles. In this thesis, the focus lies on understanding and applying the fundamental principles of biomimetic engineering by equipping colloidal nanoarchitectures (soft polymer nanoparticles and hollow sphere polymersomes) with functional biomolecules (transmembrane proteins and enzymes). First, the most important questions are addressed – why do polymer nanoarchitectures present ideal building blocks for creating novel biomimics, how do biomimics self-assemble in solution, which methods are most frequently used for their characterization, and where the applications of biomimics are in technology and medicine. Both colloidal and 2D supported/free standing polymeric nanoarchitectures structures are discussed in order to familiarize the reader with the wide range of nanoarchitectures that can be formed by polymers, however the focus primarily rests on biomimetic design of colloidal nanoarchitectures, as their colloidal nature favours them as therapeutic agents that can act on the cellular level. To develop a pH responsive protein delivery agent, a biomimetic approach is applied in equipping self-assembled poly(ethylene glycol)-b-poly(methylcaprolactone)-b-poly(2- (N,Ndiethylamino)ethyl methacrylate) (PEG-b-PMCL-b-PDMAEMA) polymer nanoparticles with a therapeutic enzyme, acid sphingomyelinase. Due to the electrostatic interactions between the negatively charged enzyme and the positively charged PDMAEMA groups present in the nanoparticle corona, the biomimetically engineered nanoparticles display a distinct protein localization on their corona and a pH dependent release behavior of the attached protein. The application of the self-assembled system as a very efficient delivery agent for catalytically active biomolecules is demonstrated in human epithelial HeLa cells. Next, a more complex nanoscale biomimic - a pH triggered catalytic nanocompartment - is built by biomimetically engineering the nanoarchitecture of poly(2-methyl-2-oxazoline)- block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b- PMOXA) polymersomes. Aqueous cavities of polymersomes are loaded with horseradish peroxidase while a chemically modified Outer membrane protein F (OmpF) is reconstituted in polymersome membranes. The chemical modification of OmpF transforms the otherwise unspecific pore into a selective and pH responsive pore, through which molecules can only diffuse once the attached molecular cap blocking the pore is cleaved. Hence, once the modified OmpF is functionally reconstituted in polymersome membranes it allows the developed biomimic to present an on demand catalytic activity. As a first proof of concept of a pH responsive catalytic nanocompartment the system demonstrates that a spatial control of a reaction inside a nanocompartment can be achieved and supports the further development of complex reaction spaces that can act in an analogous manner to cellular compartments, where in situ reactions are modulated by a plethora of responsive proteins. Finally, biomimetically engineered polymersomes are designed for an in vitro and in vivo application as artificial organelles. In order to mimic processes taking place in lipid membranes of cellular organelles, polymersome membranes are equipped with a genetically and chemically modified OmpF. The structural modifications done at the rim of the OmpF pore, limit the OmpF permeability to small molecular weight molecules, but make it capable of responding to the presence of small signaling molecules. When the modified OmpF is reconstituted in membranes of enzyme loaded polymersomes it prevents the enzyme to access enzymatic substrates. However, the presence of of glutathione, which for example is found in abundant concentrations in the cytoplasm, readily cleaves the chemical modification of OmpF and opens the pore, thereby allowing the encapsulated enzyme to catalyze a reaction. The responsiveness of the self-assembled system to glutathione, abundantly present in the cytoplasm, makes the developed biomimic a suitable candidate for intracellular functionality as an artificial organelle. To demonstrate this, we not only show that the system is functional in the cellular microenvironment of human epithelial HeLa cells but also that it is robust enough to function in vivo in Zebrafish embryos.

from # All Medicine by Alexandros G. Sfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2kXZEOe
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