**1. Introduction**

Peptide-based synthetic vaccines have attracted a significant amount of attention as a new generation of vaccines, because of their safety benefits and ease of production when compared with that of conventional whole pathogen-based vaccines [1,2]. However, poor immune responses are induced when only minimal antigenic epitopes are used without combining suitable adjuvants (immune stimulants), which are sometimes toxic. Nanocarrier-based delivery systems are a promising approach to overcome those drawbacks of peptide vaccines. In designing the nanocarrier, it is important to consider the interaction between the nanocarrier surface and cells, such as antigen presenting cells (APCs).

Over the past few decades, various nanocarriers have been developed, including liposomes [3–5], polymeric nanoparticles [6–8], and polymeric micelles [9]. In many of these systems, building block molecules for the construction of nanocarriers are first synthesized and then combined with antigenic peptides via several procedures, including nanomaterial formation and loading of antigenic peptides (encapsulation, chemical immobilization or physical adsorption), to give a nano-formulation. Recently, the use of antigenic peptides that are pre-conjugated to self-assembly motifs has attracted attention as an easier and simpler procedure to produce nano-formulations [10,11]. This self-assembly approach ensures highly efficient drug loading without laborious procedures or the use of synthetic components, which sometimes exhibit toxicity. In addition, because the resulting nanostructures consist of a single component, the physicochemical and structural features of these nanostructures can be simply tuned by the design of the building block peptide, and variation in drug loading efficiency among different nanostructures can be eliminated.

Among the various molecular blocks (e.g., lipids [12–14] and hydrophobic polymers [15,16]) used to assemble antigenic peptides into nanostructures, β-sheet-forming-peptides are extremely attractive because: (i) They can assemble in aqueous solution to give nanofibers (NFs) with highly regulated structures, even when functional molecules with a relatively large molecular weight are conjugated; (ii) the resulting well-ordered β-sheet structures allow the integration of antigens at high density; and (iii) they are relatively easy to synthesize and have high biocompatibility. These advantages make NF-vaccines a good alternative to traditional vaccines. Immune induction by NFs formed from antigenic peptides conjugated to β-sheet-forming-peptides have been reported [17–21]. For example, Rudra et al. reported that NFs composed of an antigenic epitope peptide conjugated to self-assembling peptide Q11 were subcutaneously administered, and elicited a strong antibody response [17]. They have also demonstrated that the β-sheet peptide NF system can be applied to various types of antigens, including a malaria epitope [18], a *Staphylococcus aureus* epitope [19] and a tumor-associated antigen MUC1 glycopeptide [20]. However, fundamental studies on how the hydrophilic-hydrophobic balance of NF components affects their cellular interaction—including cellular uptake, cytotoxicity, and immune stimulation response—has not been reported. Recently, studies on other particulate systems reported that surface hydrophobicity is an important factor for determining cellular response [22–32]. In addition to cellular internalization and nontoxicity (i.e., safety), the ability of nanocarriers to stimulate an immune response is an essential property in nanocarrier-based vaccine applications, because uptake of nanocarriers containing an antigen by APCs that do not induce an immune response may lead to unwanted tolerance toward the antigen. Thus, to design nanomaterial-based vaccines that elicit strong immunity without toxicity using a β-sheet assembly system requires a clear understanding of how the hydrophilic-hydrophobic balance of NF components affects their cellular interactions and response.

In previous work, we reported the preparation of antigen-loaded NFs by exploiting the self-assembly of β-sheet peptides [33,34] conjugated to antigenic peptides and hydrophilic chains, such as oligo-ethylene glycol (EG) [35–37]. MHC class I restricted epitope (SIINFEKL) from ovalbumin was selected as a model antigenic peptide. In addition, the structure of the NFs was analyzed in detail by various techniques, including wide-angle X-ray diffraction (WAXD), small-angle X-ray scattering (SAXS), Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Interestingly, structural analysis revealed that the shape of the NFs is rectangular, rather than a cylinder-like structure observed for filament micelles, possibly because of the lamination structures of β-sheets. Based on this finding, the structural model was proposed as shown in Figure 1b, which shows that the surface of the NFs is not covered with EG chains homogeneously [35,36]. Thus, we hypothesized that the EG chain length is an important parameter for tuning the cellular interactions of NFs, including cellular uptake, cytotoxicity, and immune stimulation response.

In this study, the effect of the EG chain length in building block molecules, which form peptide NFs, on their cellular interaction was investigated. The self-assembling behavior of three kinds of building block peptides with different EG lengths was evaluated by determining their critical aggregation concentration (CAC) and the critical concentration for nanofiber formation (CFC). The structures of the resulting NFs were analyzed by TEM and CD, and their surface hydrophobicity was evaluated using a hydrophobic fluorescence probe. Cellular uptake, cytotoxicity, and immune stimulation ability of the three kinds of NFs were examined in vitro using DCs. In addition, interaction of cells with micelle-like aggregates that were composed of the same building blocks as the NFs were also investigated. Cellular

interaction of the NFs was found to be significantly dependent on EG length, whereas that of micelles was independent of EG length. Notably, uptake by DC of NFs composed of EG with a moderate length was effective, and the NFs activated DC without exhibiting significant cytotoxicity. The findings provide useful design guidelines for the development of effective nanofiber-based vaccines.

**Figure 1.** (**a**) Design of the building block peptides (EG*n*) that are composed of a β-sheet-forming sequence (FVIFLD), a flexible-linker block (GSG), a model antigen sequence (SIINFEKL from OVA), and oligo-ethylene glycol. (**b**) Schematic illustration of the self-assembly process for nanofiber formation and the proposed model of highly antigen-loaded nanofibers based on previous structural study [36]. The schematic illustration was created by modification of Figures 1 and 2 of reference [35].

### **2. Results**
