*2.2. Structural Characterization of EGn Nanofibers*

NFs consisting of EG*<sup>n</sup>* peptides were prepared by incubation of the peptide solution in PBS containing 5% DMSO at 60 ◦C for 24 h. The incubation was carried out at a higher temperature in this experiment when compared with that of the ThT assay to accelerate NF formation. The resulting NFs were characterized by TEM, CD, ς-potential measurements, and the 8-anilino-1-naphtalene sulfonic acid (ANS) assay.

TEM images revealed that all peptides successfully formed NFs with a homogenous distinct width of ca. 6–8 nm and lengths of several micrometers (Figure 3). The values of the ς-potentials were −33.2 ± 10.7 mV for EG6 NFs, −33.2 ± 10.7 mV for EG12 NFs, and −32.4 ± 9.6 mV for EG24 NFs, showing that the surface of the NFs were negatively charged. The ANS assay was performed to obtain information on the surface hydrophobicity of the NFs [41,42]. The peaks shifted toward shorter wavelengths, and the intensities of the signals increased significantly for all NFs (Figure S3). These observations indicate that there are hydrophobic domains on the surface of the peptide NFs. CD was

used to examine the secondary structures adopted by peptides in the NFs. The characteristic negative Cotton peak at 217 nm was observed for the three EG*<sup>n</sup>* NFs, showing that the peptides adopt a β-sheet conformation (Figure 4) [43]. These results indicate that the three NFs possess a similar secondary structure regardless of EG length.

**Figure 3.** Negatively-stained TEM images of the nanofibers (NFs) obtained by incubation of the (**a**) EG6 peptide, (**b**) EG12 peptide, or (**c**) EG24 peptide at a concentration of 300 μM for 24 h in PBS at 60 ◦C.

**Figure 4.** CD spectra of EG*n* NFs measured in PBS at room temperature. The EG*n* NFs were prepared by incubation of EG*<sup>n</sup>* peptide solutions in PBS at 60 ◦C for 24 h. (**a**) EG6 NFs, (**b**) EG12 NFs, and (**c**) EG24 NFs.

*2.3. Cellular Uptake, Cytotoxicity, and Maturation of DCs*

We investigated the effect of the EG length of nanofibers on their cell association, cytotoxicity, and DC stimulatory activities. The peptide NFs obtained by incubation at a concentration of 1.5 mM

and 60 ◦C for 24 h were used. The length of the NFs was controlled to be 230–260 nm by an extrusion procedure using a membrane filter with a diameter of 450 nm (Figures S4 and S5). Information about the dispersion state of NFs in water was determined by dynamic light scattering (DLS) measurements of EG6 NFs, EG12 NFs, and EG24 NFs. The DLS histograms for EG12 and EG24 NFs exhibited a unimodal peak, with average diameters of 203.7 ± 119.7 nm for EG12 NFs, and 116.6 ± 68.2 nm for EG24 NFs. These values are inconsistent with the size estimates from TEM images and this is possibly because of their non-spherical morphology. Nonetheless, these unimodal histograms clearly indicate that EG12 and EG24 NFs exist as isolated NFs without aggregation in aqueous media (Figure S6). In contrast, the DLS histogram for EG6 NFs exhibited two peaks with sizes of 135.4 ± 14.5 nm and 3498.3 ± 389.0 nm, indicating that EG6 NFs formed large aggregates. The secondary aggregation of the NFs may be caused by association of surface-exposed hydrophobic domains on the NFs. Longer EG chains effectively prevent these interactions, yielding highly dispersed, stable EG12 and EG24 NFs. Conversely, the 6-mer EG is not sufficiently long to prevent secondary aggregation of the peptide NFs, resulting in the observed large aggregates. To compare the behaviors of NFs, the cellular uptake, cytotoxicity, and DC stimulatory activities of non-heat-treated EG*<sup>n</sup>* peptides (non-fiber) were also evaluated. The samples for these experiments were prepared by direct dissolution of the peptides in medium at a given concentration to avoid self-assembly into NFs.

#### 2.3.1. Cellular Association

We evaluated the effect of EG length on the cellular association of EG*<sup>n</sup>* NFs. The fluorescence-labeled EG*<sup>n</sup>* NFs were incubated with JAWS II cells for 1 h at 37 ◦C. JAWS II cells are an immortalized immature DC line that was established from bone marrow cultures of C57BL/6 p53-knockout mice [44,45]. The amount of NFs associated with the cells was evaluated by flow cytometry (FCM). For comparison, cellular association of non-heat-treated EG*n* peptides was also performed.

As shown in Figure 5a, the intensity of fluorescence signals from cells incubated with EG*n* peptides (non-fiber) increased as the concentration of the peptides increased. This trend was common to all peptides examined. Comparison of the fluorescence intensity of the three EG*<sup>n</sup>* peptides at the same concentration revealed that they were very similar, indicating that EG length had no effect on cellular association of the peptides. In contrast, cellular association of EG*<sup>n</sup>* NFs was influenced noticeably by EG length (Figure 5b). The amount of associated EG*<sup>n</sup>* NFs was larger as the EG length decreased. This trend was more apparent as the concentration of the peptide increased. This result indicates that longer EG chains may prevent interactions between cells and NFs.

**Figure 5.** Evaluation of the cellular association of various (**a**) EG*n* peptides and (**b**) EG*n* NFs labeled with fluorescein using FCM. Mean fluorescence intensity of the treated JAWS II cells is shown. Cellular treatment was performed by incubating cells with peptides or NFs in serum-free medium at 37 ◦C for 2 h. Each point is the mean ± SD (*n* = 3). \* *p* < 0.01, \*\* *p* < 0.01 compared to untreated cell.

We performed confocal laser scanning microscopic (CLSM) observations of JAWS II cells incubated with various EG*<sup>n</sup>* NFs to evaluate the association of NFs in further detail (Figure 6). The CLSM images of cells incubated with EG12 NFs clearly show that the NFs were internalized into cells. The fluorescence signals were observed as dot-like images, indicating that EG12 NFs were internalized via endocytosis. In contrast, EG24 NFs showed no fluorescence signal, indicating poor cellular uptake of EG24 NFs. The confocal images of EG6 NFs-treated cells showed large intensive fluorescence signals on the surface of cells, indicating that some aggregation of NFs were apparently adsorbed onto the surface of cells. This observation indicates that a large proportion of the fluorescence signal from EG6 NFs-treated cells observed in FCM measurements was derived from NFs that had adhered to the surface of cells. The FCM and CLSM results comprehensively showed the efficient uptake of EG12 NFs by cells.

**Figure 6.** CLSM images of JAWS II cells treated with various EG*n* NFs labeled with fluorescein. JAWS II cells were incubated with EG*n* NFs at 37 ◦C for 2 h in serum-free medium. After incubation, the cells were treated with Lyso Tracker Red and Hoechst for staining intracellular acidic compartments and nuclei, respectively. Scale bars represent 20 μM.

#### 2.3.2. Cytotoxicity

The cytotoxicity of EG*n* NFs and EG*n* peptides (non-fiber) were evaluated. The JAWS II cells were incubated with the EG*<sup>n</sup>* NFs or EG*<sup>n</sup>* peptides at 37 ◦C for 24 h at different concentrations (10–50 μM), and cell activity was evaluated. Interestingly, cell activity after incubation with all EG*<sup>n</sup>* peptides increased as the peptide concentration increased (Figure 7a). Relative cell activity was essentially 100%, independent of the EG length when peptides were co-incubated with cells at higher concentrations (40–50 μM). Co-incubation of peptides with cells at lower concentrations (10–20 μM) reduced cell activity to 70–90%. In addition, peptides with shorter EG chains showed relatively higher toxicity.

**Figure 7.** Evaluation of the cytotoxicity of EG*n* peptides (**a**) and EG*n* NFs (**b**) against JAWS II cells.

In contrast, cell activity was observed to decrease after incubation with each NF, and this reduction in cell activity was concentration-dependent (Figure 7b). In particular, the activity of EG6 NFs-treated cells was reduced to 40% at a concentration of 50 μM. For EG12 and EG24 NFs, cell activity was 70% for EG12 NFs and 80% for EG24 NFs even at a NFs concentration of 50 μM. Thus, NFs with shorter EG chains exhibited higher toxicity.

#### 2.3.3. DC Stimulatory Activity.

It is well known that DC maturation is accompanied by enhanced expression of co-stimulatory molecules (CD40, CD80, and CD86) and by an increase in the secretion of immune-stimulatory cytokines (IL-6, IL-10, IL-12, and TNF-α) [46]. We measured the amount of expressed co-stimulatory molecules (CD86) on JAWS II cells cultured in the presence of EG*<sup>n</sup>* NFs or EG*<sup>n</sup>* peptides for 24 h to determine the effect of EG length on DC maturation. As a positive control, the expression of CD86 on lipopolysaccharides (LPS)-stimulated JAWS II was also measured. The expression of CD86 on JAWS II cells after incubation with EG*<sup>n</sup>* peptides is shown in Figure 8. Even at a peptide concentration of 50 μM, the expression level of CD86 was almost the same as that of untreated JAWS II cells. These results indicate that EG*<sup>n</sup>* peptides do not stimulate DC. In contrast, co-incubation of EG*<sup>n</sup>* NFs with JAWS II cells significantly enhanced the expression of CD86, and this enhancement was dependent on the concentration of the NFs. In particular, when EG12 NFs and EG6 NFs were co-incubated with JAWS II cells, the amount of expressed CD86 was comparable to or larger than that on LPS-stimulated DC.

We also evaluated secretion of immune-stimulatory cytokines. Using enzyme-linked immunosorbent assay (ELISA) methods, we measured the amount of TNF-α and IL-6 contained in the supernatant after 24 h culturing of JAWS II in the presence of EG*n* NFs or EG*n* peptides (Figure 9). Co-incubation with EG*<sup>n</sup>* peptides did not alter the secretion levels of TNF-α and IL-6. In contrast,

interestingly, the secretion of TNF-α and IL-6 was drastically enhanced by co-incubation with EG6 NFs and EG12 NFs, but not by EG24 NFs. These results indicate that NFs with relatively short EG chains have an immune-stimulatory effect as adjuvants for DC maturation.

**Figure 8.** Evaluation of CD86 expressed on the surface of JAWS II cells co-incubated with EG*n* NFs (10–50 <sup>μ</sup>M), EG*<sup>n</sup>* peptides (10–50 <sup>μ</sup>M), the OVA peptide (SIINFEKL, 10–50 <sup>μ</sup>M), or 1 <sup>μ</sup>g·mL−<sup>1</sup> LPS at 37 ◦C for 24 h. CD86 expression was analyzed by FCM. Each result is the mean ± SD (*n* = 3). \* *p* < 0.01. a.u. represents arbitrary unit.

**Figure 9.** Quantification of immune-stimulatory cytokines secreted from JAWS II cells co-incubated with 10 <sup>μ</sup>M EG*<sup>n</sup>* NFs, 10 <sup>μ</sup>M EG*<sup>n</sup>* peptides, 10 <sup>μ</sup>M OVA peptide (SIINFEKL), or 1 <sup>μ</sup>g·mL−<sup>1</sup> LPS at 37 ◦<sup>C</sup> for 24 h. The cytokine levels (**a**,**b**) were measured by ELISA. Each result is the mean ± SD (*n* = 3). \* *p* < 0.01.

#### **3. Discussion**

In this study, we have investigated cellular uptake, cytotoxicity, and DC stimulatory activity of antigen-loaded peptide NFs with different EG lengths and their component peptides. Three building block peptide amphiphiles with different EG lengths (6-mer, 12-mer and 24-mer) were prepared. ThT assay, TEM observation, and CD measurement revealed that all type of peptide amphiphiles are successfully formed β-sheet rich nanofibers with distinct widths (Figures 2–4). The association state of EG*<sup>n</sup>* peptides was dependent on sample concentration. EG*<sup>n</sup>* peptides self-assembled into NFs above the CFC, formed spherical micelles at concentrations between the CFC and CAC, and existed as monomers in solution below the CAC. Based on these findings, we discuss separately the effect of EG length on cellular uptake, cytotoxicity, and immune stimulation for three peptide states: NFs, micelles, and monomers.

#### *3.1. E*ff*ect of EG Length of Nanofibers on Their Cellular Uptake, Cytotoxicity, and Immune Stimulation Ability*

The EG length of NFs significantly affected their cellular uptake, cytotoxicity, and DC stimulatory activity. Here, we discuss the effect of EG length on these properties of NFs using structural models derived from SAXS, WAXD, CD, and FT-IR data of a previous study [36]. FT-IR, CD, and WAXD results indicate that EG12 NFs contain β-sheet structures. In addition, synchrotron X-ray scattering profiles of EG12 NFs revealed that the morphology of the NFs is rectangular, and they do not form cylinder structures like filament micelles, presumably because of the laminated structure of β-sheets. In general, amyloid-like nanofibers have a common characteristic cross-β-sheet structure, where tightly packed β-sheets orientate themselves perpendicularly to the fiber elongation axis [47]. By combining these findings, we propose a model of EG*<sup>n</sup>* NFs (Figure S7). β-sheet structures consisting mainly of hydrophobic amino acids form the framework of NFs with EG chains facing outwards to provide water-dispersibility. The surfaces of NFs possess hydrophobic and hydrophilic domains that consist of EG chains based on this model. The ANS assay results support the notion that there are hydrophobic domains on the surface of NFs.

#### 3.1.1. Cellular Association and Internalization of NFs

The amount of NFs associated with cells increased in the order of EG24 NFs, EG12 NFs, and EG6 NFs (Figure 5b). Surface hydrophobicity of nanomaterials has been well documented to affect cellular association and uptake by phagocytic cells [24,30–32]. Surface hydrophobicity of nanomaterials facilitates interactions between nanomaterial surfaces and cellular membranes. This may lead to higher cell association of nanomaterials and occasionally increase the chance of recognition by particular receptors involved in cellular uptake. Our results show that cellular association of NFs decreased as the EG chain length increased (Figure 5b), although these NFs commonly possess hydrophobic domains on their surface, as evidenced by the ANS fluorescence assay. These results suggest that longer EG chains inhibit hydrophobic interactions between the NF surface and cell membranes, which can be explained using the model structures presented in Figure 9. The NF skeleton region, consisting mainly of hydrophobic amino acids, may facilitate the interaction with the cell membrane and the EG chain located on the lateral face of the NF may inhibit this interaction.

Results from CLSM observation revealed that EG12 NFs were more efficiently internalized by JAWS cells than EG6 NFs and EG24 NFs (Figure 6). Since the surface of EG12 NFs is negatively charged, the mechanism for internalization of EG12 NFs would be mainly via phagocytosis by scavenger receptor, which recognizes anion species, although further studies using some inhibitor for phagocytosis are required. Thus, the internalization behavior by non-phagocytic cells would be different from that by JAWS II cells. The internalization of EG6 NFs was low, whereas their association propensity to cells was high. Because the size of nanomaterials can affect cell internalization [48–51], the dispersion state of NFs in aqueous media should be considered in addition to interactions between NFs and the cell surface. The results from DLS indicate that EG12 and EG24 NFs exist as isolated NFs without

aggregation in aqueous media, whereas EG6 NFs form large aggregates. This observation is consistent with CLSM images showing large aggregates adsorbed onto the cell surfaces. Thus, it is likely that the low efficiency of cellular internalization of EG6 NFs can be attributed to their apparent size in water. The aggregation of EG6 NFs is too large for cell uptake. This interpretation is consistent with a previous study that showed that cellular uptake of microparticles with a diameter of a few micrometers or more by phagocytic cells is slow and inefficient [50,51]. Thus, for development of NFs that are efficiently taken up by cells, it is important to design a EG chain length that allows modest interactions with cell membranes while ensuring water-dispersibility.

#### 3.1.2. Cytotoxicity of NFs

Generally, nanomaterials with cationic or hydrophobic surfaces can induce significantly higher toxicities when compared with hydrophilic or anionic nanomaterials [29]. A mechanism of cytotoxicity is cell membrane perturbation, including structural alternation, pore formation, and phase transitions, which cause nonspecific entrance of extracellular components to the cytosol. An increase in hydrophobic interactions between the surface of nanomaterials and cell membranes could perturb the membrane. In the present study, the cytotoxicity of EG*<sup>n</sup>* NFs was found to increase in the order of EG24, EG12, and EG6 (Figure 7). These results indicate that longer EG chains inhibit the interaction between NFs and cell membranes, which leads to lower cytotoxicity of NFs with long EG chains. It is also possible that the cytotoxicity of NFs may be related to biological stress, e.g., induction of reactive oxygen species (ROS). The detailed mechanism of cytotoxicity by NFs is the subject of ongoing research.

### 3.1.3. DC Stimulation Ability of NFs

DC activation ability of EG6 NFs and EG12 NFs was much higher than that of EG24 NFs. Matzinger and colleagues have proposed that hydrophobic portions in various biomolecules may be involved in the activation of the immune system [22]. Hydrophobic components in molecules are usually masked from the external environment by hydrophilic components. However, when protein denaturation or cell disruption occur, these hydrophobic components become exposed and interact with particular surface receptors of immune cells, which activates the immune system. In agreement with the notion proposed by Matzinger, recently, the relationship between the surface hydrophobicity of nanomaterials and their immune stimulatory activities has been reported [23–28]. For example, Moyano and colleagues reported that the surface hydrophobicity of ligand-modified gold-nanoparticles was correlated with expression of pro-inflammatory cytokine genes in splenocytes from mice in vitro [23]. Shima and colleagues also reported that the activation ability of nanoparticles was significantly affected by the hydrophobicity of polymers constituting the nanoparticles [24]. In the present study, EG6 NFs and EG12 NFs stimulated DC maturation more effectively than EG24 NFs, as evidenced by the quantitative evaluation of expressed co-stimulatory molecules (Figure 8) and secreted immune-stimulatory cytokines, IL-6 and TNF-α (Figure 9). Based on these results, it is reasonable to consider that the hydrophobic part of NFs plays an important role in DC activation. Longer EG chains seem to inhibit the recognition of hydrophobic surfaces of NFs by DC surface receptors in a similar manner to that described above. However the mechanisms responsible for DC maturation by EG*<sup>n</sup>* NFs remain unclear and further studies are required. In addition, because IL-6 signaling cannot only promote anti-tumor-adaptive immunity, but also drive malignancy [52], the role of IL-6 in this NFs-based vaccine system should be examined further in vivo.

#### *3.2. Cellular Uptake, Toxicity, and DC Stimulatory Ability of Micelles*

In the concentration range where EG*<sup>n</sup>* peptides form micelle-like structures, their cellular association, cytotoxicity, and stimulation ability were not dependent on EG length (Figure 5a, Figure 7a, and Figure 8). These results suggest that the surface components of the micelle-like structures would be almost the same. Poly(ethylene glycol) (PEG) interactions with biological components, including cellular membranes and proteins, are weak because of their nonionic hydrophilicity and

high mobility [53]. Thus, a low-level of interaction between the surface of the micelle-like structures and cell membrane components, including receptors involved in cellular uptake and DC maturation, led to lower uptake by DC, lower cytotoxicity, and no DC activation in comparison with NFs.
