4.2.11. Evaluation of Cytotoxicity of Peptide NFs

The cytotoxicity of peptide NFs was evaluated using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's instructions. Briefly, JAWS II cells were seeded into 96-well plates (1.0 <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 nanofiber dispersion was gently added to the cells followed by incubation for 24 h. The cells were washed with PBS three times and the medium was replaced with a solution containing 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), and 1-methoxy-5-methylphenazinium methylsulfate at a 10-fold dilution. Aftera2h incubation, the absorbance was measured at 420 nm using a plate reader (Multiskan JX, Thermo Fisher Scientific, Waltham, MA, USA). The relative cellular activity was calculated using the following equation:

$$\% \text{ relative cellular activity} = \frac{A\_{420\text{ nm}} \text{ (NFs} - \text{treated cells)} - A\_{420\text{ nm}} \text{ (blank)}}{A\_{420\text{ nm}} \text{ (untreated cells)} - A\_{420\text{ nm}} \text{ (blank)}} \times 100 \tag{1}$$

where *A*420 nm is the absorbance at 420 nm, *A*420 nm (untreated cells) is the absorbance at 420 nm after incubation in the absence of peptide NFs, and *A*420 nm (blank) is the absorbance of medium containing WST-8 reagent at 420 nm. As a comparison, the cytotoxicity of building block peptides without heat treatment was investigated in a similar manner.

#### *4.3. Other Characterizations*

TEM measurements were performed using a JEM-1200EX II (JEOL, Tokyo, Japan) with an acceleration voltage of 85 keV. The samples were negatively stained with 0.1% phosphotungstate. p-potentials of NFs were measured using a Micro-Electrophoresis Zeta Potential Analyzer Model 502 (Nihon Rufuto, Tokyo, Japan). DLS analysis was performed using a particle size analyzer (ELSZ-1000, Otsuka Electronics, Osaka, Japan) at 25 ◦C. The light source was a He-Ne laser (630 nm) set at an 1ngle of 45◦. Experimental data were analyzed using the marquardt provided by the manufacturer. CD spectra were measured using a J-720 spectropolarimeter (Jasco, Tokyo, Japan) at 25 ◦C. The data were obtained using a 0.1 cm path length cell at a scan speed of 20 nm/min.

#### **5. Conclusions**

This study showed that the hydrophilic-hydrophobic balance of antigen-loaded NFs significantly impacted on their cellular uptake, cytotoxicity, and DC stimulation ability, which differs noticeably from the results observed for micelles formed from the same components of NFs. Building blocks consisting of β-sheet-forming peptides conjugated with antigenic peptides and hydrophilic EG with different lengths (6-mer, 12-mer and 24-mer) were found to successfully form NFs with homogenous widths. The uptake of NFs consisting of EG with a moderate length (12-mer) by DC was effective, and these NFs activated DC without exhibiting significant cytotoxicity. Increasing the EG chain length significantly reduced the interactions with cells. Conversely, decreasing the EG chain length enhanced DC activation ability but increased toxicity and impaired water-dispersibility, resulting in low cellular uptake. Thus, since cell entry, cytotoxicity, and the immune stimulation ability of antigen-loaded NFs can be tuned by the length of the EG moiety, the antigen-loaded NFs have potential as NF-based vaccines that can be used without additional adjuvants. In order to achieve efficient immune response in vivo, the development of intracellular environment-responsive NFs is now in progress. We believe the findings obtained in this study contribute to the understanding of the interaction between the surface of one-dimensional assemblies and cells, and provide useful design guidelines for development of effective NF-based vaccines.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/15/ 3781/s1.

**Author Contributions:** T.W. conceived and designed the experiments; S.N., S.K. (Sayaka Koeda), Y.K., and K.K. performed the experiments; all members discussed the experimental data; T.W. wrote the paper.

**Funding:** This work was partly supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number 16K01391.

**Acknowledgments:** We thank the Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. The authors would like to thank Kaeko Kamei at the Faculty of Molecular Chemistry and Engineering of Kyoto Institute of Technology for technical assistance with the ELISA assay. Also, we thank Kensuke Naka and Hiroaki Imoto at the Faculty of Molecular Chemistry and Engineering of Kyoto Institute of Technology for technical assistance with the DLS measurement.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
