The quantity of eumelanins (within melanosomes) determined the colour of human hair. Melanosomes are tightly embedded in the inter-macrofibrillar matrix of cortex . To remove those keratin, we extracted KAP and keratins from human hair adequately in advance according to Fujii et al’s protocol. the KAP and keratin had molecular weight distribution of about 10-28 kDa and 45-65 kDa, respectively (Fig. Fig. 1 b). The KAP-free hair and hair residues remained black (Fig. Fig. 1 a), indicating retention of the melanosomes. Then an enzyme esperase was used to hydrolyze the hair residues to release the melanosomes. The enzymatic hydrolysis process was highly efficient due to DTT and urea disrupted disulfide bonds and hydrogen bonds in hair residues during keratin extraction process . Therefore, melanosome isolation can also be achieved through enzymatic hydrolysis of human hair residues after keratin extraction with urea and other reducing regents like mercaptoethanol, mercaptoacetic acid, and L-cysteine, etc.
3.1 Morphology of the melanosomes
Pure, dispersed melanosomes with yields of about 1.3% (by weight of original hair fibers) were isolated successfully with centrifugation procedures as all protein components in the hair residues were degraded into soluble products. Morphological observations of the melanosomes showed the melanosomes were individually isolated and maintained their structural integrity. The melanosomes were well-defined rod-shaped and ellipsoidal particles with lengths of 904±171 nm and diameters of 341±75 nm (Fig. Fig. 2a, c). High magnification SEM and TEM images showed rough surfaces of the hair melanosomes, and melanosomes containing 10-60 nm secondary nanoparticles (Fig. Fig. 2b, d). These results are consistent with published work about human hair melanosomes, suggesting the integrity of the melanosomes was well preserved .
Takehito et al. investigated the ultrastructure of intact hair melanosomes and concluded that they were formed from a membrane-like outer structure (MS) and an inner matrix. The MS included numerous spherical vesicles (V) while the inner matrix showed a stripe-like structure (MT) in the elongated direction and a sheet-like arrays structure (SA) in the cross section. As shown in Fig. 2e, spherical vesicles with a diameter of 10-20 nm were evident, along with the MS in the longitudinal section of the melanosome. In the cross-section view, a series of concentric ring structures could be observed (Fig. 2f). All these morphological observations in the isolated melanosomes corresponded to earlier published results of intact melanosomes in human hair, suggesting that the structure of melanosomes isolated as described herein was preserved.
3.2 Structural characterization of the melanosomes
Hair fibers are mainly composed of proteins (90%) and minor components including lipids (5%) and melanin (1-3%) , while melanin mainly comprise of indole structures (Fig. 3 a. The FTIR and 13C NMR spectra of hair and melanosome reflect these differences. In Fig. 3 b, the FTIR spectra of hair shows representative absorption bands related to peptide bonds at 3,268 cm-1 (amide A), 3,068 and 3,073 cm-1 (amide B), 1,629 cm-1 (amide I), 1,523 cm-1 (amide II) and 1,240 cm-1 (amide III). The intense peaks at 1,040 cm-1 were related to the S-O stretching vibrations of the cysteic acid . In the FTIR spectra of isolated hair melanosome the absorption peaks of indolic and pyrrolic groups can be observed. The intense absorption peak at 3,248 cm-1 was ascribed as the N-H and O-H stretching vibrations in the indolic and pyrrolic structures . In the range of 2,950-2,850 cm-1, very weak peaks at 2,967, 2,940, 2,885 cm-1 were related to the C-H stretching vibration in aliphatic groups.. Aromatic C=C, C=N bending, amide C=O stretching, and N-H bending from indole, pyrrole in melanin overlapped with an absorption plateau at the region of 1700-1480 cm-1. The peaks at 1,446 cm-1, 1,375 cm-1 and 1,240 cm-1 were ascribed as aliphatic C-C bending, C-N stretching, and phenolic C-OH stretching in melanin, respectively [5, 22].
The solid-state 13C NMR spectra of intact hair and the isolated melanosomes are given in Fig. 3c. The solid state 13C NMR signals of hair proteins were composed of four typical spectral ranges including the carbonyls region mainly due to backbone carbonyls in peptide bonds and side-chain carboxyl (190-160 ppm), the aromatic region from the bulk of the aromatic residues (160-90 ppm), backbone α- and β-carbons (80-45 ppm), and aliphatic region (45-10 ppm) . The solid state 13C NMR spectra of the isolated melanosome also show complex but different spectra. The intense signals centered at 170 ppm and aliphatic region (90-10 ppm) were mostly due to residual proteinous components, whereas the signals at 160-90 ppm were related to both the melanin and the proteinous components. As melanin mainly resulted in the aromatic signals while proteins mainly contributed to the aliphatic and carboxyl signals, the ratio of the aromatic to carbonyl signal area could be used to evaluate qualitatively the relative content of melanin in the melanosomes extracted from hair . As shown in Table 1, Simona Ghiani et al had reported the ratio of aromatic-to-carbonyl signal area for intact hair as 0.15, while that for melanosomes isolated with concentrated HCl and enzymes (papain and DTT) were 2.63 and 0.75, respectively . The values were 1.88 and 0.27 for our isolated melanosomes and intact hair, respectively, suggesting a higher melanin content in our isolated melanosome due to complete removal of hair protein contaminations. In addition, the melanin-bound proteins in melanosomes from human hair have a high content of arginine, which has signal at 157 ppm . Our result also demonstrated higher intensity at 157 ppm, indicating the isolated melanosomes kept the melanin-bound proteins.
3.3 Functional properties of the melanosomes
The TGA mass loss and derivative thermogravimetric (DTG) curves of intact hair and melanosomes were shown in Fig. 3 d and 3e, respectively. The mass loss process of melanin was mainly divided into two stages (30℃-150℃, 150℃-500℃), and the weight loss rates were larger at the temperatures of 65.5 ℃, 346℃. The first weight loss stage was due to the evaporation of weakly and/or strongly bound water [37, 38]. In the second stage, the percent weight loss of about 48.8% was due to the loss of carbon dioxide, water, and ammonia during the heating of melanins at elevated temperatures [38-40]. This suggests that the hair melanosomes can be used in polymer blends such as melt extrusion and injection molding where processing temperature is lower than 150℃[37, 41].
As shown in Fig. 4 a and b, melanosomes at different concentrations showed broad absorption regions at 200-800 nm. The absorption intensities at 200-400 nm (UV region) are higher than that at 400-800 nm (visible-light region), and the absorption intensity increased with concentration of melanosomes. The UV filtering percentage of melanosome suspensions increased with concentration of melanosome, while the transparency showed an opposite trend. At concentrations up to 0.1 mg/mL, the filtering percentages of UVA, UVB, and UVC were above 99%, with a transparency of 12.8%. After that, increasing the concentration of melanosome suspensions did not improve filtering capacity further, but transparency decreased sharply (Table 2). Therefore, human hair melanosomes isolated based on our protocol retained highly effective UV filtering ability, although transparency is compromised. Regardless of transparency, the UV barrier property of the melanosomes can potentially be used as a sustainable photo-protection agent in wide ranging applications including cosmetics and packaging [24, 41].
The UV-vis transmittance spectra of PAM and PAM-M films with various concentrations of melanosome and their percentages of UV filtering were shown in Fig. 4c and Table 3, repectively. The filtering percentages of UVA and UVB in PAM film were very low (about 23.2% and 38.7%, while a higher UVC filtering percentage of 79.0% was recorded. With increasing human hair melanosome loading from 0.1% to 0.6%, the UVC filtering percentage increased to 90% and 99%. Increasing the melanosome loading concentrations to 0.4% and 1% resulted in UVA and UVB filtering percentage increase to over 90% and 99%, with the transparency sharply decreasing to 48% and 14.0% consequently. These results indicate that human hair melanosomes enhance UV-filtering performance of polymeric films such as those made with PAM.