Site-Specific In Situ Synthesis of Eumelanin Nanoparticles by an

Article pubs.acs.org/Biomac

Site-Specific In Situ Synthesis of Eumelanin Nanoparticles by an Enzymatic Autodeposition-Like Process Oliver I. Strube,*,† Anne Büngeler,† and Wolfgang Bremser‡ †

University of Paderborn, Department of Chemistry - Biobased and Bioinspired Materials, Warburger Str. 100, D-33098 Paderborn, Germany ‡ University of Paderborn, Department of Chemistry - Coating Materials and Polymers, Warburger Str. 100, D-33098 Paderborn, Germany S Supporting Information *

ABSTRACT: A method for in situ formation and controlled deposition of eumelanin nanoparticles is presented. The particles are built up by enzymatic reaction of L-DOPA with tyrosinase. The enzyme is tethered onto the support surface to get site-specific deposition of eumelanin. Due to the immediate deposition, the particles are monodisperse, with diameters of about 30−60 nm. Up to now, eumelanin particles have only been observed with sizes of about 200 nm. Deposition of those particles is site-specific on the areas where enzyme is present and results in different kinds of patterns on the support surface, including versatile monolayer structures.

1. INTRODUCTION 1.1. General Remarks. Melanins are a fascinating class of biomacromolecules with manifold functionality. They are found in a wide variety of life forms, such as humans, animals, or plants. In mammals, three predominant types of melanin are present. These are the black to brownish eumelanin, the reddish to yellow pheomelanin, and the brown neuromelanin.1,2 The first two of them are mainly found in skin and hairs, whereas the neuromelanin is found in the brain. Melanins are also found in eyes and the inner ear. Due to their high number of possible applications in physics and material sciences,3−6 as well as to their medical relevance,7,8 melanins have been subjected to intensive research activities in the last decades. High potential properties of melanins are free radical scavenging,9 paramagnetism,10 broad band absorption and very low fluorescence,11 or electrical conductivity.12 Despite this, many aspects of melanin structures remain unclear. It is known that melanins are made of oligomers with variable monomer composition. The best understood species is eumelanin, which is built by a multistep enzymatic process. The precursor L-tyrosine reacts through several steps to either 5,6-dihydroxyindole-2-carboxylic acid (DHICA) or its decarboxylated form, 5,6-dihydroxyindole (DHI). The eumelanin oligomers are mainly built from these two components at varying oxidation states. The complete mechanism is described in the literature13 and a simplified version is shown in Figure 1. Pheomelanin is formed whenever cystein is present during the monomer synthesis. The reaction then branches at the © XXXX American Chemical Society

Figure 1. Eumelanin synthetic pathway; simplified version.13

dopaquinone and forms different derivatives of benzothiazine that undergo the oligomerization.13 Neuromelanin derives from dopamine as precursor and is formed by an auto oxidative process, probably without the involvement of enzymes.14 Received: February 9, 2015 Revised: March 26, 2015

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are at least two other enzymes (tyrosinase-related proteins tyrp-1 and tyrp-2) involved in the buildup of melanin. However, it could previously be shown that tyrosinase is able to substitute the other enzymes if those are missing.13 Therefore, it can be assumed that the melanin made with our approach will be at least very similar in structure to natural eumelanin. A crucial point of our concept is the tethering of the enzyme to the support surface. The nature of immobilization has thus a great impact on the resulting deposition, as has already been shown for our previously presented system.20 Different immobilization methods result in variable sizes of the reaction zone, that is, the area from the support surface where enzymatic reactions can take place. It also indicates whether the process is self-terminating or not. A huge amount of very different immobilization techniques for enzymes are known.21 The most common methods are reversible binding via adsorption or ionic interactions and irreversible, covalent binding. In the latter, the size of reaction zone can be influenced by use of polymeric spacers, for example, end-functional PEG. The correlation between tethering method and size of reaction zone is illustrated in Figure 3.

Synthetic melanin can be made either by an enzymatic reaction with tyrosinase or via the auto oxidative approach at increased pH level.15 Natural eumelanin has been examined by various microscopy methods. These have shown that eumelanin particles have a very uniform size, that is, 200 nm in diameter.16 Synthetic melanin on the other hand is mostly described as an amorphous solid without distinctive particles,17 although there are some reports of spherical melanin-like particles from synthetic sources.18 The structural buildup from oligomeric units to the observed melanin particles is also not fully understood. A proposed architecture for eumelanin is a 3-fold aggregation of oligomers, mainly based on π-stacking.19 This model has been developed in the 1990s and has since than become quite popular, although a final proof is still to come. 1.2. Concept. We have developed a process for the controlled deposition of eumelanin nanoparticles, that are formed in situ, directly at the support surface. To this end, tyrosinase is tethered onto the support surface. The buildup of eumelanin occurs therefore in direct proximity to the support. As the particles are hydrophobic, they will deposit as soon as the buildup is complete. A model of the mechanism is shown in Figure 2. The principle of this approach is similar to the

Figure 3. Correlation between the enzyme tethering method and size of the reaction zone.

In this paper, we utilized the adsorption approach. As a consequence of this, it has to be anticipated that a certain amount of the tyrosinase might detach from the support surface and diffuse into the bulk. This would consequently lead to melanin synthesis in far distance to the support. The actual amount of uncontrolled reaction depends on the affinity of the enzyme to the support surface.

2. MATERIALS AND METHODS 2.1. Chemicals and Materials. L-DOPA, tyrosinase from mushrooms, and synthetic eumelanin were purchased from SigmaAldrich. CuSO4, NH3, H2O2, and phosphate buffer were obtained from the usual suppliers. All chemicals were used as supplied, without further purification. Microscopy glass slides (1 cm2) from Carl Roth were used as support. 2.2. Synthesis. The glass slides were cleaned with a mixture of NH3 (25%)/H2O2 (35%)/H2O in a ratio of 1/1/5 (V/V/V) for 5 min at 80 °C and washed with DI water. After that, 100 μL of a solution of tyrosinase in DI water (c = 1 g/L) was trickled onto the support and dried (i.e., 100 μg of enzyme). The slides were than washed with DI water to remove nonadsorbed enzyme. The glass slide with adsorbed enzyme was put into an aqueous solution of L-DOPA (varying concentrations) at pH 6.8 (phosphate buffer) and 40 °C. The solution contained 5 mL of a 5 μM solution of CuSO4. After 48 h the slide was removed, washed with DI water, and dried. 2.3. Scanning Electron Microscopy (SEM). Obtained samples were examined by means of electron microscopy using a Zeiss “Neon

Figure 2. Concept scheme of the autodeposition-like synthesis and subsequent deposition of eumelanin.

enzymatically mediated deposition of proteins that we introduced before.20 In contrast to that approach, which relies on the enzymatic destabilization of existing particles, here the particles are built up from low molecular components. The buildup mechanism of our eumelanin particles is very similar to the in vivo mechanism (Figure 1),13 with two differences. First, our process starts at L-DOPA, whereas, in vivo, L-tyrosine is the initial compound. This adjustment is made due to the better water solubility of L-DOPA, which allows for higher reactant concentrations. Additionally, it shortens the reaction by at least two steps. Second, a single enzyme, the tyrosinase, is utilized in our approach. In vivo there B

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Biomacromolecules 40” scanning electron microscope equipped with an EDX-detector. Pictures of the samples were obtained by applying the SE2-detector (high topography contrast) at an acceleration voltage of 2 kV. EDX spectra were measured at an acceleration voltage of 8 kV. 2.4. Atomic Force Microscopy (AFM). AFM pictures were taken with a Veeco “Dimension Icon PT” in tapping mode at a resolution of 512 points per line. Cantilever material was silicon tip on nitride lever. The 3D images were created with “Nanoscope Analysis V 1.5”. 2.5. MALDI TOF-MS. MALDI measurements were performed on a Bruker “Reflex” time of flight instrument, operating in positive linear mode. Ions formed by a pulsed UV laser beam (nitrogen laser, A = 337 nm) were accelerated to 15 keV. UV laser light (energy about 50 μJ) was focused onto the sample, using a focal diameter of about 100−300 μm. Freshly prepared melanins were suspended in double-distilled water (ca. 1 mg/mL) and finely grounded using a glass-pestle homogenizer. The resulting suspension was deposited on the stainless steel sample holder and air dried. A saturated solution of the matrix (2,5-dihydroxybenzoic acid, DHB) in 50/50 v/v water/acetonitrile (1 μL) was then added and allowed to dry in air before introduction into the mass spectrometer. Mass spectra were obtained by averaging the ions from ten laser shots. Four independent MALDI-MS measurements were made for each sample to evaluate reproducibility. Daily external calibration was provided by the [M + H]’ ions of Angiotensin II (m/z 1046) and DHB (m/z 155). Mass accuracy was always about 0.05%. 2.6. ATR-FTIR Spectroscopy. The ATR-FTIR spectra have been measured using a Bruker Alpha-FT-IR spectrometer with a ZnSe-ATRcrystal. The attenuated total-reflectance spectrum was obtained by subtracting the background IR spectrum of air from the spectrum of the dried melanin between 375 and 7500 cm−1 at a resolution of 2 cm−1. 2.7. UV−vis Spectroscopy. UV−vis-absorption spectroscopy was performed with a Thermo Scientific Evolution 600 spectrometer using a scan rate of 3.80 nm/min. The bandwidth of the light source in the UV−vis region was chosen between 200 and 800 nm. The measured melanin solutions were freshly prepared in 0.1 mol NaOH (1 mg/10 mL). The same glass cuvette was used for obtaining the spectrum and the background subtraction.

particles is not possible. Therefore, dynamic light scattering could not be applied, and the particle size is defined as the range where 95% of the particles fit in (measured under the SEM). AFM analytics affirm the results from the SEM investigations and can be found in the Supporting Information (Figure S3). The small size of the particles is highly exiting, as previous investigations of isolated natural eumelanin particles have shown them to be always of about 200 nm in size. Synthetic melanin on the contrary is mostly described as an amorphous solid without any distinctive structure. There are reports of synthetic melanin-like polydopamine nanoparticles with roughly the same size as our particles.22,23 However, these particles are made via an auto oxidative process at alkaline pH value, with dopamine as precursor, and without the use of enzyme. To our best knowledge, enzymatically synthesized nanoparticles of melanin were not known up to now. Referring to the recommended nomenclature,2 the presented product should be named (synthetic) L-DOPA eumelanin. To ensure, that our nanoparticles are indeed similar to eumelanin, we applied several analytical methods to compare our particles with a commercial synthetic eumelanin as reference. Via EDX measurement under the SEM, we obtained an elemental composition that is visualized in Figure 5a. The chemical compositions of sample and reference are very similar, which indicates that our particles are indeed made of some kind of melanin. The high oxygen content is due to the SiO2 support. Additionally, we compared UV−vis-spetra (Figure 5b) and ATR-FTIR-spectra (Figure 5c) of our nanoparticles with the purchased synthetic eumelanin as reference. Both analytics showed no significant discrepancy between the samples. The UV−vis spectrum shows broad band adsorption, as is typical for melanin structures. The FTIR spectrum of the sample shows all significant vibration signals for the most frequent monomeric units DHI and DHICA (Table 1). The reference shows mostly the same signals. However, the signal of the carboxylic acid (1038 cm−1) is not found. This indicates a lower DHICA content in the reference melanin. In summery, the results speak for the fact, that the obtained nanoparticles consist at least of a melanin-like structure. To get a more profound insight into the chemical structure of the particles, we applied MALDI-TOF MS (Figure 6). The signals with high intensities are identified in Figure 6c. It is revealed that the oligomeric structures consist of both DHI and DHICA at variable oxidation states. Several fragmented structures are also found. These are the same monomeric units that were found previously by MALDI investigations of natural sepia eumelanin.24 Furthermore, identical signals are found in the spectrum of the synthetic eumelanin reference (Figure 6b). As the combined results illustrate that our particles exhibit a high similarity to natural eumelanin2,24 and the synthetic eumelanin reference, it is very likely that they are in fact synthetic L-DOPA eumelanin. Therefore, the presented autodeposition-like technique enables, for the first time, an enzyme mediated synthesis of eumelanin-like particles with diameters below 100 nm. 3.2. Site Specificity of the Process. To examine the site specificity of the method, we adsorbed the enzyme in a circular pattern as shown in Figure 7. If the method works as intended, melanin formation and subsequent deposition should occur only in the enzyme-functionalized areas.

3. RESULTS AND DISCUSSION 3.1. Shape, Size, and Structure of Eumelanin Nanoparticles. As a proof of concept, we adsorbed tyrosinase continuously on a glass support which resulted in a continuous enzyme film (Figure S1). We than immersed the support into a solution of L-DOPA in DI water with a small amount of CuSO4 to activate the enzyme. After 24 h reaction time, SEM investigation revealed deposition of distinguishable spheric particles with diameters ranging from 30 to 60 nm (Figure 4). The particles undergo only partial coalescence or none at all. Due to this deposition and coalescence, redispersing of the

Figure 4. Eumelanin nanoparticles on a support surface (glass). C

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Figure 5. Spectra of (a) EDX, (b) UV−vis, and (c) FTIR analysis for the obtained melanin particles (straight lines) and a reference eumelanin (dashed lines).

Table 1. Assignment of Vibration Signals from FTIR Analyses wavenumber (cm−1) signal

sample

reference

−OH, −NH, and water band (broad signal) quinone (CO str) indole (ring vib) pyrrole (CC str) pyrrole (N−C str) -COOH substituted pyrrole (DHICA monomer)

3198 1700 1601 1440 1255 1038

3196 1705 1607 1440 1250

Figure 7. Site specific deposition of eumelanin nanoparticles.

support surface, due to the above-mentioned leaking of enzyme into the solution. However, this leakage seems to happen only to a small amount, as the predominant quantity of deposition is found on the enzyme functionalized areas. This is also validated by AFM measurements that show similar results (Figure S4). Additionally, we also applied a linear pattern, which confirms the results (Figure S5).

The results of this experiment are shown in Figure 8. It becomes obvious that indeed only the areas that have been functionalized with the tyrosinase show a high amount of melanin deposition. The nonfunctionalized areas show only very little deposition of particles. Those particles most likely derive from reactions that occur in a higher distance from the

Figure 6. MALDI-TOF MS spectra of (a) sample and (b) reference; (c) Identified structures. D

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A more precise examination of the monolayers shows that different structures can be obtained (Figure 10). First, it is

Figure 8. Representative SEM pictures from the site-specific deposition of eumelanin nanoparticles.

This result proves a high site-specificity of the process even with the simple enzyme immobilization via adsorption. It also proves the similarity of this process to autodeposition techniques, as the production of destabilized particles (here, melanin) occurs in direct proximity to the support surface and results in immediate deposition. 3.3. Structuring of Deposition. A main goal of this work was to gain a high level of control over the deposition of melanin. In this context, it could be observed that the process enables formation of different structures made from the eumelanin nanoparticles, by variation of reaction conditions. In this way, it is possible to get a variable amount of layers of melanin particles. Figure 9 shows some examples. Up to now, the thickness of deposition ranges from about 1 μm down to monolayers with about 30−50 nm (measured under SEM). The amount of layers is influenced by enzyme concentration and reaction time. Quantitative examination of the effects is currently under research, but it can be stated that higher values yield more layers. It is remarkable that the surface of the deposition is mostly very smooth. This speaks for a very uniform deposition and supports the claim of high sitespecificity, as stated above. The coating generally exhibits a high level of homogeneity. Some minor defects are found in the monolayer sample and rarely a small accumulation of single particles is found on top of the layer. This is probably caused by the chosen enzyme tethering approach (i.e., adsorption). More specific binding methods (e.g., covalent immobilization) might result in fewer defects.

Figure 10. SEM pictures of accessible monolayer structures from eumelanin nanoparticles and respective model pictures: (a) Partially coalesced particles, (b) laterally attached particle chains, (c) isolated particles.

possible to get randomly distributed, partially coalesced particles, as expected from the previous results (Figure 10a). Sometimes though, the particles arrange in a way that is best described as lateral stacking of “particle chains” (Figure 10b). It appears that the particles arrange linearly and then laterally attach to each other. At least it seems that a preferred direction for the particles is present. Finally, at very low deposition levels, isolated particles can be obtained (Figure 10c). Interestingly, they appear to have a preferred direction as well, as indicated in the model picture. A final explanation for the existence of such different monolayer structures is not possible at the current state of research. A plausible cause for the preferred direction may be found in the properties of the support surface. Moreover, the

Figure 9. Control over film thickness: (a) multilayer deposition; (b) monolayer deposition. E

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(10) Fisher, O. Z.; Larson, B. L.; Hill, P. S.; Graupner, D.; NguyenKim, M.-T.; Kehr, N. S.; De Cola, L.; Langer, R.; Anderson, D. G. Adv. Mater. 2012, 24, 3032−3036. (11) Nighswander-Rempel, S.; Riesz, J.; Gilmore, J.; Meredith, P. J. Chem. Phys. 2005, 123, 194901−194901−6. (12) Mostert, A. B.; Powell, B. J.; Pratt, F. L.; Hanson, G. R.; Sarna, T.; Gentle, I. R.; Meredith, P. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 8943−8947. (13) Ito, S. Pigment Cells Res. 2003, 16, 230−236. (14) Ikemoto, K.; Nagatsu, I.; Ito, S.; King, R. A.; Nishimura, A.; Nagatsu, T. Neurosci. Lett. 1998, 253, 198−200. (15) Hearing, V. J. J. Invest. Dermatol. 2011, DOI: 10.1038/ skinbio.2011.4. (16) Clancy, C. M. R.; Simon, J. D. Biochemistry 2001, 40, 13353− 13360. (17) Nofsinger, J. B.; Forest, S. E.; Eibest, L. M.; Gold, K. A.; Simon, J. D. Pigment Cell Res. 2000, 13, 179−184. (18) Kim, D. J.; Ju, K. Y.; Lee, J. K. Bull. Korean Chem. Soc. 2012, 33, 3788−3792. (19) Zajac, G. W.; Gallas, J. M.; Cheng, J.; Eisner, M.; Moss, S. C.; Alvarado-Swaisgood, A. E. Biochim. Biophys. Acta 1994, 1199, 271− 278. (20) Strube, O. I.; Rüdiger, A. A.; Bremser, W. J. Biotechnol. 2015, 201, 69−74. (21) Barbosa, O.; Torres, R.; Ortiz, C.; Berenguer-Murcia, Á .; Rodrigues, R. C.; Fernandez-Lafuente, R. Biomacromolecules 2013, 14, 2433−2462. (22) Ju, K. Y.; Lee, Y.; Lee, S.; Park, S. B.; Lee, J. K. Biomacromolecules 2011, 12, 625−632. (23) Kim, D. J.; Ju, K.-Y.; Lee, J.-K. Bull. Korean Chem. Soc. 2012, 33, 3788−3792. (24) Pezzella, A.; Napolitano, A.; Ischia, M.; Prota, G.; Traldi, P. Rapid Commun. Mass Spectrom. 1997, 11, 368−372.

exact conditions to obtain a specific monolayer structure are not yet fully understood and are subject to current and future research. The mere existence of such defined monolayer structures, though, speaks for a higher affinity of the melanin particles to the support surface than to itself and is a promising result for various applications.

4. CONCLUSION We successfully applied an autodeposition-like process for the synthesis and immediate deposition of L-DOPA eumelanin nanoparticles. The resulting particle sizes, of about 30−60 nm, are observed for the fist time, in an enzyme-mediated buildup of melanin. Investigation of the chemical structure and composition has shown a high similarity to natural eumelanin, with monomeric units consisting of DHI and DHICA at different oxidation states. The presented approach enables a high level of control over the deposition, even with the relatively rough enzyme tethering via adsorption. Deposition occurs very site specific on enzyme functionalized areas. The amount of deposed particles can be controlled by variation of reaction conditions, and ranges from the μm level down to different monolayer structures on the nm level. The process may open up many possible applications, as it combines the versatile properties of melanin with the benefit of very small nanoparticles, such as a large surface area, and highly site-specific deposition of monolayers or even isolated particles. Conceivable may be the utilization for biomaterials, nanosensors, UV protection, corrosion protection, or light harvesting.

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ASSOCIATED CONTENT

S Supporting Information *

SEM pictures of tyrosinase coverage and the linear patterning experiment, alongside AFM pictures of eumelanin nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49 5251 602133. Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.biomac.5b00187 Biomacromolecules XXXX, XXX, XXX−XXX