Aqueous self-assembly of extracted cyclotides from<i> Viola</i><i> odorata</i> into novel stable supramolecular structures

Abstract

Cyclotides are categorized as cyclic peptides consisting of 28-37 amino acids. The cyclic nature, as well as the presence of three intramolecular disulfide bonds, resulted in the extraordinary stability of the cyclo-tides. In addition, they have the ability to self-associate. In this study, we investigated the ability of the cyclotides to be self-assembled by UV-visible spectroscopy. Then after, the isoelectric points of cyclotides were determined by potentiometer titration and the zeta potential method. Also, the effects of time (1, 2, 3, 5, 7, 10 days), temperature (25 degrees C, 37 degrees C), pH (2, 4, 6), and peptide concentration (100, 1000, 5000 lg/ ml) on size, poly dispersity index (PDI) and zeta potential of the self-assembled nanostructures were investigated. The stability of the cyclotide nanotubes after freeze-drying, 50-fold dilution with phosphate buffered saline (PBS), and exposure to the fetal bovine serum (FBS) were studied too. The results demon-strated that the cyclotides can self-assemble in different studied media. The potentiometer titration and zeta potential measurements confirmed that the isoelectric points of cyclotides were near the pH of 4. From day 0 to 10, the size of nanostructures increased and at pH 4, the largest nanostructures were obtained at day 10, which is due to the fact that the isoelectric points of the cyclotides were near this pH. Also, increasing the cyclotide concentration increased the size of the assemblies. Furthermore, the effect of temperature is largely depending on pH in which it seems that at pH 2 and 37 degrees C, the aggregation of cyclotides were facilitated than 25 degrees C. An interesting finding was that the cyclotide nanotubes showed exceptional stability after freeze-drying, exposure to FBS, and dilution by PBS, suggesting that the pre-pared cyclotide nanotubes are excellent candidates to be used in biological fields, for example, in drug delivery purposes. (c) 2023 Elsevier B.V. All rights reserved.