Fine tuning the morphology of peptide amphiphile nanostructures via co-assembly

Abstract

The self-assembly of peptide amphiphiles (PAs) in aqueous solution yields nanoconstructs displaying a rich spectrum of sizes and morphologies, including micelles, fibers, and lamellar ribbons. The morphology impacts the bioactivity of the PAs and, thus, efforts have been made to control it by tuning their molecular structure or the solution pH. However, synthesizing new PAs is time consuming and biomedical applications limit the pH to physiologically relevant ranges. This work demonstrates that the composition of a binary mixture of co-assembled PAs serves as a powerful approach to exert rational control over the morphology, size and transition pHs of the supramolecular nanostructures. We combined light scattering, SAXS, TEM and AFM experiments and theoretical predictions using a Molecular Theory (MOLT) to construct composition-pH morphology diagrams for three relevant PA mixtures. For C₁₆KK/C₁₆KKK mixtures (C₁₆: palmitoyl and K: lysine), we demonstrate fine tuning of the micelle-to-fiber transition pH by varying the composition of the system. For a mixture of oppositely charged PAs, C₁₆EEE/C₁₆KKK (E: glutamic acid), theory and experiments reveal interesting composition-driven micelle-to-fiber-to-micelle transitions. The C₁₆KK/C₁₆EE mixture exhibits three different morphologies-micelles, fibers, and lamellae-and regions of the morphology diagram showing coexistence between fibers and lamellae. MOLT calculations also provide insights into the internal organization of the assemblies and predict that the nanostructure radius can also be tuned by the composition of the mixture, in agreement with SAXS observations.

Fig. 1. Scheme of the free energy per PA (ω) as a function of the molar fraction of PA₁ in the mixture, x₁, for the spherical micelle (M) and cylindrical nanofiber (F) morphologies. The solid red line is tangent to the M and F curves at points x^M₁ and x^F₁, respectively. For all global compositions between x^M₁ and x^F₁, there is coexistence between micelles with composition x^M₁ and fibers with composition x^F₁.

Fig. 2. (a) Structures of the PAs used in the C₁₆KK/C₁₆KKK mixture. (b) Free energy per PA for this mixture at different pH values determined through MOLT calculations assuming ideal cylindrical fibers (red solid lines) or spherical micelles (green solid lines). For pH = 9, the dashed line (tangent to the free-energy curves) indicates micelle–fiber coexistence (the inset shows an enlargement of the region indicated with a box). (c) Predicted morphology diagram. M and F indicate the region where fibers and micelles are stable, respectively, and M–F is the coexistence region.

Fig. 3. (a) Experimental verification of the morphology diagram of C₁₆KK/C₁₆KKK mixtures. The figure shows the boundaries for the M → F transition predicted by MOLT (red and green solid circles, same as in Fig. 2c) and measured by LS (solid black circles and dashed gray lines, which indicate regions of high LS). The TEM, AFM and SAXS observations under specific conditions were categorized as micelles (blue symbols), fibers (red) or micelles + fibers (blue and red). (b) LS normalized intensity as a function of pH for mixtures with different ratios of C₁₆KK and C₁₆KKK. Solid lines show the best fit using the sigmoid function in eqn (1). The LS intensity, I, was normalized using the fitting parameters as I^norm = (I − I^min)/(I^max − I^min). (c) TEM images for x₁ = 0.5 and different pHs. (d) AFM at pH 8.7 and different values of x₁. (e) SAXS curves at pH 9 and different x₁ values.

Fig. 4. Morphology behavior of C₁₆KKK/C₁₆EEE mixtures. (a) Structures of the PAs in the mixture. (b) Free energy per PA at different pH values determined through MOLT calculations assuming ideal cylindrical fibers (red solid lines) or spherical micelles (green solid lines). (c) Comparison of the boundaries of the M ↔ F transition predicted by MOLT (solid red and green circles), measured by LS (solid black circles and dashed gray lines, which indicate regions of high LS) and obtained from SAXS experiments, which were categorized as micelles (blue symbols) or fibers (red). (d) Normalized LS intensity vs. x₁ for pH 8. Solid lines show the best fit using a product of two sigmoid functions. (e) SAXS curves at pH 8 and different x₁ values. (f) AFM images at pH 8 and different x₁ values.

Fig. 5. (a) Coarse-grain strategy for C₁₆KKK and C₁₆EEE used in MOLT calculations. The C₁₆ alkyl chain is represented by four alkyl-tail beads (blue). The amino acid backbone (magenta) and side chains (orange for lysines and green for glutamic acid) are represented by a single bead each. (b) Volume fraction of the beads vs. distance from the center of a spherical micelle composed of a C₁₆KKK/C₁₆EEE mixture for different values of x₁ (molar fraction of C₁₆KKK).

Fig. 6. Morphology behaviour of C₁₆KK/C₁₆EE mixtures. (a) Structures of the PAs used in the mixtures. (b) Normalized LS intensity vs. x₁ at pH values, 6 and 8. Solid lines show the best fit using a sigmoid function. (c) SAXS curves at pH 9 and different x₁ values. (d) Comparison of the boundaries of the M ↔ F transition predicted by MOLT (solid red, green and black circles) and measured by LS (solid gray circles and dashed gray lines, which indicate regions of high LS) and SAXS experiments for specific conditions that were categorized as micelles (blue symbols), fibers (red) or fibers + lamellae (red and yellow). The symbols M, F and L indicate the regions of stability for micelles, fibers and lamellae, respectively.