Small amphipathic peptides are responsible for the assembly of cruciferin nanoparticles

Hui Hong,Ali Akbari,Jianping Wu

doi:10.1038/s41598-017-07908-z

Abstract

Amphipathic peptides are versatile building blocks for fabricating well-ordered nanostructures, which have gained much attention owing to their enormous design possibilities and bio-functionalities. However, using amphipathic peptides from natural proteins to create tunable nanostructures is challenging because of their heterogeneity and great tendency to form aggregates. Here we fabricated two well-defined nanoparticles from cruciferin amphipathic peptides by integrating top-down and bottom-up approach. Alkali hydrolysis (pH 12, 120 °C for 30 min) was introduced to break down intact cruciferin into peptides (top–down). The cruciferin peptides and their fractions were then assembled into nanoparticles (bottom–up) in the presence of calcium ions. The permeate fraction from 10 kDa cut-off membrane formed smaller nanoparticles (F1-NPs) (around 82 nm) than that of unfractionated cruciferin peptides (CRU-NPs, around 185 nm); the electrostatic and hydrophobic interactions were the main driving forces for particle formation. LC-MS/MS analysis characterised that the small amphipathic peptides (Xn1Zn2Xn3Zn4, n1–4 = 0~5), composed of alternating hydrophobic (X) and hydrophilic (Z) amino acid with a length of 5–15 and 5–20 residues for F1-NPs and CRU-NPs, respectively, were responsible for particle formation. Our study established the mechanism of particle formation of the cold gelation is through assembly of amphipathic peptides.

Highlights

Polymeric nanocarriers are extensively explored as vehicles for encapsulation and delivery of bioactive compounds and therapeutic molecules[1,2,3]
To investigate the effects of peptide size on particle formation, several fractions of cruciferin peptides were obtained for fabricating particles
Cruciferin fraction < 10 kDa (F1) showed the greatest increase in turbidity after addition of Ca2+ (Table 1); the nanoparticles were deemed ‘formed’ when the suspension turbidity increased by at least 50% of the original dispersion, and when no precipitate formed after adding calcium ions[30]

Summary

Introduction

Polymeric nanocarriers are extensively explored as vehicles for encapsulation and delivery of bioactive compounds and therapeutic molecules[1,2,3]. Despite the successful fabrication of cruciferin-based nanoparticles in the previous study[27], the fundamental question remains in regard to the mechanisms of particle assembly. In this previous study, after heat treatment at 120 °C, cruciferin were broken down into peptides. Increased turbidity (%) — 106.05 ± 3.17l 98.51 ± 1.60j — 25.79 ± 0.54c 103.54 ± 0.78k — 7.30 ± 0.52a 85.15 ± 1.46i — 36.12 ± 0.39e 54.29 ± 1.24g — 42.37 ± 0.11 f 22.29 ± 0.79b — 30.88 ± 0.14d 26.68 ± 0.39c — — 29.42 ± 0.37d — 62.60 ± 0.21h 62.79 ± 5.12h investigate (1) what peptides exactly assembled the nanoparticles; (2) what were the main driving forces that contributed to this assembly; (3) whether the primarily β-sheet-containing cruciferin will generate peptides to form β-sheet-stabilized nanostructure. We aimed at elucidating the underlying mechanism of the particle development and identifying the sequence of peptides that were responsible for the nanoparticle formation

Objectives

Methods

Results