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Abstract
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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 cyclotides. 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 C, 37 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 demonstrated 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 C, the aggregation
of cyclotides were facilitated than 25 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 prepared cyclotide nanotubes are excellent candidates to be used in biological fields, for example, in drug
delivery purposes
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