De novo-designed bacterial polypeptides: synthesis, assemblies and biomedical applications

© 2012 Dr. Denison H. C. ChangIn recent years, nano-engineered functional and biocompatible materials have been the focus of widespread research as building blocks for the assembly of nano- and micro-scaled therapeutic delivery vehicles. Such delivery vehicles have the dual benefit of protecting the therapeutic cargo from being degraded by the body, and preventing unwanted side effects by limiting delivery to healthy cells. Successful assembly and application of these vehicles requires control over properties of the building blocks such as structure, chemical and bio-functionality, and degradation behaviour. Polypeptides represent an excellent class of materials to assemble into delivery vehicles due to their inherent biodegradability and biocompatibility, as well as the flexibility to be engineered to contain a variety of functional groups. The focus of this thesis was the synthesis of an array of novel polypeptides via bacterial peptide expression for the assembly of various delivery vehicles. Bacterial expression affords the ability to genetically ‘design’ polypeptides, offering facile and precise control over molecular weight, sequence, and chemical properties of the expressed product. As bacteria reproduce at an exponential rate, large quantities of identical polypeptides can be synthesised in a short span of time, minimizing batch-to-batch variation and cost. Synthesis of monodisperse and precisely defined polypeptides in terms of peptide composition and sequence is reported in the first section of this thesis. That precise control over composition and sequences, particularly that of crosslinking groups, influenced the rate of degradation of an assembled delivery system as reported in the subsequent section. The use of bacterial expression to produce sequence specific polypeptides also enabled the functionalisation of complex polypeptides, such as poly(L-histidine) (PLH) which lack reactive chemical groups and thus are impossible to chemically post-functionalise. Assembly of these functionalised PLH into biodegradable and biocompatible histidine-based particles with thiol and amine functionalities is reported. Finally, the precision of bacterial synthesis was exploited to fabricate a pH-responsive di-block copolypeptide, which self-assembled into stable vesicles in physiological conditions and disassembled in simulated endosomal conditions. These self-assembled vesicles exhibited a reversible multilamellar phase triggered by pH. This thesis demonstrates the benefits of using polypeptides (biocompatibility and biodegradability) and bacterial biosynthesis (precise control over sequence, size, structure and chemistry) to synthesise a range of nano-engineered materials with precisely defined properties. This in turn presents new opportunities for the development of delivery vehicles with tailored functionalities