Control of peptide aggregation and fibrillation by physical PEGylation

Peptide therapeutics have the potential to self-associate, leading to aggregation and fibrillation. Non-covalent PEGylation offers a strategy to improve their physical stability; an understanding of the behaviour of the resulting polymer:peptide complexes is, however, required. In this study we have performed a set of experiment with additional mechanistic insight provided by in silico simulations to characterise the molecular organisation of these complexes. We used palmitoylated vasoactive intestinal peptide (VIP-palm) stabilized by methoxy-poly(ethylene glycol)5kDa-cholane (PEG-cholane) as our model system. Homogeneous supramolecular assemblies were found only when complexes of PEG-cholane:VIP-palm exceeded a molar ratio of 2:1; at and above this ratio the simulations showed minimal exposure of VIP-palm to the solvent. Supra-molecular assemblies formed, composed of, on average, 9-11 PEG-cholane:VIP-palm complexes with 2:1 stoichiometry. Our in silico results showed the structural content of helical conformation in VIP-palm increases when it is complexed with the PEG-cholane molecule; this behaviour becomes yet more pronounced when these complexes assemble into larger supra-molecular assemblies. Our experimental results support this: the extent to which VIP-palm loses helical structure as a result of thermal denaturation was inversely related to the PEG-cholane:VIP-palm molar ratio. The addition of divalent buffer species and increasing the ionic strength of the solution both accelerate the formation of VIP-palm fibrils, which was partially and fully suppressed by 2 and >4 mole equivalents of PEG-cholane, respectively. We conclude that the relative freedom of the VIP-palm backbone to adopt non-helical conformations is a key step in the aggregation pathway.