Effect of the Hydrophilic-Hydrophobic Balance of Antigen-Loaded Peptide Nanofibers on Their Cellular Uptake, Cellular Toxicity, and Immune Stimulatory Properties

Abstract

Recently, nanofibers (NFs) formed from antigenic peptides conjugated to β-sheet-forming peptides have attracted much attention as a new generation of vaccines. However, studies describing how the hydrophilic-hydrophobic balance of NF components affects cellular interactions of NFs are limited. In this report, three different NFs were prepared by self-assembly of β-sheet-forming peptides conjugated with model antigenic peptides (SIINFEKL) from ovalbumin and hydrophilic oligo-ethylene glycol (EG) of differing chain lengths (6-, 12- and 24-mer) to investigate the effect of EG length of antigen-loaded NFs on their cellular uptake, cytotoxicity, and dendritic cell (DC)-stimulation ability. We used an immortal DC line, termed JAWS II, derived from bone marrow-derived DCs of a C57BL/6 p53-knockout mouse. The uptake of NFs, consisting of the EG 12-mer by DCs, was the most effective and activated DC without exhibiting significant cytotoxicity. Increasing the EG chain length significantly reduced cellular entry and DC activation by NFs. Conversely, shortening the EG chain enhanced DC activation but increased toxicity and impaired water-dispersibility, resulting in low cellular uptake. These results show that the interaction of antigen-loaded NFs with cells can be tuned by the EG length, which provides useful design guidelines for the development of effective NF-based vaccines.

Investigation on the Interactions between Self-Assembled β-Sheet Peptide Nanofibers and Model Cell Membranes

“…Previously, we stated that the EG length of NFs significantly influences their cellular association and toxicity [ 40 ]. For further understanding on the effect, we investigated herein the interactions between the DPPC Langmuir membrane and three types of NFs with different EG lengths (6-, 12- and 24-mer).…”

“…In a previous study, we reported that EG n NFs with shorter EG chains were more efficiently associated with JAWS II cells and exhibited higher cytotoxicity [ 40 ]. The EG-length dependency for cellular interaction had the same tendency as that for the interaction with the DPPC membrane.…”

“…EG n peptides ( n = 6, 12, and 24) were prepared using L-Fmoc amino acids and PEG that having carboxyl group and Fmoc-protected amino group at their terminal, Fmoc- N -amido-dPEG n acid, by Fmoc solid-phase peptide synthesis, according to our previous reports [ 40 ]. Molecular weight was analyzed by MALDI-TOF mass (autoflex speed system, Bruker, Billerica, MA, USA).…”

“…The three types of NFs with different EG lengths were prepared by the previously reported procedure [ 40 ]. EG n peptide was dissolved in HFIP and dried with N 2 flow to allow for film formation.…”

“…In a previous study, we have reported the preparation of antigenic peptide-loaded NFs by using self-assembled β-sheet peptides conjugated with model antigen peptides from ovalbumin and oligo(ethylene glycol) (EG) for antigenic peptide delivery ( Figure 1 ) [ 36 , 37 , 39 , 40 ]. Therein, the effect of EG length on the cellular association and toxicity of the NFs was investigated, and we had observed that those with shorter EG length exhibited both higher cellular association and toxicity [ 40 ]. However, the EG length-dependency on their cellular association and cytotoxicity are well not understood in terms of the interactions between NFs and cell membranes.…”