Hair dye-incorporated poly-γ-glutamic acid/glycol chitosan nanoparticles based on ion-complex formation

Figures & data

Figure 1 Schematic illustrations of PDA-incorporated nanoparticles based on ion-complex formation between PDA and PGA. Weight ratio of PDA/PGA was 20/50 (mg/mg). The opaque solution of PGA was readily changed to a transparent solution by addition of PDA.

Abbreviations: PDA, p-phenylenediamine; PGA, poly(γ-glutamic acid).

Figure 2 Typical particle size (A) and SEM photo (B) of PDA-incorporated PGA nanoparticles.

Note: Composition of PDA/PGA is PDA5 in Table 1.

Abbreviations: PDA, p-phenylenediamine; PGA, poly(γ-glutamic acid); SEM, scanning electron microscopy.

Figure 3 FT-IR analysis of PDA-incorporated nanoparticles.

Notes: PDA and PGA were used as a solid powder, and lyophilized PDA-incorporated nanoparticles (PDA5 in Table 1) were used for FT-IR measurement. Carboxylic groups of PGA were decreased by complexation with PDA at PDA5 measurement.

Abbreviations: FT-IR, Fourier-transform infrared; PDA, p-phenylenediamine; PGA, poly(γ-glutamic acid).

Table 1 Characterization of PDA-incorporated submicroparticles of PGA/GC complexes

Formulation no.	Weight ratio of PGA/PDA/GC (mg)a	Drug contents (%, w/w)	Particle size (nm)			Statusb
			Intensity average	Weight average	Number average
PDA1	50:5:5	8.3	253.8 ± 108.5	242.3 ± 78.5	230.2 ± 61.8	+
PDA2	50:10:5	15.4	336.2.4 ± 105.5	313.4 ± 90.3	280.9 ± 81.2	+
PDA3	50:20:5	26.7	554.3 ± 130.5	536.4 ± 121.2	465.7 ± 110.8	++
PDA4	50:40:5	42.1	689.6 ± 121.4	680.0 ± 115.8	523.5 ± 98.7	++
PDA5	50:10:0	16.7	121.3 ± 60.9	117.9 ± 58.7	76.9 ± 42.0	−
PDA6	50:10:5	15.4	312.5 ± 103.7	290.6 ± 93.6	262.4 ± 63.7	+
PDA7	50:10:10	14.3	320.7 ± 96.7	298.9 ± 102.8	278.6 ± 82.6	++
PDA8	50:10:20	12.5	328.7 ± 150.9	295.6 ± 121.3	281.8 ± 102.9	+++

a Notes: Final volume of nanoparticle solution was adjusted to 10 mL;

b fine distribution (+, small amount of aggregates; ++, moderate amount of aggregates; +++, significant amount of aggregates or precipitation).

Abbreviations: GC, glycol chitosan; PDA, p-phenylenediamine; PGA, poly(γ-glutamic acid).

Figure 4 Photos of the PDA-incorporated nanoparticles. Photo in top-left box is of ion complexes of PDA-incorporated PGA nanoparticles: PDA/PGA weight ratio (w/w) (a) 50/5; (b) 50/10; (c) 50/20; and (d) 50/40. The top-right box is of nanoparticles after addition of GC, and the bottom photo is of PDA-incorporated nanoparticles.

Note: The composition of PDA/PGA/GC is illustrated in Table 1.

Abbreviations: GC, glycol chitosan; PDA, p-phenylenediamine; PGA, poly(γ-glutamic acid).

Figure 5 FT-IR analysis of PDA-incorporated nanoparticles, showing the effect of PDA content on the chemical properties of nanoparticles.

Notes: The composition of PDA/PGA/GC is illustrated in Table 1. PDA, PGA, and GC were used as a solid powder, and lyophilized PDA-incorporated nanoparticles (PDA1–5 in Table 1) were used for FT-IR measurement. As PDA content increased, the carboxylic acid peak of PGA gradually decreased.

Abbreviations: GC, glycol chitosan; FT-IR, Fourier-transform infrared; PDA, p-phenylenediamine; PGA, poly(γ-glutamic acid).

Figure 6 FT-IR analysis of PDA-incorporated nanoparticles showing the effect of GC addition on the chemical properties of nanoparticles.

Notes: The composition of PDA/PGA/GC is illustrated in Table 1. GC was used as a solid powder, and lyophilized PDA-incorporated nanoparticles (PDA5–8 in Table 1) were used for FT-IR measurement. The carboxylic acid peak of PGA decreased with increasing GC content.

Abbreviations: GC, glycol chitosan; FT-IR, Fourier-transform infrared; PDA, p-phenylenediamine; PGA, poly(γ-glutamic acid).

Figure 7 XRD analysis of PDA-incorporated nanoparticles.

Notes: The composition of PDA/PGA/GC is illustrated in Table 1. PDA, PGA, and GC were used as a solid powder, and lyophilized PDA-incorporated nanoparticles (PDA1–8 in Table 1) were used for XRD analysis. The physical mixture was PGA/PDA (weight ratio = 5:1). PDA itself has sharp crystalline peaks, while nanoparticles showed broad peaks for all compositions.

Abbreviations: GC, glycol chitosan; PDA, p-phenylenediamine; PGA, poly(γ-glutamic acid); XRD, X-ray diffractometry.

Figure 8 PDA release from nanoparticles: the effects of (A) PDA content and (B) the addition of GC.

Note: The composition of PDA/PGA/GC is illustrated in Table 1.

Abbreviations: GC, glycol chitosan; PDA, p-phenylenediamine; PGA, poly(γ-glutamic acid).

Figure 9 Cytotoxicity of PDA and PDA-incorporated nanoparticles (PDA2 in Table 1) against HaCaT cells. HaCaT cells (1 × 10⁴ cells) were seeded in 96-well plates, and then the cells were exposed to PDA or PDA-incorporated nanoparticles for 3 days. The cell viability was assayed using the MTT colorimetric assay.

Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NP, PDA-incorporated nanoparticles; PDA, p-phenylenediamine.

Figure 10 Flow cytometry analysis of PDA and PDA-incorporated nanoparticles (PDA2 in Table 1) against HaCaT cells. FITC-annexin V (Annexin V) and PI were used for apoptosis and necrosis of cells, respectively. Apoptosis or necrosis of cells were analyzed using a FACScan (Becton Dickinson Biosciences, San Jose, CA) flow cytometer.

Abbreviations: FITC, fluorescein isothiocyanate; NP, PDA-incorporated nanoparticles; PDA, p-phenylenediamine; PI, propidium iodide.