4.2.1. Synthesis of Building Block Molecules

Loading of resin: Fmoc-*N*-amido-dPEG12 was dehydrated by azeotropy with benzene prior to use. A solution of Fmoc-*N*-amido-dPEG12 acid (0.238 mmol) and DIPEA (0.952 mmol) in CH2Cl2 (2.6 mL) was added to 2-chlorotrityl chloride resin (0.397 mmol, 1.5 mmol/g loading max) for 12 h.

Peptide synthesis: Coupling reactions were performed using a standard Fmoc protocol. The coupling cycle included 3 repeats of Fmoc deprotection (20% piperidine in DMF, 5 min), a wash in DMF, two repeats of amino acid coupling: L-Fmoc amino acids (4 eq.), HBTU (3.6 eq.), HOBt (4 eq.), and DIPEA (8 eq.) for 30 min, and a final DMF wash. After all coupling reactions, the obtained peptides were cleaved from the resin using a solution of H2O/TFA/triisopropylsilane (100:5:2 volume ratio) containing 500 mM phenol for 2 h. The resulting peptides were precipitated in ice cold diethyl ether, filtered, centrifuged, and washed with diethyl ether. The crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC, SPD-10A and LC-10A, Shimadzu Scientific Instruments, Kyoto, Japan). Other building blocks with different EG lengths were synthesized by a similar procedure. Molecular weight was analyzed by MALDI-TOF mass (autoflex speed system, Bruker, Billerica, MA, USA). MS (MALDI-TOF): EG6; Cald. MASS: 2234.92, Obsd. MASS: 2234.359, EG12; Cald. MASS: 2499.12, Obsd. MASS: 2498.11, EG24; Cald. MASS: 3027.52, Obsd. MASS: 3027.04.

Fluorescence-labeled building block peptides were synthesized using Fmoc-Lys(5-FAM)-OH as the first amino acid residue by a similar procedure. MS (MALDI-TOF): EG6-FAM; Cald. MASS: 2721.41, Obsd. MASS: 2720.84, EG12-FAM; Cald. MASS: 2985.61, Obsd. MASS: 2985.26, EG24-FAM; Cald. MASS: 3514.01, Obsd. MASS: 3514.65.

#### 4.2.2. Preparation of Antigen-Loaded Peptide NFs

EG*<sup>n</sup>* peptide was dissolved in HFIP and dried with nitrogen flow to allow film formation. The obtained film was re-dissolved at a concentration of 30 mM in DMSO. The resulting solution (15 μL) was added to PBS (285 μL) to a final concentration of 1.5 mM and incubated at 60 ◦C for 24 h. Following incubation, the resulting peptide nanofiber dispersion was dialyzed against PBS for 24 h using dialysis membrane (MWCO 8,000, GE Healthcare, Chicago, IL, USA) to remove DMSO and free peptides. For cell-based experiments (cytotoxicity, DC maturation), the length of NFs was controlled by filtration using a syringe filter with a pore size of 0.45 μM (GE Healthcare).
