Morphological studies of self-assembled cyclotides extracted from Viola odorata as novel versatile platforms in biomedical applications

Abstract

Self-assembling peptides have attracted researchers' attention recently. They are classified as biomedical materials with unique properties formed in response to environmental conditions. Cyclotides are macrocyclic plant-derived peptides containing 28-37 amino acids that have the ability to self-assemble. Herein, we investigated the effect of pH, time, and temperature on the self-assembling properties of the cyclotides extracted from Viola odorata. For this purpose, the cyclotides were dispersed in aqueous trifluoroacetic acid at pH 2, 4, or 6 and incubated at 25 or 37 °C for 1, 2, 3, 5, 7 or 10 days, and the morphology of the self-assembled structures was identified by optical microscopy, polarized optical microscopy, scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. At pH 2 and 4, the self-assembly process of cyclotides comprises a number of steps, starting with the formation of spherical peptide nanostructures followed by hierarchically assembled nanotubes, and then shifting to nanofibers after 10 days. At pH 6, amorphous structures were produced even after 10 days. The temperature also could affect the self-assembly mechanism of the cyclotides. At 25 °C, the spherical peptide micelles formed firstly and then merged to form nanotubes, while at 37 °C the cyclotides showed crystallization followed by an increase in length with time. The fluorescence microscopy images showed that the nanotubes could efficiently entrap the hydrophobic molecules of coumarin. This comparative study on the self-assembly of the cyclotides extracted from Viola odorata is the first example exploring the capacity of these cyclotides to adopt precise nanostructures. The nanotubes and nanofibers obtained with these cyclotides might find interesting applications in drug delivery and tissue engineering.