4.2.3. Preparation of Fluorescence-Labeled Antigen-Loaded Peptide NFs

The HFIP-treated mixture of EG*<sup>n</sup>* and EG*n*-FAM peptides was dissolved in DMSO; then, 15 μL of the solution was added to 285 μL PBS to give final concentrations of 1.425 mM for EG*<sup>n</sup>* and 0.071 mM for EG*n*-FAM. The solution was incubated at 60 ◦C for 24 h, and then the resulting peptide nanofiber dispersion was dialyzed against PBS for 24 h using dialysis membrane (MWCO 8,000, GE Healthcare) to remove DMSO and free peptides. The length of NFs was controlled by filtration using a syringe filter with a pore size of 0.45 μM (GE Healthcare).

#### 4.2.4. Determination of Critical Aggregation Concentration

CAC was determined using the pyrene 1:3 method [56]. First, a saturated solution of pyrene was prepared by mixing an excess of pyrene with PBS, and using the supernatant to dissolve EG*<sup>n</sup>* peptides at a concentration of 150 mM (the stock solution). A concentration range of EG*<sup>n</sup>* peptides from 2.5 μM to 100 μM was then prepared using serial dilutions of the stock solution with the saturated solution of pyrene. The final concentration of pyrene was equal in each solution. The fluorescence emission of pyrene was monitored using a fluorescence spectrometer (RF5300 PC, Shimadzu Scientific Instruments, Kyoto, Japan) with an excitation wavelength of 335 nm at 37 ◦C. The ratio of the emission intensities at 376 nm and 392 nm were then plotted as a function of the EG*n* peptide concentration (log scale). The CAC was determined from an abrupt change in the slope of the plot using the least-squares fitting technique.

#### 4.2.5. ThT Assay

PBS containing 10 μM Thioflavin T (ThT) was dispensed into a 96-well plate. EG*<sup>n</sup>* peptides solution (6 mM) was prepared and added to the 96-well plate, giving a final concentration of 300 μM. ThT fluorescence intensities at 480 nm (excitation; 440 nm) were monitored using a Genios microplate reader (TECAN, Männedorf, Switzerland) at 37 ◦C.

#### 4.2.6. Measurement of Surface Hydrophobicity

The surface hydrophobicity of NFs in the solution was determined using an ANS fluorescent probe as previously reported [41,42]. A concentration range of EG*<sup>n</sup>* NFs in PBS from 9.4 μM to 200 μM was prepared. The nanofiber dispersion was mixed with the equivalent volume of PBS containing 20 μM ANS. The intensities of ANS fluorescence ranging from 400 nm to 600 nm (excitation; 370 nm) were monitored using a fluorescence spectrometer (RF5300 PC) at 37 ◦C.

#### 4.2.7. Cell Culture

JAWS II, a DC line derived from mouse bone marrow, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown in EMEM supplemented with 20% FBS, 5 ng/mL murine GM-CSF, and antibiotics at 37 ◦C, 5% CO2.

#### 4.2.8. Evaluation of Cellular Association of Peptide NFs

JAWS II cells were seeded into 12-well plates (2.5 <sup>×</sup> 105 per well) and cultured for 12 h at 37 ◦C in a humidified atmosphere (5% CO2). After 12 h, the cells were washed with PBS and serum-free culture medium. The fluorescence-labeled peptide NF dispersion was added gently to the cells followed by incubation for 2 h at 37 ◦C. Following incubation, the cells were washed with PBS and 0.2% trypan blue aqueous solution, which was used to quench the flourescence from surface-adsorbed NFs [57]. The cells were then detached using trypsin and subsequently analyzed by FCM (Guava EasyCyte Plus, Millipore, Burlington, MA, USA). As a comparison to peptide NFs, the cellular uptake of the building block peptides without heat treatment was investigated under the same conditions.
