Metal-Chelation-Assisted Deposition of Polydopamine on Human Hair

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Metal-chelation-assisted deposition of polydopamine on human hair: a ready-to-use eumelanin-based hair dyeing methodology Kyung-Min Im, Tae-Wan Kim, and Jong-Rok Jeon ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00031 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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ACS Biomaterials Science & Engineering

- Title Page -

Title: Metal-chelation-assisted deposition of polydopamine on human hair: a ready-to-use

eumelanin-based hair dyeing methodology

Name of Authors: Kyung Min Im,† Tae-Wan Kim,†,‡ and Jong-Rok Jeon*,†,§

Addresses of Institutions: †

Department of Agricultural Chemistry and Food Science & Technology, ‡Division of Applied Life

Science (BK21Plus), §IALS, Gyeongsang National University, Jinju 52727, Republic of Korea.

*Correspondence: Prof. Jong-Rok Jeon, Ph. D. E-mail - [email protected]

Tel - +82-55-772-1962 Fax - +82-55-772-1969

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ABSTRACT Permanent dyeing of gray hair has become an increasingly active area in the cosmetics industry because of the increasingly aging population in developed countries. So far, p-phenylenediamine (PPD) and related diamine-based monomeric compounds have been widely used for the dyeing processes, but toxicological studies have revealed such compounds to be carcinogenic and allergenic. Here, we for the first time demonstrated that polydopamine, a mimic of human eumelanin, gives rise within a commercially acceptable period of time (i.e. 1 hour) to deep black colors (i.e. natural hair colors of Asian) in human keratin hairs in the presence of ferrous ions. The dyed hairs showed excellent resistance to conventional detergents, and the detailed color was readily varied by changing the kind of metal ion used. SEM images and FT-IR-ATR spectra suggested that the extent of polydopamine aggregation was crucial for the dyeing efficiency. High-resolution (15 T) FT-ICR mass spectrometry performed on the products detached from hairs with either 0.1 N HCl or NaOH indicated that similar polydopamine products were recruited into the hair matrices whether in the presence or absence of metal-based chelating. Polydopamine chains were determined using EPR and ICP-OES to use chelation of ferrous ions to self-assemble as well as to bind keratin surfaces in the dyeing conditions. Also, mice subjected to skin toxicity tests showed much greater viability and much less hair loss with our dyeing agents than with PPD. In conclusion, this study showed that a safe eumelanin mimic may be used to permanently dye gray hair, and showed three kinds of deposition mechanisms (i.e. innate binding ability of polydopamine, metal-assisted self-assembly of polydopamine, and metal-related bridging between keratin surface and polydopamine) to be involved.

Keywords: polydopamine; permanent hair dyeing; polyphenol-metal complex; toxicity

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INTRODUCTION Natural hair colors and the melanin-based polymeric pigments that determine them often differ for different ethnic groups. Human melanin can be classified into eumelanin and pheomelanin, whose monomeric subunits differ.1 Eumelanin is produced via oxidative polymerization of tyrosine, while cysteine is additionally involved in in vivo pheomelanin synthetic pathways. The ratio of the concentration of eumelanin to that of pheomelanin varies between ethnic groups. Eumelanin gives rise to deep black hair, which is found, for example, in Japanese, Korean, and Mongolian people, while Celtic red hair derives mainly from pheomelanin. The aggregation pattern and size of the melanin polymer also significantly affect hair color.2,3 Beyond the color appearances, melanin have been widely studied due to its multifunctional physicochemical properties that are applicable to bioelectronics, adhesives, drug delivery and sunscreens.4

What people across ethnic groups do generally share is a tendency to develop gray hair as they age. Melanogenesis for hair coloring is associated with tyrosinase activity in hair follicle cells, but the activity of this enzyme reduces with age, thus resulting in the formation of gray hair.5 Breakdown of melanocyte stem cells also leads to the graying of hair.6 Consumer demand for age-concealing products and services has led to the successful commercialization of methods for permanently dyeing gray hair. Small diamine-based organic molecules such as p-phenylenediamine (PPD) have been widely used in oxidative permanent hair dyeing.1,7 Oxidizing agents (e.g., hydrogen peroxide) allow the diamine compounds to co-polymerize with coupling agents (e.g., resorcinol), generating various shades of many colors. Small organic molecules are able to penetrate into hair matrices during this polymerization and this penetration contributes to the tight binding of the resulting polymeric dyes to hair layers and to the strong resistance of the dyeing to external stimuli including shampooing. However, organics with molecular masses of less than 500 Dalton have been shown to easily cross the skin barrier.8 PPD, whose molecular mass is less than 500 Da, has in fact been regarded as an allergen and carcinogen.9,10 Some developed countries such as Japan also consider hair dyeing ingredients to be “quasi-drugs” owing to the potential harm these ingredients may pose to human health and safety.1 The in situ oxidation reactions that color hair as well as the dyeing ingredients themselves cause hair and the scalp to become bleached and damaged.11

What are the safest dyeing agents for human gray hair? Melanin extracted from biological sources may be the best candidate because the melanin is an in vivo hair color agent in human. However, melanin constituents spontaneously 3



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aggregate to form spheres and rods with dimensions in the micron range.12 Objects of such size do not very efficiently penetrate into hair matrices, and thus hair colors resulting from the use of such melanin tend to be neither deep nor permanent. In fact, several cosmetic companies in Korea had a decade ago used squid melanin as an ingredient of oxidation-based products advertised to provide permanent dyeing, but later shown scientifically to have negligible coloration activity towards human hair due to the melanin particles being too large to penetrate into the hair; the Korea Food & Drug Administration finally prohibited these companies from making such dazzling claims in their advertisements.13

To overcome this problem with extracted melanin, the aggregation properties of the hair dye need to be manipulated and additional binding agents allowing for the efficient deposition of the dye onto the hair matrices are required. Polydopamines are black-colored products derived from high pH- or oxidant-induced polymerization of dopamine, and are structurally very similar to human eumelanin.14-16 The structures of dopamine and 3,4dihydroxyphenylalanine, which is the precursor for human eumelanin, are very similar — with the only difference being the lack of a carboxylic group in dopamine — and the mechanisms for both of their oxidative polymerizations (i.e., in vivo melanogenesis and in vitro polydopamine synthesis) are also very similar. In contrast to extracted melanin, polydopamine shows the benefit of being able to be made in such a way that its self-aggregation can be controlled. This control results from the transformation of dopamine to polydopamine proceeding in a bottom-up manner wherein the reaction time and precursor concentration can be finely tuned. Polydopamines, due to their catechol groups,17 can bind other molecules in various ways, including by chelation and using hydrogen bonds, and are thus capable of binding to hair surfaces and to employ other binding agents such as metal ions.

To date, application of polydopamine-based coating on synthetic and natural fibers has been extensively studied. Stem cell differentiation was effectively modulated through polydopamine-coated poly(L-lactide).18 Conductivity of aramid fibers was enhanced with the coating.19 Inorganic and organic hybrids were readily manufactured with polydopamine coating of poly(vinyl alcohol) nanofibers.20 Beyond the organic fiber structures, inorganic ones such as steel fibers were also a coating subject of polydopamine.21 However, no articles have dealt with polydopamineinvolved color quality, which is a critical factor for cosmetic applications. In addition to polydopamine coating ability, low toxicity of the melanin mimetic has been highlighted. Cytotoxicity, inflammation and immunological responses22,23 were attenuated with polydopamine treatments, suggesting that the material can be valuable to 4


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research and development areas needing low toxicity and irritation. Simultaneous involvement of both catecholamine-induced binding and metal-polyphenol chelating has been not yet demonstrated in a field of materialindependent surface engineering.

Here we employed polydopamine and ferrous ions (Fe2+) to manufacture size-controlled eumelanin particles capable of providing permanent gray hair coloration in a commercially acceptable period of time (i.e. 1 hour). After confirming that these eumelanin particles successfully dyed human hair, several analytical tools such as electron paramagnetic resonance (EPR), scanning electron microscope (SEM), Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry, inductively coupled plasma-optical emission spectrometer (ICP-OES), high performance liquid chromatography (HPLC) and Fourier transform-infrared spectroscopy-attenuated total reflection (FT-IR-ATR) were used to decipher the mechanisms by which the polydopamine and ferrous ions were deposited on the human hair. Finally, the toxicity of polydopamine-based dyeing conditions was compared with that of PPD (which is the most widely used dyeing precursor in commercial products)1 by spreading the dyeing agents on the skin of ICR mice.

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Materials and methods Chemicals and materials Iron (III) sulfate n-hydrate, iron (II) sulfate heptahydrate and copper (II) chloride were purchased from Junsei chemical. Aluminum (III) potassium sulfate, sulfuric acid and 60% perchloric acid were obtained from Daejung chemical, Samchum chemical and J. T. Baker, respectively. Dopamine hydrochloride and PPD were purchased from Sigma-Aldrich. Sodium dodecyl sulfate (SDS) was obtained from Promega. Human gray hair was kindly donated from Amorepacific Corporation (Yongin, Republic of Korea). Female ICR mice were purchased from KoaTech, and they were cared for in accordance with the guidelines of the University animal center. All protocols regarding mice were reviewed and approved by the animal care and use committee of the University before conducting the experiments.

Gray hair dyeing and detergent resistance test Dopamine hydrochloride (0.4 g) was completely dissolved in 40 mL of 100 mM Tris-HCl buffer (pH 8.1) to initiate a pH-induced oxidative polymerization as described previously.24,25 After 6-h incubation for the pre-polymerization of dopamine in the Tris-HCl buffer, a mass of 0.2 g of human gray hair (4 cm long) was additionally soaked in a volume of 6 mL of the 6-h incubated solution for a 1-h dyeing process at room temperature. Various incubation times for the pre-polymerization of dopamine were also employed to help decipher the mechanisms by which products of polydopamine reactions were deposited during the 1-h dyeing process with hair fibers. For the samples treated with ferrous ions, a mass of 0.33 g of iron (II) sulfate heptahydrate was dissolved immediately when the gray hair was added in the pre-synthesized polydopamine solutions; other samples were treated with the same amounts of other metallic ions derived from iron (III) sulfate n-hydrate, copper (II) chloride, and aluminum (III) potassium sulfate instead of ferrous ions.

After the 1-h dyeing of each gray hair sample at room temperature, the dyed hair was washed with tap water and dried completely with an electric hair dryer. Color characteristics of the dyed hairs were measured with a conventional colorimeter (SPEC, China). The color parameters measured from seven randomly chosen different locations on each hair tress were averaged. ∆E was calculated with the formula: [(100 - L*)2 + (a*)2 + (b*)2]1/2.26

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SDS (1 g) dissolved in distilled water (40 mL) was employed to test whether the dyed hairs would retain their colors in the presence of a detergent. Dyed hair samples were completely soaked with SDS-containing solutions for 10 min. Following this treatment, each hair tress was removed from the SDS-containing solution and then washed with tap water. Color parameters of the completely dried hairs were evaluated with a conventional colorimeter as described above. These procedures were repeated twice more with the same hair tress.

Insoluble products, SEM, ICP-OES and HPLC analyses Insoluble products from pH-induced polymerization of dopamine for weighing were obtained with centrifugation (20,000 g) for 10 min. The products were washed three times with repeated centrifugation and vigorous vortexing followed by complete drying at 50℃. Dyed and virgin hairs were coated with gold using a sputtering process. Their ultrastructures were then obtained by using a field-emission SEM (Philips, XL30S FEG). To acquire the SEM image of the polydopamine aggregates, a small volume (15 µL) of the reaction solution was spread onto a cover glass and dried at room temperature. The Fe ion contents of the samples were determined by using an ICP-OES (OPTIMA 5300DV). Before the instrumental analyses, a mass of 0.2 g of each tested hair was digested to completion with a volume of 10 mL of an aqueous solution containing distilled water, 60% perchloric acid, and sulfuric acid (1:9:5 v/v/v ratio).27 The solutions containing hairs were heated with a conventional hot plate whose surface temperature gradually increased up to 300℃ during 2 hours. The heating at 300℃ continued for further 4 hours. The volume of each sample was adjusted to 100 mL with distilled water after confirming the digestion. The diluted solutions were finally filtrated with a filter paper (AVANTEC, No. 2, 9 cm diameter) for further Fe elemental analyses. Residual dopamine was analyzed with reverse phase HPLC (Agilent 1200 Series) equipped with a ZORBOX Bonus-RP column (4.6 × 150 mm). Mobile phase for initial 10 min was used with 100% distilled water (flow rate, 1.0 mL/min). Gradient flow from 100% distilled water to 100% acetonitrile during 10 min was then applied. Absorbance was measured at 280 nm.

FT-ICR MS, FT-IR-ATR and EPR analyses Ultrahigh-resolution mass spectrometric analysis was performed using a 15 Tesla FT-ICR mass spectrometer equipped with an electrospray ionization source (solariXTM system, Bruker Daltonics, Billerica, MA) at the Korea Basic Science Institute (KBSI). The polydopamine products were detached from the dyed hairs by sequential soaking in 0.1 N HCl and 0.1 N NaOH solutions. The products detached from the hair were themselves observed 7



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with the naked eye to be colorful. The 0.1 N HCl and 0.1 N NaOH solutions were then neutralized by adding to them 0.1 N NaOH and 0.1 N HCl, respectively. Finally, the above two solutions were mixed with methanol (1:1 v/v ratios) and formic acid (0.1% to total volume), thus facilitating electrospray ionization. The samples were directly infused into the 15 T FT-ICR mass spectrometer using a TriVersa NanoMate (Advion BioSciences, Ithaca, NY) with a flow rate of approximately 300 nL/min and analyzed in positive ion mode within a mass-to-charge (m/z) ratio range of 200–1500. The mass resolving power was set at 540,000 (at m/z 400) for all spectra, and 40 scans per sample were collected with a 4 M transient. The other MS parameters were as follows: a capillary volt age of 3 kV, drying gas flow rate of 1.5 L/min, drying gas temperature of 180°C, ion accumulation time of 0.001 s, and transient length of 1.89 s. External calibration was performed with quadratic regression using an arginine solution (10 µg/mL in methanol). Data acquisition was controlled by FTMSControl 2.0 software (Bruk er Daltonics), and the raw spectra were imported to DataAnalysis 4.2 (Bruker Daltonics) to detect their peaks.

EPR (Bruker EMX Plus 6/1 spectrometer equipped with a dual-mode cavity) measurements were taken at KBSI as described previously.28 The 6 h-incubated polydopamine solutions with or without ferrous ions were lyophilized to obtain solid powders before EPR analyses. The experiments were conducted at room temperature and used a microwave frequency of 9.64 GHz, microwave power of 1 mW, and modulation amplitude of 1 G. FT-IR spectra of dyed and virgin hairs were obtained by using the ATR mode (iS50, ThermoFisher).

Mouse skin toxicity tests Female ICR mice were allowed to freely breed for one week and then their hip regions were treated with a commercial depilatory agent. The mice were classified into four groups based on dosage (high, 0.6 g; low, 0.3 g) and the kind of dyeing agent (PPD and 6 h-incubated polydopamine products). Ferrous ions (0.33 g of iron (II) sulfate heptahydrate) were mixed with the polydopamine products, and this mixture was then immediately applied to the depilated (hair-free) hips. A solution of PPD in 100 mM Tris-HCl buffer (pH 8.1) was also applied. The solutions containing either PPD or polydopamine products complexed with ferrous ions were repeatedly applied every day for 16 days by employing conventional brushes.

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Results and discussion Metal-chelation-assisted coloration of human gray hair with polydopamine Oxidative polymerization of dopamine to polydopamine gave rise to a black coloration, indicating that diverse chromophore structures were synthesized. Such coloration has been reported in several cases for the polymerization of small aromatics.25,29 In these cases, delocalized pi-bonds resulting from covalent linkages between phenolic structures may have allowed for the absorption of visible light. We first determined the feasibility of attaching preformed polydopamine to gray keratin surfaces by observing whether the hair got colored. As shown in Figures 1A and 1D, a significant increase in ∆E and a visually obvious color change were observed, respectively. The dyed hair (Figure 1C) showed a rougher keratin surface than did the virgin gray hair, which was also consistent with the dyeing results. Detergent resistance was evaluated by repeatedly soaking hair samples with SDS-containing solutions. ∆E was found to be stable over the course of these soaking treatments (Figure 1B), which suggested that detachment of polydopamine hardly happened in the presence of detergents. Several studies have highlighted the binding capabilities of polydopamine. The catechol and amine groups of polydopamine are believed to simultaneously promote binding to several solid surfaces.14,30 Similar binding mechanisms may have also been involved in our dyeing experiments.

Despite of the innate ability of polydopamine to bind gray hair and hence dye it, the extent of the coloration provided by polydopamine alone was not nearly sufficient for commercial purposes. The amount of polydopamine deposited on solid surfaces has been reported by Lee et al.24 to plateau at relatively low levels after a certain dipping time. This result indicated additional agents to be required for recruiting polydopamine products more efficiently to keratin surfaces, and hence enhancing the absorption of visible light. Ferrous ions are known to induce the formation of metal-polyphenol complexes through chelation. It is thus not unreasonable for ferrous ions to act as a bridge between polydopamine products. Moreover, ferrous ions are relatively non-toxic compared with other heavy metals. In fact, the Korea Ministry of Food and Drug Safety permits, according to its regulatory guidelines, the use of ferrous ions at a limited concentration on human hair and scalps. Interestingly, treatment of polydopamine products with ferrous ions resulted in dramatic enhancement of ∆E (Figure 1A). Visual inspection of hair samples treated with polydopamine and ferrous ions (see photograph in Figure 1D) was also consistent with the ∆E change. The black hair colors we produced (Figure 1D) were observed to be similar to the natural hair colors of Korean and Japanese people, suggesting our methods to be especially valuable for coloring the hair of Asians. The dyeing speed 9



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(See Materials and Methods) and the strong resistance to detergents (Figure 1B) also indicated our method to nearly meet commercial requirements.

Why did combining polydopamine with ferrous ions so effectively yield deep colorations? As indicated above, keratin hairs dyed with polydopamine and ferrous ions showed the roughest surfaces of the various samples inspected (Figure 1C). This higher roughness may have been due to the recruitment of more products from the polydopamine solutions to the keratin surfaces as a result of Fe2+-based bridging. Moreover, the formation of novel chromophores by phenols that chelate metal ions is well defined.31 Different metal ions were employed to confirm this possibility in our dyeing methods. As seen in Figures 2A and 2B, a significant color change and resistance to detergents were observed regardless of the kind of metal ion tested. Moreover, differences in the detailed colors were observed for different metal ions, indicating that the use of different metals yielded different chromophore structures and different sets of assembled polydopamine products. An ability to choose the detailed hair color is important to meet the preferences that vary by individual but also by ethnic group. While this approach of using different metals with polydopamine provides a diverse range of colors, the toxicity of the metals to human skin must first be strictly tested.

Mechanisms for the deposition of polydopamine on human gray hair We sought to determine why extracted melanin-based keratin hair dyeing failed to be commercialized by those Korean cosmetic companies that tried to do so. We first investigated whether the size of the melanin macromolecule is a critical factor for the ability to change hair color. As the extent of dopamine polymerization was proportional to reaction time, the 1-h dyeing processes with ferrous ions and virgin gray hairs were evaluated at various reaction times of dopamine pre-polymerization. Interestingly, no significant difference in ∆E values was observed up to reaction times of 10 h, as seen in Figure 3. But longer reaction times yielded lower ∆E values. SEM analyses were then conducted to evaluate the morphology of eumelanin mimetics at the various reaction times of dopamine prepolymerization. Nanometer-sized granules formed, as seen in Figure 4A. The number of granules increased with increasing reaction time, and the granules were observed at all reaction times to be linked together like grapes. Granule size, however, was observed to be independent of reaction time after the nanometer-sized granules formed.

The commercial failure of extracted melanin-based keratin hair dyeing may also have been due to the observed 10


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granule aggregation resulting in a decreased diffusion rate of the polydopamine products into hair matrices. Consistent with the results shown in Figure 4A, the weight of the insoluble products obtained using centrifugation was found to be directly correlated with reaction time (Figure 4B). Through these results, co-presence of the nanosized granules and soluble products was expectable in the pre-synthesized polydopamine solutions. Which, between the two portions, was more actively involved in the dyeing processes of gray hairs? SEM images of the dyed hairs (Figure 1C) showing no attached granules strongly indicate that the soluble portion not participating melanin aggregation was crucial for inducing keratin hair colorations. Polydopamine products in the soluble portion may penetrate into hair matrices with or without the aiding of ferrous ions, thus contributing to permanent hair dyeing observed in this study.

IR spectra (Figure 5A) of keratin hairs dyed with the polydopamine products also suggested that the products of long reaction times of dopamine pre-polymerization hardly penetrated the keratin surfaces. The spectra resulting from the 3-d and 7-d reactions were quite similar to that of virgin hairs, suggesting rare attachment of polydopamine products. However, the IR signals of virgin hairs were largely absent from the spectrum resulting from the 6-h prepolymerization of dopamine, suggesting that the entire surfaces of the keratin hairs were modified. Curiously, the previously reported polydopamine-related IR signals32 were negligible for our 6-h incubated solution (Figure 5A) although significant color changes on hair surfaces appeared. This phenomenon may be attributable to complex interaction between polydopamine products and keratin matrices during the penetration. Overall, the dependence of our results on reaction time for the dopamine pre-polymerization suggested that aggregation of polydopamine granules inhibited recruitment of the reaction products to the keratin surfaces. Delicately controlling the selfassembly during in vitro melanization is expected to be key for maximizing the extent of keratin hair coloration. The presence of additional reactive agents such as ammonia33 may accelerate the self-assembly, thus controlling the time to be taken by hair dyeing. In addition, major IR peaks near 3300, 1511, 1623 and 1200 cm-1 in virgin gray hairs (Figure 5A) may correspond to amide groups widespread in protein backbone.34 It is noticeable that amide groups can act as a chelating site for metal ions.35 Indeed, it was proved in this study that ferrous ions could deposit on human gray hair without polydopamine products (See below).

To evaluate the exact role of ferrous ions in our dyeing methods, we first examined the affinity of these ions for keratin hairs in the presence and absence of the polydopamine products. Significant amounts of the ferrous ions 11



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were found to bind the keratin surface without polydopamine, suggesting the roles of ferrous ions as a primer capable of recruiting polydopamine products at the keratin surfaces. However, more ferrous ions bound as more of the polydopamine products were included (Figure 5B), indicating that Fe-based complexes resulted in the formation of connections of polydopamine products to each other as well as to the keratin surfaces. More direct evidence for the cross-linking of the polydopamine products to each other was revealed in EPR spectra, as seen in Figure 6. The g-values of polydopamine with or without ferrous ions were evaluated to be about 2.00, supporting the presence of semiquinone-type free radicals. Catechol groups of polydopamine readily oxidize into quinones.14 The line widths of both peaks were almost the same, while their signal intensities were significantly different. The lower intensities of the peaks corresponding to polydopamine complexed with ferrous ions suggested that chelating bonds between the ferrous ions and catechol moieties of polydopamine products may interfere with the formation of semiquinone-type free radicals.

Polydopamine products are generally diverse, and supramolecular interactions between them have been observed.14,36 Ultra-high-resolution FT-ICR mass spectrometry was therefore employed to determine the masses of the polydopamine products deposited on the keratin surfaces. Note that FT-ICR mass spectrometry has been previously used to characterize complex radical-based polymerization of small aromatics,37 as in the current work. First, we collected deposited products from the dyed hairs by soaking the samples in pH-adjusted water. We hypothesized Fe2+-independent and –dependent recruitments of polydopamine products to be feasible based on our dyeing methods and results (Figure 1). The former type of deposition (i.e. layers formed through innate binding ability of polydopamine) has been reported to lead to a destabilized connection with increasing pH.38 In contrast, the latter type of deposition (i.e. ferrous ion-polydopamine complexes) has been shown to lead to a destabilized connection with decreasing pH.39 In our current work, we found an overlap between major m/z peaks of the polydopamines detached from the hair samples by applying low pH conditions and those peaks resulting from a high pH-induced detachment (See the denoted m/z values in Figure 7A). This overlap indicated that similar assemblies of polydopamine products were deposited in the presence and absence of ferrous ion-based chelating. In addition, orderly change in m/z values through Fe2+-based adducts was confirmed (Figure 7A and Table S1 in the supporting information), indicating that Fe2+ chelation was indeed involved in the recruitment of polydopamine products, thus resulting in the dyeing of the gray hairs. Compared with previously reported high resolution mass spectrometry of polydopamine products,40 exactly same m/z values were not identifiable. This kind of mismatching may be due to 12


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complex modifications of functional groups observed in oxidative polymerization of small aromatics.37 An HPLC analysis (data not shown) also showed that any dopamine was hardly present after a 1-h pre-polymerization reaction. This observation is important because the use of dopamine in cosmetics and hair care products is currently banned in Korea.

Based on the results of several analytical tools, three kinds of deposition mechanisms were proposed, as summarized in Figure 8. Metal-assisted cross-linking between keratin hairs and polydopamine would be an initial step to form thin layers just above the keratin, followed by induction of a layer-by-layer deposition via the metal ion chelating the polydopamine products to each other. Innate adhesive action of polydopamine that was independent with metal assistance was proven to contribute to the coloring of gray hairs in our reaction conditions.

Toxicity of dyeing agents to ICR mice We expected our dyeing agents to be less toxic than currently used PPD and diamine-based small aromatics because polydopamine is a human eumelanin mimetic. To test this prediction, we assessed the skin toxicity of our dyeing methods using ICR mice and PPD. PPD is still used in permanent hair dyeing products that are distributed in Asian countries. Expected skin troubles such as edema were rarely observed. Instead, a complete blockage of hair growth appeared in PPD-treated mice (Figure 9A), raising the possibility that PPD-containing hair dyeing agents may also damage human hair follicles. In fact, PPD-induced human hair loss has been previously reported.41 Allergic and toxic reactions driven by PPD may damage hair cells. In contrast to these results with PPD, the side effects of treatments involving polydopamine and ferrous ions were negligible (Figure 9A). Previous reports dealing with tissue engineering and MTT assay42,43 have indicated polydopamine products themselves to be cytocompatible and nontoxic.

More interesting results came from experiments testing the viability of the mice exposed to these agents. Most mice whose skin was subjected to PPD died within 16 days, while those subjected to polydopamine and ferrous ions remained alive in this time period (Figure 9B). These results suggested that PPD can penetrate effectively into skin layers and reach the bloodstream, thus causing systemic toxicity leading to death. Precise measurements of the molecular masses of polydopamine-ferrous ion complexes were difficult due to their low solubility in any solvent, but it is not unreasonable to expect that the polymers aggregated with ferrous ions would show a much lower rate of 13



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skin penetration. That is, the size and innate chemical properties of polydopamine would be expected together to contribute to its non-toxicity to humans.

Further studies showing the rates of skin invasion including involvements of keratinocytes and immune cells44 will be necessary to fully characterize the detailed toxicity mechanisms. The standardized human gray hairs here we used derived from a cosmetic company (See materials and methods), but characteristics of human hair surfaces are variable from person to person and also depends on previous hair treatments such as a perm and a bleaching. Various kinds of human hairs should be further tested with our dyeing technique. Natural phenolics containing catechol groups45 are readily identifiable and extractable from plants. Such plant phenolics have been proven to show cytoprotective activity to human cells46 and to transform into more high-molecular-weight products with mild oxidants.29 Currently, we are employing several pretreatment methods to overcome low efficiency of plant phenol variant-based hair dyeing.47

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Conclusions Here we developed novel methods for dyeing human gray hair that are safer and more eco-friendly than currently used methods involving small diamine-based aromatics. We applied polydopamine products together with ferrous ions on gray hair to achieve black colors similar to the natural hair colors of Asians. Moreover, this treatment was accomplished within a commercially acceptable period of time (i.e., less than one hour). In addition, we were able to vary the detailed color by simply changing the kind of metal ion used. The coloring resulting from our methods was shown to be resistant to detergents, and our methods could therefore be used for permanent hair dyeing, which is the main source of income in the hair dyeing industry. Experiments involving analytical tools such as FT-IR, highresolution mass spectrometry, SEM, and EPR revealed metal-dependent and –independent depositions of sizecontrolled polydopamine aggregates to be involved in the coloration. Also, ICR mice subjected to skin toxicity tests showed much greater viability and much less hair loss with our polydopamine/ferrous ion dyeing agents than with diamine-based permanent dyeing agents. Taken together, these results suggest our methods for coloring human hair to be potentially superior to those currently used.

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ASSOCIATED CONTENT Supporting information Full data set of mass spectrometry obtained from FT-ICR analyses of polydopamine products detached through either 0.1 N HCl or 0.1N NaOH treatments to dyed hairs.

AUTHOT INFORMATION Corresponding Authors *E-mail: [email protected] (J.-R. Jeon). Funding This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Grant number 115085-2). Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We thank Dr. Kyoung-Soon Jang (KBSI) for FT-ICR analyses.

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Figure caption Figure 1. Color difference values (i.e. ∆E) of virgin and dyed hairs (A) before and (B) after repeated SDS treatments. (C) SEM and (D) photographical images of virgin and dyed hairs before SDS treatments. PD derived from dopamine pre-polymerization for 6 hours. The dyeing processes took 1 hour with virgin gray hairs. Abbreviation: VH, virgin hairs; PD, hairs dyed with polydopamine; PD + Fe, hairs dyed with polydopamine and ferrous ions.

Figure 2. Photographical images of (A) hairs dyed with polydopamine and different metal ions. (B) Color difference values (i.e. ∆E) of dyed hairs after repeated SDS treatments. Polydopamine derived from dopamine prepolymerization for 6 hours. The dyeing processes took 1 hour with virgin gray hairs.

Figure 3. Color difference values (i.e. ∆E) of hairs dyed with ferrous ions and different polydopamine products formed in a time-dependent manner. Different time values (i.e. X-axis) indicate incubation times for dopamine prepolymerization. The dyeing processes took 1 hour with virgin gray hairs, ferrous ions and the corresponding polydopamine products.

Figure 4. (A) SEM images of distribution patterns of polydopamine granules formed in a time-dependent manner and (B) a weight increase of insoluble portion of polydopamine products formed in a time-dependent manner.

Figure 5. (A) FT-IR-ATR spectra of virgin and dyed hairs with different polydopamine products manufactured in a time-dependent manner. The dyeing processes took 1 hour with virgin gray hairs. (B) Fe contents of hairs after soaking with ferrous ions, polydopamine, or both the agents. Polydopamine derived from dopamine prepolymerization for 6 hours. The soaking continued for 1 hour.

Figure 6. EPR spectra of polydopamine with or without ferrous ions.

Figure 7. Mass spectrometric analyses (Electrospray ionization-FT-ICR) of molecules detached from hairs dyed with polydopamine and ferrous ions. Polydopamine derived from dopamine pre-polymerization for 6 hours. The dyeing processes took 1 hour with virgin gray hairs. Two classes of detached molecules were obtained depending on sequential low (0.1 N HCl) and high pH (0.1 N NaOH) treatments. 21



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Figure 8. A scheme showing a mechanism of polydopamine-ferrous ion complex deposition to keratin hair surfaces.

Figure 9. (A) Representative photographical images of mouse skins applied daily with p-phenylenediamine (PPD) (low, 0.3 g; high, 0.6 g) and polydopamine-ferrous ion complex (PD + Fe) (low, 0.3 g of polydopamine with 0.33 g of FeSO4·6H2O; high, 0.6 g of polydopamine with 0.33 g of FeSO4·6H2O). Abbreviation: 1,

just after use of a

commercial depilatory; 2, 16-d incubation without any treatments; 3. 16-d incubation applied with a low-dosage PPD; 4. 16-d incubation applied with a high-dosage PPD; 5, 16-d incubation applied with a low-dosage PD + Fe; 6, 16-d incubation applied with a high-dosage PD + Fe. (B) The number of dead mice caused by daily treatments of PPD (low, 0.3 g; high, 0.6 g) and polydopamine-ferrous ion complex (PD + Fe) (low, 0.3 g of polydopamine with 0.33 g of FeSO4·6H2O; high, 0.6 g of polydopamine with 0.33 g of FeSO4·6H2O). Polydopamine used in mice derived from dopamine pre-polymerization for 6 hours.

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Figure Page 23 of1.32

E value

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 7.

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548.6122

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490.6534

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606.5712

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430.6974

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Figure 8. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Metal chelating between polydopamine components

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Metal chelating between polydopamine layers and keratin surfaces


PageFigure 31 of 329.

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For Table of Contents Use Only Metal-chelation-assisted deposition of polydopamine on human hair: a ready-to-use eumelanin-based hair dyeing methodology Kyung Min Im, Tae-Wan Kim and Jong-Rok Jeon

One-hour dyeing Eumelaninlike assembly

Fe2+

Non-toxicity


Metal-Chelation-Assisted Deposition of Polydopamine on Human Hair

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