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Supramolecular Biomedical Materials

Final Report Summary - SUPRABIOMAT (Supramolecular Biomedical Materials)

Using polycarboxylic acid functionalised dendritic peptide scaffolds, we have achieved and reported in a first set of papers the multistimuli-responsive self- assembly into 1D supramolecular polymers. We have designed a dendritic nonaphenylalanine as a supramolecular synthon, since oligophenylalanines are known to be reliable self-assembly motifs in water, due to their relatively large hydrophobic character and potential for shielding hydrogen-bonding motifs. The amphiphilic dendritic peptide monomers are based on a C3-symmetrical hydrophobic core that directs their supramolecular polymerisation into strictly one-dimensional morphologies with negligible secondary aggregation.
The branched nonaphenylalanine equipped with nine carboxylic acid groups turned out to be highly soluble in aqueous buffers at neutral pH. First, we studied the self-assembly in phosphate buffer using circular dichroism (CD) spectroscopy. At pH 7.4 in 10 mM phosphate buffer, the CD spectra suggest the presence of molecularly dissolved species. The weak negative CD band at 210 nm and the weak positive band at 220 nm are identical to the bands observed in buffer–CH3CN 1:1 mixtures. The addition of CH3CN is known to disrupt supramolecular polymerisation in water, because it diminishes the hydrophobic shielding of the hydrogen bonding amide sequences. Coulombic repulsive interactions from the negatively charged dendritic carboxylate groups in the hydrophilic rim of the peptides prevent aggregation into large macromolecular structures. Importantly, the range and strength of the charge–charge repulsive interactions can be modulated simply by adjusting the ionic strength of the aqueous buffer. The addition of a 1:1 electrolyte, like NaCl, leads to a significant increase in the intensity of the negative CD bands. This increase is a strong indication of the self-assembly into ordered chiral supramolecular polymers, which becomes more favourable when the negative charges in the periphery of the self-assembled peptidic nanorods are screened electrostatically. These findings strongly suggest that the self-assembly of the polyanionic dendritic peptide at neutral pH is largely dictated by a mechanism of frustrated growth: attractive supramolecular interactions, hydrogen- bonding, π–π interactions and hydrophobic effects that drive the monomers to assemble are opposed by repulsive electrostatic interactions that disfavour polymerisation.
Based on this observation the self-assembly should not only be triggered by changes in the ionic strength, but also by acidifying the buffered environment. Indeed when we lowered the pH from 7.4 to 5.0 at 0 M added NaCl self-assembly was induced, resulting in a very sharp increase in the CD bands. Partial protonation of the dendritic carboxylates and reduction of the charge–charge repulsive interactions, due to a lower net charge of the monomeric unit, effectively decrease the molecular frustration and trigger the supramolecular polymer- isation. The self-assembly of these unique multistimuli- responsive peptidic supramolecular polymers can be most conveniently visualised in an ionic strength and a pH dependent contour plot. Remarkable is the extremely sharp increase from a disassembled state at pH 7.4 to a fully poly- merised one at pH 6, a transition that occurs at near physiological NaCl concentration of 100 mM. This monomer to polymer transition between pH 7.4 and pH 6.0 is reversible, and can be switched on and off repeatedly by adding HCl and NaOH. We are thereby able to show that frustrated growth into ordered supramolecular polymers can be independently fine-tuned, by adjusting both the ionic strength and the pH.
Small angle X-ray scattering (SAXS) experiments were conducted to complement the spectroscopic data with insight into the morphology of the supramolecular assemblies in solution. The hallmark of rigid cylindrical structures can clearly be observed in all of the scattering profiles. All profiles are reasonably well described with a form factor for homogeneous rigid cylinders enabling us to set a lower limit for their length, L > 20 nm, and to determine their cross-sectional radius, RCS = 2.8 nm. The latter value agrees well with the length of the stretched out dendritic peptide side arms, corresponding to 3 nm. The length of the self-assembled one-dimensional nanorods cannot be determined from these measurements as it is beyond the experimental resolution.
To visualize the effect of the ionic strength on the length of the nanorods we turned to cryogenic transmission electron microscopy (cryo-TEM). In agreement with the SAXS results, nanorods with a diameter of about 4.5 nm can clearly be observed. At 0 M added NaCl the nanorods are about 20–50 nm long. When increasing the ionic strength to 0.8 M NaCl the nanorods grow larger than 200 nm in length. As expected, aggregation into larger morphologies is favoured at high ionic strength when repulsive electrostatic interactions are effectively screened. Similarly, ionic strength dependent length profiles have also been seen in worm-like micellar solutions of charged surfactants. Cryo-TEM micrographs of charge-neutral dendritic peptide amphiphile 3b show the presence of micrometer long nanorods. In this case control over the 1D supramolecular polymerisation process is lost and the growth occurs uninhibited, to produce nanorods of near infinite length, compared to the frustrated self-assembly system. These results corroborate our proposed strategy to (partially) compensate attractive with repulsive interactions to control the size and shape of self-assembled nanomaterials.
Furthermore we have developed a series of anionic dendritic peptide amphiphiles of increasing hydrophobic character. By establishing state diagrams mentioned above, we can describe their pH and ionic strength triggered self-assembly into supramolecular nanorods in water and highlight the impact of hydrophobic shielding in the supramolecular polymerisation process. Via the incorporation of fluorinated peptide side chains the pH-triggered monomer to polymer transition at physiological ionic strength is shifted from pH 5.0 to pH 7.4. We thereby show that compensating attractive non-covalent interactions and hydrophobic effects with repulsive electrostatic forces, a concept we refer to as frustrated growth, is a sensitive tool in order to manipulate one-dimensional supramolecular polymerisation processes in water.
A unique example of supramolecular polymerisation in water was finally based on monomers with nanomolar affinities, which yield rod-like materials with extraordinarily high thermodynamic stability, yet of finite length, is reported. A small library of charge-neutral dendritic peptide amphiphiles was prepared, with a branched nonaphenylalanine-based core that was conjugated to hydrophilic dendrons of variable steric demand. Below a critical size of the dendron, the monomers assemble into nanorod-like polymers, whereas for larger dendritic side chains frustrated growth into near isotropic particles is observed. The supramolecular morphologies observed by electron microscopy, X-ray scattering and diffusion NMR spectroscopy studies are in agreement with the mechanistic insights obtained from fitting polymerisation profiles: non-cooperative isodesmic growth leads to degrees of polymerisation that match the experimentally determined nanorod contour lengths of close to 70 nm. The reported designs for aqueous self-assembly into well-defined anisotropic particles has promising potential for biomedical applications and the development of functional supramolecular biomaterials, with emerging evidence that anisotropic shapes in carrier design outperform conventional isotropic materials for targeted imaging and therapy.

• Advancement beyond the state of the art in the field:
We managed to tuned the ‘reactivity’ of the weakly acidic or neutral supramolecular monomers via electrostatic and steric repulsive forces, which could be independently varied using pH, ionic strength and concentration triggers. This has allowed us to study the stimuli-responsive self-assembly and supramolecular polymerisation processes. Combining all of these external triggers has allowed us to build state diagrams, well-known for natural systems, like the tobacco mosaic virus.

• Impact:
Responsive well-defined nanomaterials that can undergo morphological transitions induced by specific changes in a physiological environment hold promise for biomedical applications, such as drug delivery carriers and imaging agents. For example, the pH triggered self-assembly of small monomers into long supramolecular nanorods in the acidic micro-environment of tumour or inflamation tissues could favour their accumulation as diffusion is decelerated upon the pH-triggered local increase in size.

• Transfer of knowledge:
The fellow and his students have been actively involved in group seminars of his mentor, which has been an excellent platform in order to share knowledge between the groups. Furthermore the fellow has set-up a new teaching course on supramolecular chemistry for MSc students, given talks in workshops and seminars for PhD students in Münster and organised two conferences on supramolecular chemistry and (bio)organic chemistry, held in Münster in 2013. More details are given in section 6 and 7. Finally the Fellow has built up a portfolio that was strong enough to secure a Professorship at the University of Mainz. The fellow and his whole group moved in January 2015, the request of change for the grant was successfully reviewed external by REA and granted.