Characterization of double-layered peptide nanoparticles. (A) Schematic diagram of the NP peptide (NPep) and 4M2e. 4M2e is composed of four tandem repeats of M2e epitopes connected with short flexible linker sequences (blue arrowheads). (B) Western blotting analysis of 4M2e, NP peptide, and nanoparticles using anti–His-tag monoclonal antibody. (C) Schematic diagram of core nanoparticle fabrication and double-layered nanoparticle generation. An additional layer of the 4M2e peptide was cross-linked onto the desolvated NP-peptide particulate core surface via DTSSP cross-linking. (D) Size distribution of NP-peptide nanoparticles (NPep nano), NP-peptide/4M2e–layered nanoparticles (NPep/4M2e nano), NP-protein nanoparticles (NP nano), and NP-protein/4M2e–layered nanoparticle NP/4M2e nano). (E) Scanning electron microscopy of NP-peptide/4M2e–layered nanoparticles and NP-protein/4M2e–layered nanoparticles. (Scale bars, 200 nm.)