*2.1. Self-Assembly Behavior of EGn Peptides*

We have reported previously the preparation of NFs in aqueous solution by heat-treatment of the EG12 peptide (EG*n*, where *n* is the length of the EG chain), which is composed of a β-sheet-forming sequence, an antigen sequence, and a hydrophilic oligo-ethylene glycol (12-mer). In this study, three building block peptide amphiphiles with different EG lengths (6-mer, 12-mer, and 24-mer, which are termed EG6, EG12, and EG24, respectively) were prepared for assessing the effect of EG length on cellular uptake of NFs, cytotoxicity, and immune stimulatory activities.

Initially, we investigated the effect of EG length on the self-assembly behavior of these peptides by estimating the CFC of each peptide. EG*n* peptides were incubated in the presence of the thioflavin T (ThT) dye at 300 μM in phosphate buffered saline (PBS) containing 5% dimethylsulfoxide (DMSO) at 37 ◦C for 24 h, and a time course of change in ThT fluorescence was measured. The ThT assay is often used to monitor the growth of amyloid-like nanofibers from their component peptides or proteins. A remarkable increase in ThT fluorescence was observed, indicating the formation of NFs by each peptide (Figure 2 and Figure S1). After a certain period of time, the intensity of the ThT fluorescence reached a plateau value. The systems were in a dynamic equilibrium between fibrils and the peptide monomer when the ThT fluorescence intensity reached a plateau value. The concentration of the free peptide monomer at equilibrium corresponds to the CFC [38,39]. The peptide concentration in the supernatant following ultracentrifugation of the EG*<sup>n</sup>* peptide solutions incubated at 300 μM and 37 ◦C for 24 h provided estimates of the CFC values, which were 96.0 ± 3.6 μM for EG6, 72.4 ± 3.7 μM for EG12, and 83.8 ± 1.0 μM for EG24.

**Figure 2.** Time-dependent change in the ThT fluorescence intensity of solutions containing the (**a**) EG6 peptide, (**b**) EG12 peptide, and (**c**) EG24 peptide when incubated at 37 ◦C.

Below the CFC, EG*n* peptides could either form spherical micelle-like structures or exist as isolated molecules in water because of their amphiphilic structures [40]. The CAC was determined for EG*<sup>n</sup>* peptides in PBS (pH 7.4) using the pyrene 1:3 method to gain information on the association state of EG*<sup>n</sup>* peptides at relatively low concentrations. The CAC was estimated to be 16.6 μM for EG6, 21.7 μM for EG12, and 29.9 μM for EG24 (Figure S2). The CAC value increased as the EG length increased. Because CAC values were smaller than the CFC values, EG*<sup>n</sup>* peptides self-assembled into spherical micelle-like structures over the concentration range between the CAC and the CFC, and existed as monomers at concentrations below the CAC.
