*3.4. Design of NF-based Vaccines*

Recently, various types of NFs for immunotherapy have been reported [54,55]. In particular, NFs formed from antigenic peptides conjugating to β-sheet-forming peptides have been recognized as very promising candidates for next-generation nanoparticle-based vaccines. In the present study, we demonstrated that the hydrophilic-hydrophobic balance of peptide NFs affects their cellular uptake, cytotoxicity, and DC activation ability. NFs consisting of EG with a moderate length (12-mer) showed the most balanced character: Highly efficient cell entry, low cytotoxicity, and high DC activation ability, indicating that the NFs have significant potential as NF-based vaccines, which can be used without additional adjuvants. In general, the relationship between toxicity and DC stimulation ability is a trade-off. It is important to improve the stimulation ability, while simultaneously reducing the cytotoxicity of the NFs. Our results demonstrate that such balance can be simply tuned by the length of the EG. This feature is important for designing safe NF-based vaccines with high immune stimulatory ability. In contrast to NF uptake, the uptake of micelles and monomeric peptides by DC cells inefficient and showed no DC stimulation ability independent of EG length. This result indicates that the assembly style of building block peptide molecules influences the properties of the nanoassembly formed from these building blocks. Finally, to develop NFs with strong immunity-inducing ability, it is necessary to precisely adjust the EG length and introduce intracellular environment-responsive links for efficient release of antigens in cells.

Although we focused on the effect of hydrophilic and hydrophobic balance of nanofibers on their interaction with cells, surface charge of nanofibers is also an important factor in determining the interaction. In general, positively charged nanomaterials are more effectively internalized to cells than negatively charged ones, but they are more toxic. Thus, for design of nanofiber vaccines, it is necessary to address the role of their surface charge. In addition to surface charge, the length of NFs is also an important factor determining their property as a nano-vaccine. In a previous study, we investigated the effect of nanofiber length on their cellular uptake using various NFs with different lengths (40 nm, 120 nm, 280 nm, 800 nm). The study demonstrated that nanofibers with a length of 280 nm were most effectively uptaken by phagocytic cells compared to the others (unpublished data). Based on this finding, we used NFs with a length of 230–260 nm for the cell experiments in the present study. However, other properties—e.g., cytotoxicity and the ability to stimulate immune cells etc.—could exhibit different size-dependencies. Therefore, the optimization of nanofiber length is also required for developing effective nanofiber vaccine.

The important attributes of a vaccine, which are antigen processing, antigen presentation, T-cell stimulation, and successful activation of adaptive immune response against target antigen, should also be evaluated. However, because the NFs used in this study comprised the minimum required block (β-sheet forming peptide, antigenic peptide, oligo(ethylene glycol)) and the antigen could not be released in the cells, the effective antigen presentation via MHC class I pathway and subsequent induction of immunity are not expected. Therefore, we are addressing the development of intracellular environment-responsive NFs for efficient release of antigens in cells and the characterization of their function to induce immunity in vivo.

### **4. Materials and Methods**
