Development of a novel stimuli responsive filtration membrane using self-assembling peptides.
Thesis DisciplineChemical Engineering
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Membrane based separation of specific constituents from a complex mixture is a wellestablished technology. Numerous filtration membranes have been developed for separations based on size, charge and hydrophobicity. Alternatively, synthetic membranes have been modified using various materials (e.g. charged polymers), to induce stimuli responsiveness and achieve specific separation goals. However, controlling finer separations based on more complex properties has been a challenge. Further improvements in separations could be made with a universal membrane that could be used for a wide range of applications. This thesis describes the development of a novel stimuli-responsive membrane whose pores and surface are functionalised with self-assembling peptides (SAPs). SAPs are molecules that spontaneously organize into ordered structures through non-covalent interactions, in response to external stimuli. The use of reversible SAPs to control membrane permeability could possibly result in a more effective separation process, whereby a condition such as pH could be used to enable or block passage of certain solutes through the membrane. To progress towards achieving this goal, the following sub-objectives were addressed, i) to identify a suitable reversible SAP and examine the effect of conjugating it with a poly(ethylene glycol) (PEG) polymer spacer on self-assembly, ii) to functionalise a membrane surface and subsequently tether the SAP via the spacer molecule, whilst retaining the self-assembling behaviour on the membrane surface.
Initially, peptide P11-4 (QQRFEWEFEQQ) was chosen as the candidate for membrane tethering, because it can spontaneously switch molecular conformation from random coil at high pH to β sheet at low pH. P11-4 was conjugated to a 2 kDa N-hydroxy succinimideactivated PEG via N-terminal amine coupling to form P11-4-PEG-2K and the conjugation was validated using MALDI-tof spectrometry. However, P11-4-PEG-2K did not retain self-assembly of the peptide P11-4 but instead retained its random coil structure at pH < 3, in contrast to the behaviour of native P11-4, which formed β sheets at similar pH. P11-4-PEG- 2K retained its random coil conformation across various pH conditions (pH 2.5, 7 and 11) and incubation periods (2 min-20 days). Additional investigations on the effect of unreacted free PEG on P11-4 self-assembly indicated that the presence of free PEG in solution did not hinder self-assembly. Further, conjugation carried out in 3 mM sodium phosphate buffer and water also resulted in P11-4-PEG-2K that lost its ability to form β sheets at pH 2.5. Thus, P11-4-PEG-2K was deemed unsuitable for membrane modification and an alternative peptide EL-5F (ELELELELELF), with pH responsiveness, was used for further investigations on bioconjugate self-assembly in solution and upon being tethered to a membrane surface.
For the first time, rapid and reversible pH-regulated self-assembly of EL-5F and its conjugates with 2 and 5 kDa PEG (EL-5F-PEG-2K and EL-5F-PEG-5K) was demonstrated. Circular dichroism indicated the formation of β sheet structures at pH < 5.9, 5.8 and 5.4 and disassembly to random coils above those pH values for EL-5F, EL-5F-PEG-2K and EL-5FPEG- 5K, respectively. β sheets were confirmed by the thioflavin T assay and transmission electron microscopy revealed the existence of extended fibrillar structures below the above pH values. pH-induced secondary structure conversion in both directions was reproducible for over fifteen cycles, even at salt concentrations of up to 200 mM NaCl, and the amounts of sheet formed were quantitatively related to pH below the transition points. Self-supporting hydrogelation after self-assembly was observed at concentrations as low as 0.2 wt%, which is 15-fold lower than previously reported concentrations with PEGylated SAPs.
Subsequent work, involving tethering of EL-5F-PEG-2K to an Anodisc alumina membrane surface and/or pores and investigations on the effect of reversible self-assembly on the permeability of the membrane was carried out. COOH groups were immobilised on the membrane surface to prepare it for the coupling of EL-5F-PEG-2K, using multilayer deposition of poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH). EL-5FPEG- 2K was then successfully amine-coupled to the COOH groups on the surface and this was confirmed using ATR-FTIR. Subsequent flux tests indicated no pH-dependant variation in buffer flux properties of the EL-5F-PEG-2K modified membrane. However, MWCO experiments using 72 and 29 kDa PEGs indicated reversible self-assembly of membrane tethered EL-5F-PEG-2K, upon changes in pH, thereby affecting macromolecule permeation. Furthermore, investigations on the reversible, pH-regulated self-assembly of peptide EL-5F and conjugate EL-5F-PEG-2K tethered to polystyrene nanoparticle (NP) surfaces, showed size transitions and aggregation of NPs upon changes in pH, indicating retention of peptide and bioconjugate self-assembly after surface tethering.
The results provide a tentative but consistent proof-of-concept, giving the first steps towards the development of a novel switchable SAP-based, stimuli-responsive membrane. Further investigations suggested to validate reversible peptide self-assembly on the membrane surface, are expected to pave the way towards achieving finely controlled membrane separations.