Mechanistic understanding of peptide binding and translocation across native like membrane model systems

Therapeutic peptides are widely applied for treatment of chronic diseases, e.g. type 1 diabetes mellitus, however the current administration methods use needle injection which have several drawbacks, with low patient compliance being the most important. Oral peptide delivery harbors great promise with its ease of administration, however the efficiency of the current delivery systems is still fairly low. This can, at least to some extent, be tied back to a lack of fundamental knowledge of how peptide and peptide delivery systems cross the membranes they encounter when trafficking from the intestine and into the blood stream. Today when researchers try to elucidate mechanistic insights for peptide cross membrane translocation they are often hampered by the use of bulk ensemble read out, which has been shown to mask underlying molecular mechanistic behavior.
Thus, there is a need for developing methodologies capable for of delineating the membrane permeation and translocation mechanism of peptides and permeation enhancers, such to enable the reengineering of these peptides and permeation enhancers to deliver high oral bioavailability.
In this thesis I outline the gastro intestinal tract, identifying the barriers which prevent oral delivery of therapeutic peptides and discuss a selection extensively researched peptide permeation enhancers. Based on the lack of methods delivering detailed mechanistic insights on peptide permeation and translocation across membranes I develop a range of advanced fluorescence microscopy assays. I apply these assays to investigate the molecular membrane interaction mechanisms of peptide lipidation, cell penetrating peptides and polymer coacervates as permeation enhancers. I use giant unilamellar vesicles (GUVs) as the main membrane model in my fluorescence microscopy studies, which provide a highly level of flexibility and control in regards to membrane composition, allowing for detailed understanding of membrane interactions. To ease the analysis of the GUV fluorescence imaging data, I established in-house semi-automatic analysis tools, capable of tracking GUVs and extracting parameters for each individual GUV, which are used to quantify the molecular processes occurring. This experimental methodology was implemented for studying how lipidation enhances peptide translocation across membranes, here we provided for the first time mechanistic evidence that small changes in lipidation length can shift therapeutic peptides from having the ability to introduce minor perturbations of the membrane to being able to produce membrane pores. Illustrating how lipidation, if finetuned correctly can bestow therapeutic peptides with a strong intrinsic ability to translocate across membranes. Secondly, we also looked at the mode of action of membrane active peptides, a more classical permeation enhancer class typically co-administered with the peptide drug. Thirdly, we attempt to improve current oral delivery by designing, optimizing and characterizing a new drug delivery systems I investigated the application of acrylamide based heteropolymers as combined peptide carrier and permeation enhancer. I show that finetuning the physio-chemical properties of the chosen heteropolymer we can produce polymer coacervated capable of being loaded with semaglutide and display membrane interaction/disruption capabilities. Finally I implemented live cell 3D timelapse fluorescence imaging to assisting in the investigation of reactive oxygen species regulating extracellular vesical secretion.
This provided me the foundation for firstly developing future in vitro assays for studying peptide permeation in an increased biological context and secondly the associated analytical tools for quantifying cellular data.
Overall the work presented here delivers methodologies and analytical tools for examining membrane permeation properties of potential oral peptide delivery compounds. The work furthermore provides fundamental insights for the potential development of improved oral peptide delivery concepts and systems.