Cyclotide-rich fractions containing nanofibers by electrospinning: preparation, characterization and examination of antimicrobial activity

Abstract

In this study, antimicrobial nanofibers were produced with the mixtures of polyvinyl alcohol (PVA) and cyclotide-rich fractions by electrospinning method. After extraction, the first separation was carried out with C18 flash chromatography and then fractioned into five separate parts by reversed-phase high-pressure liquid chromatography (RP-HPLC). The molecular weights of cyclotides in each fraction were determined by quadrupole time-of-flight liquid chromatography-mass spectrometry (Q-TOF LC-MS). Cyclotide-rich fractions were mixed with 10% of PVA solution and nanofibers were produced from this biocomposite mixture by electrospinning method. The nanofibers were characterized by field emission scanning electron microscopy (FE-SEM), and it was observed that 100% peptide-containing nanofibers (cyclotide-rich fraction/10% PVA, w/v) had more regular fiber textures. The presence of the peptides in the nanofiber was also confirmed by analytical RP-HPLC, as the peptides in both peptide fractions and nanofiber solutions have the same retention times. The nanofibers produced with the fourth cyclotide-rich fraction showed activity against gram-positive bacteria (Bacillus cereus) in antimicrobial susceptibility test. As a result of these findings, cyclotide-containing nanofibers with antimicrobial activity can be produced for pharmaceutical research and development studies.

Keywords: Cyclotides; antimicrobial activity; drug delivery; electrospinning; nanofibers.

Figure 1

A. Three-dimensional structure of cyclotide kalata B1 (PDB code: 1NB1) with a cyclic cystine knot motif. The peptide backbone is shown in red, and the three disulfide bonds forming the cystine knot motif are shown in orange. Kalata B1 structure was prepared with MOLMOL [30]. B. Schematic illustration of an electrospinning device. It has a syringe with a metallic needle and a rotating collector. An automated pump is used to control the flow rate of the polymer solution and a high voltage is applied between the syringe needle and the collector to form the charged jet.

Figure 2

RP-HPLC chromatogram of the lyophilized cyclotide-rich part after C18 flash chromatography. Five different fractions were collected into different containers every 10 min. After lyophilization of the fractions, only Fractions 2, 3, and 4 were used for further studies as there was enough peptide sample for nanofiber production.

Figure 3

Deconvoluted mass spectra of A. Fraction 2, B. Fraction 3, and C. Fraction 4 after RP-HPLC. LC-MS chromatograms and spectra were analyzed by Agilent Qualitative Analysis B.06.00 software. Deconvoluted mass spectra are shown in the mass range of 2500–4000 Da since cyclotides fall within this mass range as in the previous literature [11].

Figure 4

FE-SEM images of 100% w/v nanofibers (25 mg of cyclotide-rich fraction + 25 mL of 10% of PVA solution) of Fractions 2, 3, 4 and the nanofibers produced only with 10% PVA. A. FE-SEM image of nanofiber NF2-100 (Mag = 10000, fiber diameter range: 254.5–906.9 nm). B. FE-SEM image of NF3-100 (Mag = 10000, fiber diameter range: 210.7–513.3 nm). C. FE-SEM image of NF4-100 (Mag = 10000, fiber diameter range: 134.9–755.4 nm). D. FE-SEM image of PVA nanofiber (Mag = 10000, fiber diameter range: 196.4–567.2 nm).

Figure 5

Analytical RP-HPLC chromatograms of the PVA solution, peptide fractions and the nanofibers (NF2-100, NF3-100, and NF4-100). A. Analytical RP-HPLC chromatogram of the nanofiber of 10% PVA solution alone. B. Analytical RP-HPLC chromatogram of Fraction 2. C. Analytical RP-HPLC chromatogram of NF2-100. D. Analytical RP-HPLC chromatogram of Fraction 3. E. Analytical RP-HPLC chromatogram of the NF3-100. F. Analytical RP-HPLC chromatogram of Fraction 4. G. Analytical RP-HPLC chromatogram of NF4-100. The similar retention times of the peptides in both the fractions and the nanofibers show that the nanofibers contain cyclotides.

Figure 6

Antimicrobial activities of nanofibers NF4-50, NF4-100, and NF4-150 produced from Fraction 4/10% PVA solution and nanofiber produced only with 10% PVA solution. Cyclotide containing nanofibers did not show antimicrobial activity on S. Typhimurium (gram-negative) bacteria, but they showed activity on B. cereus (gram-positive) bacteria. NF4-150 nanofiber showed the highest activity compared to nanofibers NF4-100 and NF4-50 as it has the highest cyclotide content.