Influence of Electrostatic Interactions on the Self-Assembly of Charged Peptides

Abstract

Peptides can be designed to self-assemble into predefined supramolecular nanostructures, which are then employed as biomaterials in a range of applications, including tissue engineering, drug delivery, and vaccination. However, current self-assembling peptide (SAP) hydrogels exhibit inadequate self-healing capacities and necessitate the use of sophisticated printing apparatus, rendering them unsuitable for 3D printing under physiological conditions. Here, we report a precisely designed charged peptide, Z5, with the object of investigating the impact of electrostatic interactions on the self-assembly and the rheological properties of the resulting hydrogels. This peptide displays salt-triggered self-assembly resulting in the formation of a nanofiber network with a high β-sheet content. The peptide self-assembly and the hydrogel properties can be modified according to the ionic environment. It is noteworthy that the Z5 hydrogel in normal saline (NS) shows exceptional self-healing properties, demonstrating the ability to recover its initial strength in seconds after the removal of shear force, thus rendering it an acceptable material for printing. In contrast, the strong salt shielding effect and the ionic cross-linking of Z5 hydrogels in PBS result in the bundling of peptide nanofibers, which impedes the recovery of the initial strength post-destruction. Furthermore, incorporating materials with varied charging properties into Z5 hydrogels can alter the electrostatic interactions among peptide nanofibers, further modulating the rheological properties and the printability of SAP hydrogels.

Keywords: 3D printing; electrostatic interactions; hydrogel; self-assembling peptide; self-healing.

Figure 1

Salt-triggered self-assembly of Z5. TEM images of 100 μM Z5 in PBS (a), in NS (b), and in DI water (c). (d) CD spectra of 100 μM Z5 in DI water, PBS, and NS obtained in a CD cuvette with a 1 mm path length. (e) CD spectra of 7 mM Z5 hydrogels in PBS and NS obtained in a CD cuvette with a 0.1 mm path length.

Figure 2

SEM images of Z5 hydrogels (2 wt%) in PBS (a) and in NS (b).

Figure 3

Rheological properties of 2 wt% Z5 hydrogels. Time sweeps of Z5 hydrogels in 0.2×, 0.5×, and 1× PBS (a) and in 1×, 2×, and 4× NS (d) monitoring the gelation process. Frequency sweeps of Z5 hydrogels in 0.2×, 0.5×, and 1× PBS (b) and in 1×, 2×, and 4× NS (e). Time sweeps with alternating low–high strains of Z5 hydrogels in 0.2×, 0.5×, and 1× PBS (c) and in 1×, 2×, and 4× NaCl (f) for the evaluation of self-healing properties.

Figure 4

The co-assembly of Z5 and Z4. TEM images of 100 μM Z4 in DI water (a) and in PBS (b). TEM images of peptide mixture solutions with 50 μM Z5 and 50 μM Z4 in DI water (c) and in PBS (d). Scale bar: 500 nm. CD spectra of 100 μM Z4 in DI water and in PBS (e). CD spectra of peptide mixture solutions with 50 μM Z5 and 50 μM Z4 in DI water and in PBS (f). Turbidity test of peptide solutions or hydrogels (g). Rheologicdal frequency sweeps of peptide hydrogels (h).

Figure 5

Rheological time sweeps with alternating low–high strains of the composite hydrogels with 1 wt% Z5 and 1 wt% nanoclay in DI water (a) and in NS (b).

Figure 6

The printing performance of Z5 hydrogel at 2 wt% in NS. (a) The extrusion of the Z5 hydrogel from a nozzle with a diameter of 0.4 mm. The effect of air pressure on the printed line width at a printing speed of 4 mm/s with a 0.15 mm nozzle (b) and a 0.4 mm nozzle (d). (c) The effect of printing speed on the printed line width with air pressure at 3.5 psi and a 0.15 mm nozzle. (e) The effect of air pressure on the printed line width at a printing speed of 6 mm/s with a 0.4 mm nozzle. (f) The effect of nozzle diameter on the printed line width at a printing speed of 4 mm/s with air pressure at 2.5 psi.

Figure 7

The printed 2D graphics (a–d) and 3D circle rings (e,f) using Z5 hydrogels at 2 wt% in NS with a needle diameter of 0.15 mm.

Figure 8

The printing performance of Z5–gelatin composite hydrogels. The printed 2D graphics (a), 10 layers of 3D circle rings (b), and 30 layers of 3D circle rings (c) using the composite hydrogels with 1.5 wt% Z5 and 5 wt% gelatin. The printed 2D graphics (d), 4 layers of 3D grid structure (e), 10 layers of 3D circle rings (f), and 30 layers of 3D circle rings (g) using the composite hydrogels with 1.5 wt% Z5 and 7 wt% gelatin. The diameter of the printing nozzle was 0.15 mm.