Artificial Melanogenesis by Confining Melanin/Polydopamine Production inside Polymersomes.

Melanin and polydopamine are potent biopolymers for the development of biomedical nanosystems. However, applications of melanin or polydopamine-based nanoparticles are limited by drawbacks related to a compromised colloidal stability over long time periods and associated cytotoxicity. To overcome these hurdles, a novel strategy is proposed that mimics the confinement of natural melanin in melanosomes. Melanosome mimics are developed by co-encapsulating the melanin/polydopamine precursors L-DOPA/dopamine with melanogenic enzyme Tyrosinase within polymersomes. The conditions of polymersome formation are optimized to obtain melanin/polydopamine polymerization within the cavity of the polymersomes. Similar to native melanosomes, polymersomes containing melanin/polydopamine show long-term colloidal stability, cell-compatibility, and potential for cell photoprotection. This novel kind of artificial melanogenesis is expected to inspire new applications of the confined melanin/polydopamine biopolymers.

Propionic acid produced by Cutibacterium acnes fermentation ameliorates ultraviolet B-induced melanin synthesis.

Ultraviolet irradiation induces melanin accumulation, which can be reduced by the use of chemical whitening products. However, the associated safety concerns of such products have prompted the search for natural and harmless alternatives. This study aimed to identify a natural acidic formulation to reduce skin pigmentation. The metabolite propionic acid (CH3CH2COOH, PA) was the most abundant fatty acid in the filtrate from Pluronic F68 (PF68) fermentation of Cutibacterium acnes (C. acnes) and reduced the DOPA-positive melanocytes by significantly inhibiting cellular tyrosinase activity via binding to the free fatty acid receptor 2 (FFAR2). Moreover, 4 mM PA treatment did not alter melanocyte proliferation, indicating that it is an effective solution for hyperpigmentation, causing no cellular damage. The reduced DOPA-positive melanocytes and tyrosinase activity were also observed in mice ear skin tissue injected with a mixture of C. acnes and PF68, supporting that the inhibition of melanogenesis is likely to be mediated through fermentation metabolites from C. acnes fermentation using PF68 as a carbon source. Additionally, PA did not affect the growth of its parent bacteria C. acnes, hence is a potent fermentation metabolite that does not disrupt the balance of the skin microbiome.

Synthetic Porous Melanin.

Commonly known as a skin pigment, melanin has a vital role in UV radiation protection, primarily acting as a radical scavenger. However, a lesser known natural property of melanin, observed in some melanized organisms, is its capacity to adsorb toxins, including metals and organic molecules. Inspired by this, we set out to generate a synthetic porous melanin that would pave the way to enhancing the natural adsorbent properties of melanin and melanin-like materials. Here, we developed a method for the synthesis of porous polydopamine-based melanin utilizing a mesoporous silica (MS) nanoparticle template and characterized its physical properties. Through the oxidative polymerization of dopamine, followed by the etching of silica, we generated synthetic porous melanin (SPM) with the highest measured surface area of any known polydopamine-based material. The prepared SPM was effective for the uptake of various gases and organophosphate toxins, with the material exhibiting high selectivity for CO2 over CH4 and high potential for ammonia capture. Given the demonstrated advantages provided by synthetic porous melanin and melanin's role as an adsorbent in nature, we anticipate the discovery of porous analogues in biological systems.

Study of tyrosine and dopa enantiomers as tyrosinase substrates initiating l- and d-melanogenesis pathways.

Tyrosinase starts melanogenesis and determines its course, catalyzing the oxidation by molecular oxygen of tyrosine to dopa, and that of dopa to dopaquinone. Then, nonenzymatic coupling reactions lead to dopachrome, which evolves toward melanin. Recently, it has been reported that d-tyrosine acts as tyrosinase inhibitor and depigmenting agent. The action of tyrosinase on the enantiomers of tyrosine (l-tyrosine and d-tyrosine) and dopa (l-dopa and d-dopa) was studied for the first time focusing on quantitative transient phase kinetics. Post-steady-state transient phase studies revealed that l-dopachrome is formed more rapidly than d-dopachrome. This is due to the lower values of Michaelis constants for l-enantiomers than for d-enantiomers, although the maximum rates are equal for both enantiomers. A deeper analysis of the inter-steady-state transient phase of monophenols demonstrated that the enantiomer d-tyrosine causes a longer lag period and a lower steady-state rate, than l-tyrosine at the same concentration. Therefore, d-melanogenesis from d-tyrosine occurs more slowly than does l-melanogenesis from l-tyrosine, which suggests the apparent inhibition of melanin biosynthesis by d-tyrosine. As conclusion, d-tyrosine acts as a real substrate of tyrosinase, with low catalytic efficiency and, therefore, delays the formation of d-melanin.

Facile Synthesis of Melanin-Dye Nanoagent for NIR-II Fluorescence/Photoacoustic Imaging-Guided Photothermal Therapy.

BACKGROUND: Laryngeal cancer is the second most common type of primary epithelial malignant tumor in the head and neck region, and the development of therapies that are more precise, efficient, and safe is necessary to preserve patient speech and swallowing functions as much as possible. Multi-modal imaging-guided photothermal therapy (PTT) can precisely delineate tumors, monitor the real-time accumulation of photothermal agents at the tumor site, accurately select the optimal region for irradiation, and predict the best time for laser treatment. Compared with exogeneous photothermal agents, endogenous melanin materials have better biosafety in vivo, in terms of native biocompatibility and biodegradability, as well as good near-infrared (NIR) absorbance. An NIR-II dye can be attached to melanin via a facile method, and applying a melanin-dye-based nanoprobe could be an excellent choice for the elimination of superficial laryngeal cancer while avoiding total laryngectomy. METHODS: In this work, a promising nanoprobe was constructed using a facile EDC/NHS strategy involving an NIR-II dye and melanin nanoparticles. RESULTS: The nanoprobe exhibited good water solubility, dispersibility, strong NIR-II fluorescence and photoacoustic (PA) signals, and higher photothermal performance. Cellular studies showed that the nanoprobe had low toxicity, excellent biocompatibility, and significantly enhanced imaging properties. After the nanoprobe was intravenously injected into Hep-2 laryngeal xenografts, superior dual-modal images were obtained at various time points, which revealed that the optimal photothermal treatment time was 8 h. Subsequently, PTT was carried out in vivo, and laryngeal tumors were completely eliminated after laser irradiation without any obvious side effects. CONCLUSION: These results indicate the immense potential of nanoprobes for the NIR-II fluorescence/PA imaging-guided photothermal therapy of laryngeal cancer.

Mimicking neuromelanin nanoparticles as a selective Pb2+ probe.

Exposure to even very low concentration of Pb2+ can cause neurological and developmental disorders, and affects children more severely. Neuromelanin (NM) in the substantial nigra can trap endogenous and environmental toxins and immobilize them as stable adducts, thus protecting brain against toxicity. Until now, the structure and Pb2+-chelating ability of NM is still little understood. Here, we prepare a mimicking NM by amino acid and dopamine. The prepared NM has strong fluorescent and its fluorescence can be quenched by Pb2+. This study offers a novel way to synthetic NM and provides an effective method to detect Pb2+.

Multifaceted Influences of Melanin-Like Particles on Amyloid-beta Aggregation.

The properties of eumelanin-like particles (EMPs) and pheomelanin-like particles (PMPs) in regulating the process of amyloid formation of amyloid-beta 42 (Aß42) were examined. EMPs and PMPs are effective both in interfering with amyloid aggregation of Aß42 and in remodeling matured Αß42 fibers. The results suggest that some (but not all) molecular species consisting of melanin-like particles (MPs) are responsible for their inhibiting property toward amyloid formation, and the influence is likely manifested by long-range interactions. Incubating preformed Aß42 fibers with catechols or MPs leads to the formation of mesh-like, interconnected Aß42 fibers encapsulated with melanin-like material. MPs are kinetically more effective than catechol monomers in this process, and a detailed investigation reveals that 4,5-dihydroxyindole, a major intermediate in the formation of melanin-like species, and its derivatives are mainly responsible for remodeling amyloid fibers.

A new ratiometric electrochemical immunoassay for reliable detection of nuclear matrix protein 22.

Ratiometric signal transducing strategies can improve the precision of immunoassay, which possess the unique merits of spatial-resolved signal readout and self-correcting towards possible false positive results. In this work, a new ratiometric electrochemical immunosensor has been developed for reliable detection of Nuclear matrix protein 22 (NMP22). Bioinspired synthetic melanin nanospheres (SMNPs) were elaborately chosen for the following considerations: 1) SMNPs can supply good biocompatible biorecognition interface for antibody anchoring; 2) SMNPs can chelate a large amount of lead ions (Pb2+) and copper ions (Cu2+) to fabricate two spatial-resolved electrochemical signals with different response manners towards NMP22. SMNPs chelated with Pb2+ were used for the immobilization of captured primary anti-NMP22. And SMNPs chelated Cu2+ were employed to prepare signal labels after anchored with anti-NMP22 antibody. After sandwich-type immunoreaction, with the increasing concentration of NMP22, the stripping peak current of Pb2+ decreases while the stripping peak current of Cu2+ increases. Thus, ratiometric electrochemical signals could be provided for NMP22 detection. The immunosensor presented a linear concentration range from 0.013 U mL-1 to 6.7 U mL-1 with a limit of detection of 0.005 U mL-1 towards NMP22. Meanwhile, satisfactory reproducibility, stability and selectivity of the immunosensor were demonstrated.

Application of transition metal ions in a study of photoinduced modifications of melanin.

Short wavelength visible light is viewed as the main agent responsible for oxidative modification of melanin in the human retinal pigment epithelium (RPE). The aim of this research was to study light-induced modifications of melanin using iron and zinc as molecular probes. A synthetic model of eumelanin was treated by intense violet light. The interaction of melanin with metal ions was examined by electron paramagnetic resonance (EPR) spectroscopy and a thiocyanate assay. Weak photodegradation of melanin was shown to increase exposure of melanin subunits, while stronger photodegradation caused a loss of melanin subunits. Iron-binding in such melanin was weak and nonspecific.

Melanin production by tyrosinase activity on a tyrosine-rich peptide fragment and pH-dependent self-assembly of its lipidated analogue.

We investigate the self-assembly of a palmitoylated (C16-chain at the N terminus) peptide fragment in comparison to the unlipidated peptide EELNRYY, a fragment of the gut hormone peptide PYY3-36. The lipopeptide C16-EELNRYY shows remarkable pH-dependent self-assembly above measured critical aggregation concentrations, forming fibrils at pH 7, but micelles at pH 10. The parent peptide does not show self-assembly behaviour. The lipopeptide forms hydrogels at sufficiently high concentration at pH 7, the dynamic mechanical properties of which were measured. We also show that the tyrosine functionality at the C terminus of EELNRYY can be used to enzymatically produce the pigment melanin. The enzyme tyrosinase oxidises tyrosine into 3,4-dihydroxyphenylalanine (DOPA), DOPA-quinone and further products, eventually forming eumelanin. This is a mechanism of photo-protection in the skin, for this reason controlling tyrosinase activity is a major target for skin care applications and EELNRYY has potential to be developed for such uses.

Understanding the Role of Aggregation in the Broad Absorption Bands of Eumelanin.

In this work, we investigate the relationship between the complex hierarchical assembly structure of eumelanin, its characteristic broad absorption band, and the highly unusual nonlinear dynamics revealed by pump-probe or transient absorption microscopy. Melanin-like nanoparticles (MelNPs), generated by spontaneous oxidation of dopamine, were created with uniform but adjustable size distributions, and kinetically controlled oxidation was probed with a wide range of characterization methods. This lets us explore the broad absorption bands of eumelanin models at different assembly levels, such as small subunit fractions (single monomeric and oligomeric units and small oligomer stacks), stacked oligomer fractions (protomolecules), and large-scale aggregates of protomolecules (parental particles). Both the absorption and pump-probe dynamics are very sensitive to these structural differences or to the size of intact particles (a surprising result for an organic polymer). We show that the geometric packing order of protomolecules in long-range aggregation is key secondary interactions to extend the absorption band of eumelanin to the low energy spectrum and produce drastic changes in the transient absorption spectrum.

Synthetic melanin bound to subunit vaccine antigens significantly enhances CD8+ T-cell responses.

Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8-35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8+ lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant.

Synthesis, Structure Characterization, and Evaluation in Microglia Cultures of Neuromelanin Analogues Suitable for Modeling Parkinson's Disease.

In the substantia nigra of human brain, neuromelanin (NM) released by degenerating neurons can activate microglia with consequent neurodegeneration, typical of Parkinson's disease (PD). Synthetic analogues of NM were prepared to develop a PD model reproducing the neuropathological conditions of the disease. Soluble melanin-protein conjugates were obtained by melanization of fibrillated ß-lactoglobulin (fLG). The melanic portion of the conjugates contains either eumelanic (EufLG) or mixed eumelanic/pheomelanic composition (PheofLG), the latter better simulating natural NMs. In addition, the conjugates can be loaded with controlled amounts of iron. Upon melanization, PheofLG-Fe conjugates maintain the amyloid cross-ß protein core as the only structurally organized element, similarly to human NMs. The similarity in composition and structural organization with the natural pigment is reflected by the ability of synthetic NMs to activate microglia, showing potential of the novel conjugates to model NM induced neuroinflammation. Thus, synthetic NM/microglia constitute a new model to develop anti-Parkinson drugs.

Energetics of Radical Formation in Eumelanin Building Blocks: Implications for Understanding Photoprotection Mechanisms in Eumelanin.

The supramolecular structure of melanin pigments is characterized by a high concentration of radical species. Therefore, the energetics of the radical formation in melanin building blocks is key for understanding the structure and the electronic properties of the pigments at the molecular level. Nevertheless, the radical energetics of even the simplest melanin building blocks are largely unknown. In order to address this fundamental issue, the bond dissociation enthalpies (BDEs) for the melanin monomers 5,6-dihydroxy-1H-indole-2-carboxylic acid (DHICA), 1H-indole-5,6-diol (DHI), and 1H-indole-5,6-dione (IQ) were determined through high-accuracy ab initio quantum chemistry methods. Our results provide strong evidence of the importance on BDEs for explaining the experimentally observed dependence of the antioxidant properties of eumelanin pigments on the DHICA/DHI ratio, and the role that these two species play on the photoprotection mechanism.

Superior Performance of Polyurethane Based on Natural Melanin Nanoparticles.

Melanin, a kind of well-known multifunctional biomacromolecules that are widely distributed in natural sources. In this work, polyurethane (PU)/melanin nanocomposites with enhanced tensile strength and toughness were successfully fabricated via in situ polymerization. It was found that the tensile strength (σ), elongation-at-break (εmax), and toughness (W) were improved from 5.6 MPa, 770%, and 33 MJ/m3 for PU to 51.5 MPa, 1880%, and 413 MJ/m3 for PU/melanin (2 wt %) nanocomposite, respectively. Micromorphology indicated that individualized melanin nanoparticles were specifically linked to the hard domains of PU chains and fine dispersed in matrix. FTIR, DSC, and AFM results suggested melanin induced an improvement in degree of phase separation, which resulted in remarkable enhancements in mechanical properties of PU. However, with further increasing content of melanin, a relatively large-scale phase separation was formed and led to a decrease in mechanical properties of PU. In addition, interactions between melanin and hard segments of PU were increased, leading to a higher TgHS. Moreover, the dynamic mechanical properties and rheological behavior of PU/melanin nanocomposites were further investigated.

Saturation Binding of Nicotine to Synthetic Neuromelanin Demonstrated by Fluorescence Spectroscopy.

Neuromelanin (NM) has long been considered as an aging pigment, perhaps an unavoidable and undesirable byproduct of dopaminergic neural transmission. However, NM is carefully packaged into double membrane-bound structures within cells of the substantia nigra and other neural tissues, suggesting a beneficial function to maintaining these stores. It is well established that NM is able to concentrate toxic xenobiotics within pigmented cells due to its unique chemical environment. In doing so, such agents may confer susceptibility to Parkinson's disease (PD) as illustrated by model PD-inducing neurotoxins such as methyl-phenyl-pyridinium ion. It is possible that high-affinity binding interactions toward NM may contribute to the adverse effects of PD-inducing toxins, as well as neuroprotective agents. Here we aim to develop a generalized assay capable of elucidating the binding constants of chemical agents to synthetic and natural neuromelanins. Toward this end, a model neuromelanin synthesized from dopamine and cysteine was prepared according to published procedure. Using a UV/Visible spectroscopic assay, we show that dopamine, 6-hydroxy dopamine, and nicotine bind to the synthetic neuromelanin, while caffeine did not. More importantly, nicotine was further found to induce a fluorescence signal in the presence of NM which was used to establish a binding constant estimated at 0.65 mM. Dopamine appears to enhance this signal, also in a saturable manner, with an estimated Kd of 0.05 mM in our isolated chemical system. In summary, the micro-scale fluorescence assay described herein will allow us to overcome many of the problems inherent in the study of chemical interaction with NM through traditional spectroscopic means. Using a single standardized signal, it should now be possible to rank a number of PD-related toxins based on NM-binding affinity and shed further light on this important problem.

A simple one-step synthesis of melanin-originated red shift emissive carbonaceous dots for bioimaging.

Carbonaceous dots (CDs) are superior nanomaterials owing to their promising luminescence properties and good biocompatibility. However, most CDs have relatively short excitation/emission, which restrict their application in bioimaging. In this study, a simple one-step procedure was developed for synthesis of melanin-originated CDs (MNPs). The MNPs showed two long red shift emissions at 570nm and 645nm with broad absorptions from 200nm to 400nm and 500nm to 700nm, suggesting the great potential of MNPs in bioimaging. Besides, several experiments indicated that MNPs possessed good serum stability and well blood compatibility. In vitro, MNPs could be taken up by C6 cell in a concentration- and time-dependent manner with endosomes involved. In conclusion, MNPs were prepared using a simple one-step method with unique optical and good biological properties and could be used for bioimaging.

Bio-Inspired Structural Colors Produced via Self-Assembly of Synthetic Melanin Nanoparticles.

Structural colors arising from interactions of light with submicron scale periodic structures have been found in many species across all taxa, serving multiple biological functions including sexual signaling, camouflage, and aposematism. Directly inspired by the extensive use of self-assembled melanosomes to produce colors in avian feathers, we set out to synthesize and assemble polydopamine-based synthetic melanin nanoparticles in an effort to fabricate colored films. We have quantitatively demonstrated that synthetic melanin nanoparticles have a high refractive index and broad absorption spanning across the UV-visible range, similar to natural melanins. Utilizing a thin-film interference model, we demonstrated the coloration mechanism of deposited films and showed that the unique optical properties of synthetic melanin nanoparticles provide advantages for structural colors over other polymeric nanoparticles (i.e., polystyrene colloidal particles).

Site-specific in situ synthesis of eumelanin nanoparticles by an enzymatic autodeposition-like process.

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.