Self-assembled or mixed peptide amphiphile micelles from Herpes simplex virus glycoproteins as potential immunomodulatory treatment

Figures & data

Figure 1 Amino acid sequences of gB_498–505 and gD_301–309 peptide epitopes, the schematic representation of the corresponding peptide amphiphilic derivatives and of (C18)₂-L adjuvant, and their molecular weights (Mw).

Note: The amino acid sequences are reported using the three-letter abbreviation.

Abbreviations: AdOO, 8-amino-3,6-dioxaoctanoic acid; Ala, alanine; Arg, arginine; Asp, aspartate; Glu, glutamic acid; Gly, glycine; Ile, isoleucine; Leu, leucine; Phe, phenylalanine; Pro, proline, Ser, serine; Val, valine.

Table 1 List of the investigated aggregated systems, with the ratio (R) between the amphiphilic monomers, and their structural properties

Aggregated system	R	CMC (mol ⋅ kg^−1)	D ⋅ 10^−12 (m^2 ⋅ s^−1) ± SD	RH (nm) ± SD	ζ (mV) ± SD
(C18)2-L-gB498–505	100%	3.0 ⋅ 10^−7	3.7±1.8	67±32	−37±9.3
(C18)2-L-gD301–309	100%	4.3 ⋅ 10^−7	3.6±1.4	68±27	−38±8.5
(C18)2-L-gB498–505/(C18)2-L-gD301–309	50/50	4.3 ⋅ 10^−7	3.2±1.6	78±40	−47±6.4
(C18)2-L	100%	2.5 ⋅ 10^−6	3.5±1.1	70±22	−16±4.8
(C18)2-L/(C18)2-L-gB498–505	80/20	4.1 ⋅ 10^−7	3.3±1.1	74±25	−35±6.4
(C18)2-L/(C18)2-L-gB498–505	50/50	6.8 ⋅ 10^−6	5.0±1.5	49±14	−32±6.8

Abbreviations: ζ, zeta potential; CMC, critical micellar concentration values; D, diffusion coefficients; R_H, hydrodynamic radii; SD, standard deviation.

Figure 2 Structural characterization of the aggregates, by fluorescence and dynamic light scattering.

Notes: (A) Fluorescence intensity of the ANS fluorophore at 480 nm as a function of PAs concentration: (C18)₂-L-gB_498–505 (□), (C18)₂-L-gD_301–309 (▲), and (C18)₂-L-gB_498–505/(C18)₂-L-gD_301–309 (•). CMC values were established from the graphical break point; (B) DLS spectra of (C18)₂-L-gB_498–505/(C18)₂-L-gD_301–309 mixed aggregates at 25°C and 1 × 10⁻⁴ M concentration.

Abbreviations: ANS, 8-anilino-1-naphthalene sulfonic acid ammonium salt; CMC, critical micelle concentration; DLS, dynamic light scattering; PA, peptide amphiphile.

Figure 3 Far-UV CD spectra of: (A) gD_301–309 peptide at 0, 40%, and 80% of TFE in TRIS buffer solution, after subtraction of gD_301–309 spectrum at 0% TFE; similar spectra were observed for the gB_498–505 peptide; (B) pure and mixed (50/50) amphiphilic epitopes at 2 × 10⁻⁴ M concentration, well above the CMC values to ensure the presence of aggregates in solution.

Abbreviations: CMC, critical micelle concentration; MRE, mean residue ellipticity; TFE, trifluoroethanol; TRIS, tris(hydroxymethyl)aminomethane; UV-CD, ultraviolet circular dichroism.

Figure 4 Effects of PAs (gB, gD, gB/gD) and PA-micelles on cell viability.

Notes: Cell viability was evaluated by MTT assay at 24 hours posttreatment in U937 (A) and RAW 264.7 macrophages (B). Cisplatin (20 μM) was used as a positive control of cell death. Values are expressed as the mean ± SD of three independent experiments.

Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PA, peptide amphiphile; SD, standard deviation.

Figure 5 IL-17- and IL-23-release after treatment with 10 μM of each peptide and peptide-micelles for 24 hours.

Notes: LPS was used as a positive control. The results shown are the average of three independent experiments, and the error bars indicate the standard errors of the means. *P≤0.01 indicates statistically significant difference between untreated cells versus single/mixed peptide pretreated cells (Student’s t-test).

Abbreviations: IL, interleukin; LPS, lipopolysaccharide.

Figure 6 IL-6-, IL-8- or MIP-2-, and TNF-α-release after treatment with 10 μM of each peptide and peptide-micelles, for 24 hours.

Notes: The results shown are the average of three independent experiments, and the error bars indicate the standard errors of the means. *P≤0.01 indicates a statistically significant difference between untreated cells versus single/mixed peptides pretreated cells (Student’s t-test).

Abbreviations: IL, interleukin; MIP, macrophage inflammatory protein; TNF, tumor necrosis factor.