Sub-50 fs Formation of Charge Transfer States Rules the Fate of Photoexcitations in Eumelanin-Like Materials.

Eumelanins play a crucial role as photoprotective agents for living organisms, yet the nature of the stationary and transient species involved in the light absorption and deactivation processes remains controversial. Moreover, the critical sub-100 fs time scale, which is key to the characterization of the primary excited species, has remained unexplored. Here, we study the eumelanin analogue polydopamine (PDA) and employ a combination of steady-state and transient optical spectroscopies to reveal the presence of spectrally broad coupled electronic transitions with, at least partial, charge-transfer (CT) character. We monitor the CT state dynamics using tunable sub-20 fs pulses. We find that high photon energy excitation results in accelerated (sub-20 fs) CT formation times while activating pathways, which lead to long-lived (≫1 ns), possibly reactive CT species. On the other hand, visible light excitation results in a slower (≈45 fs) formation of bound CT states, which, however, recombine on the ultrafast sub-2 ps time scale.

The dual nature of biomimetic melanin.

Melanin-inspired nanomaterials offer unique photophysical, electronic and radical scavenging properties that are widely explored for health and environmental preservation, or energy conversion and storage. The incorporation of functional melanin building blocks in more complex nanostructures or surfaces is typically achieved via a bottom-up approach starting from a molecular precursor, in most cases dopamine. Here we demonstrate that indeed, the oxidative polymerization of dopamine, for the synthesis of melanin-like polydopamine (PDA), leads to the simultaneous formation of more than one nanosized species with different compositions, morphologies and properties. In particular, a low-density polymeric structure and dense nanoparticles (NP) are simultaneously formed. The two populations could be separated and analyzed in real time using a chromatographic technique free of any stationary phase (flow field fractionation, FFF). The results following the synthesis of melanin-like PDA showed that the NP are formed only during the first 6 hours as a result of a supramolecular self-assembly-driven polymerization, while the formation of the polymer continues for about 36 hours. The two populations were also separated and characterized using TEM, UV-vis absorption spectroscopy, fluorescence and light scattering spectroscopy, DLS, FTIR, ζ-potential measurements, gel electrophoresis and pH titrations. Interestingly, very different properties between the two populations were observed: in particular the polymer contains a higher number of catechol units (8 mmol g-1 -OH) with respect to the NP (1 mmol g-1 -OH) and presents a much higher antioxidant activity. The attenuation of light by NP is more efficient than that by the polymer especially in the Vis-NIR region. Moreover, while the NP scatter light with an efficiency up to 27% they are not fluorescent, and the polymer does not scatter light but shows an excitation wavelength-dependent fluorescence typical of multi-fluorophoric uncoupled systems.

Polydopamine-Based Nanoprobes Application in Optical Biosensing.

Polydopamine (PDA), the synthetic counterpart of melanin, is a widely investigated bio-inspired material for its chemical and photophysical properties, and in the last few years, bio-application of PDA and PDA-based materials have had a dramatic increase. In this review, we described PDA application in optical biosensing, exploring its multiple roles as a nanomaterial. In optical sensing, PDA can not only be used for its intrinsic fluorescent and photoacoustic properties as a probe: in some cases, a sample optical signal can be derived by melanin generation in situ or it can be enhanced in another material thanks to PDA modification. The various possibilities of PDA use coupled with its biocompatibility will indeed widen even more its application in optical bioimaging.

Insight into the Antioxidant Activity of 1,8-Dihydroxynaphthalene Allomelanin Nanoparticles.

Melanins are stable and non-toxic pigments with great potential as chemopreventive agents against oxidative stress for medical and cosmetic applications. Allomelanin is a class of nitrogen-free melanin often found in fungi. The artificial allomelanin obtained by the polymerization of 1,8-dihydroxynaphthalene (DHN), poly-DHN (PDHN), has been recently indicated as a better radical quencher than polydopamine (PDA), a melanin model obtained by the polymerization of dopamine (DA); however, the chemical mechanisms underlying this difference are unclear. Here we investigate, by experimental and theoretical methods, the ability of PDHN nanoparticles (PDHN-NP), in comparison to PDA-NP, to trap alkylperoxyl (ROO•) and hydroperoxyl (HOO•) radicals that are involved in the propagation of peroxidation in real conditions. Our results demonstrate that PDHN-NP present a higher antioxidant efficiency with respect to PDA-NP against ROO• in water at pH 7.4 and against mixed ROO• and HOO• in acetonitrile, showing catalytic cross-termination activity. The antioxidant capacity of PDHN-NP in water is 0.8 mmol/g (ROO• radicals quenched by 1 g of PDHN-NP), with a rate constant of 3 × 105 M-1 s-1 for each reactive moiety. Quantum-mechanical calculations revealed that, thanks to the formation of a H-bond network, the quinones in PDHN-NP have a high affinity for H-atoms, thus justifying the high reactivity of PDHN-NP with HOO• observed experimentally.

Optical absorption and dichroism of single melanin nanoparticles.

Melanin nanoparticles (NPs) have important biological functions including photoprotection and colouration, and artificial melanin-like NPs are relevant for catalysis, drug delivery, diagnosis and therapy. Despite their importance, the optical properties of single melanin NPs have not been measured. We combine quantitative differential interference contrast (qDIC) and extinction microscopy to characterise the optical properties of single NPs, both naturally sourced from cuttlefish ink, as well as synthetic NPs using polydopamine (PDA) and L-3,4-dihydroxyphenylalanine (L-DOPA). Combining qDIC with extinction, we determine the absorption index of individual NPs. We find that on average the natural melanin NPs have a higher absorption index than the artificial melanin NPs. From the analysis of the polarisation-dependent NP extinction, the NP aspect ratio is determined, with mean values at 405 nm wavelength in agreement with transmission electron microscopy. At longer wavelengths, we observe an additional optical anisotropy which is attributed to dichroism by structural ordering of the melanin. Our quantitative analysis yields a dichroism of 2-10% of the absorption index, increasing with wavelength from 455 nm to 660 nm for L-DOPA and PDA. Such an in-depth quantification of the optical properties of single melanin NPs is important for the design and future application of these ubiquitous bionanomaterials.

Functionalization of and through Melanin: Strategies and Bio-Applications.

A unique feature of nanoparticles for bio-application is the ease of achieving multi-functionality through covalent and non-covalent functionalization. In this way, multiple therapeutic actions, including chemical, photothermal and photodynamic activity, can be combined with different bio-imaging modalities, such as magnetic resonance, photoacoustic, and fluorescence imaging, in a theragnostic approach. In this context, melanin-related nanomaterials possess unique features since they are intrinsically biocompatible and, due to their optical and electronic properties, are themselves very efficient photothermal agents, efficient antioxidants, and photoacoustic contrast agents. Moreover, these materials present a unique versatility of functionalization, which makes them ideal for the design of multifunctional platforms for nanomedicine integrating new functions such as drug delivery and controlled release, gene therapy, or contrast ability in magnetic resonance and fluorescence imaging. In this review, the most relevant and recent examples of melanin-based multi-functionalized nanosystems are discussed, highlighting the different methods of functionalization and, in particular, distinguishing pre-functionalization and post-functionalization. In the meantime, the properties of melanin coatings employable for the functionalization of a variety of material substrates are also briefly introduced, especially in order to explain the origin of the versatility of melanin functionalization. In the final part, the most relevant critical issues related to melanin functionalization that may arise during the design of multifunctional melanin-like nanoplatforms for nanomedicine and bio-application are listed and discussed.

Effect of Size, Shape and Surface Functionalization on the Antibacterial Activity of Silver Nanoparticles.

Silver nanoparticles (AgNPs) are the most investigated antibacterial agents against multidrug resistant (MDR) pathogens. They can lead to cellular death by means of different mechanisms, damaging several cell compartments, from the external membrane, to enzymes, DNA and proteins; this simultaneous attack amplifies the toxic effect on bacteria with respect to traditional antibiotics. The effectiveness of AgNPs against MDR bacteria is strongly correlated with their chemical and morphological properties, which influence the pathways involved in cellular damage. In this review, AgNPs' size, shape and modification by functional groups or other materials are reported, both to investigate the different synthetic pathways correlated with nanoparticles' modifications and to evaluate the related effect on their antibacterial activity. Indeed, understanding the synthetic conditions for obtaining performing antibacterial AgNPs could help to tailor new and improved silver-based agents to combat multidrug resistance.

Recent Applications of Melanin-like Nanoparticles as Antioxidant Agents.

Nanosized antioxidants are highly advantageous in terms of versatility and pharmacokinetics, with respect to conventional molecular ones. Melanin-like materials, artificial species inspired by natural melanin, combine recognized antioxidant (AOX) activity with a unique versatility of preparation and modification. Due to this versatility and documented biocompatibility, artificial melanin has been incorporated into a variety of nanoparticles (NP) in order to give new platforms for nanomedicine with enhanced AOX activity. In this review article, we first discuss the chemical mechanisms behind the AOX activity of materials in the context of the inhibition of the radical chain reaction responsible for the peroxidation of biomolecules. We also focus briefly on the AOX properties of melanin-like NP, considering the effect of parameters such as size, preparation methods and surface functionalization on them. Then, we consider the most recent and relevant applications of AOX melanin-like NPs that are able to counteract ferroptosis and be involved in the treatment of important diseases that affect, e.g., the cardiovascular and nervous systems, as well as the kidneys, liver and articulations. A specific section will be dedicated to cancer treatment, since the role of melanin in this context is still very debated. Finally, we propose future strategies in AOX development for a better chemical understanding of melanin-like materials. In particular, the composition and structure of these materials are still debated, and they present a high level of variability. Thus, a better understanding of the mechanism behind the interaction of melanin-like nanostructures with different radicals and highly reactive species would be highly advantageous for the design of more effective and specific AOX nano-agents.

Luminescent Gold Nanoclusters for Bioimaging: Increasing the Ligand Complexity.

Fluorescence, and more in general, photoluminescence (PL), presents important advantages for imaging with respect to other diagnostic techniques. In particular, detection methodologies exploiting fluorescence imaging are fast and versatile; make use of low-cost and simple instrumentations; and are taking advantage of newly developed powerful, low-cost, light-based electronic devices, such as light sources and cameras, used in huge market applications, such as civil illumination, computers, and cellular phones. Besides the aforementioned simplicity, fluorescence imaging offers a spatial and temporal resolution that can hardly be achieved with alternative methods. However, the two main limitations of fluorescence imaging for bio-application are still (i) the biological tissue transparency and autofluorescence and (ii) the biocompatibility of the contrast agents. Luminescent gold nanoclusters (AuNCs), if properly designed, combine high biocompatibility with PL in the near-infrared region (NIR), where the biological tissues exhibit higher transparency and negligible autofluorescence. However, the stabilization of these AuNCs requires the use of specific ligands that also affect their PL properties. The nature of the ligand plays a fundamental role in the development and sequential application of PL AuNCs as probes for bioimaging. Considering the importance of this, in this review, the most relevant and recent papers on AuNCs-based bioimaging are presented and discussed highlighting the different functionalities achieved by increasing the complexity of the ligand structure.

Reversible Supramolecular Noncovalent Self-Assembly Determines the Optical Properties and the Formation of Melanin-like Nanoparticles.

The role of noncovalent supramolecular self-assembly in the formation of melanin-like NP, as well as the nature of the electronic transition at the basis of their unique optical properties, is strongly debated. Here we demonstrate that, during the first stage of formation of synthetic melanin, polydopamine (PDA), a small fraction of the molecular precursor dopamine (DA) is oxidized to quinone (Q) and a simple supramolecular charge-transfer (CT) adduct is formed thanks to the electron donor and electron acceptor properties of DA and Q, respectively. This adduct, also detectable by HPLC-MS, presents the broad absorption band in the red-NIR region typical of melanin-like materials. Importantly, its disaggregation upon dilution can be easily detected since it leads to the disappearance of the CT band, indicating the reversibility of the process. Moreover, the stability constant K of the CT adduct could be obtained using a simple association model.

Local detection of pH-induced disaggregation of biocompatible micelles by fluorescence switch ON.

Fluorogenic nanoparticles (NPs) able to sense different physiological environments and respond with disaggregation and fluorescence switching OFF/ON are powerful tools in nanomedicine as they can combine diagnostics with therapeutic action. pH-responsive NPs are particularly interesting as they can differentiate cancer tissues from healthy ones, they can drive selective intracellular drug release and they can act as pH biosensors. Controlled polymerization techniques are the basis of such materials as they provide solid routes towards the synthesis of pH-responsive block copolymers that are able to assemble/disassemble following protonation/deprotonation. Ring opening metathesis polymerization (ROMP), in particular, has been recently exploited for the development of experimental nanomedicines owing to the efficient direct polymerization of both natural and synthetic functionalities. Here, we capitalize on these features and provide synthetic routes for the design of pH-responsive fluorogenic micelles via the assembly of ROMP block-copolymers. While detailed photophysical characterization validates the pH response, a proof of concept experiment in a model cancer cell line confirmed the activity of the biocompatible micelles in relevant biological environments, therefore pointing out the potential of this approach in the development of novel nano-theranostic agents.

Photothermal motion: effect of low-intensity irradiation on the thermal motion of organic nanoparticles.

The effect of local photo-triggered heat release on the motion of organic nanopartcles (NP), a process that is itself thermal, is largely unexplored under low-intensity irradiation. Here, we develop organic NP specifically tailored for this study and demonstrate, comparing three different irradiation intensity regimes, that indeed the NP undergo "acceleration" upon light absorption (Photothermal Motion). These NP have a well-defined chemical composition and extremely high molar absorbance coefficient, and upon excitation, they deactivate mostly non radiatively with localized heat dissipation. The residual fluorescence efficiency is high enough to allow the detection of their trajectory in a simple wide field fluorescence microscope under low-intensity irradiation, a typical condition for NP bio-applications. The NP were characterized in detail from the photophysical point of view using UV-VIS absorption, steady-state and time-resolved fluorescence spectroscopy and ultra-fast transient absorption (UF-TA). A detailed analysis of the trajectories of the NP reveals a strong dependency of the diffusion coefficient on the irradiation intensity even in a low power regime. This behavior demonstrates the inhomogeneity of the environment surrounding the NP as a result of local heat generation. Upon irradiation, the effective temperature increase, that emerges from the analysis, is much larger than that expected for plasmonic NP. Anomalous diffusion object-motion analysis (ADOMA) revealed that, in the more intense irradiation regime, the motion of the NP is a fractional Brownian motion, which is a simple generalization of Brownian motion where the steps are not independent of each other.

The Photophysics and Photochemistry of Melanin- Like Nanomaterials Depend on Morphology and Structure.

Melanin-like nanomaterials have found application in a large variety of high economic and social impact fields as medicine, energy conversion and storage, photothermal catalysis and environmental remediation. These materials have been used mostly for their optical and electronic properties, but also for their high biocompatibility and simplicity and versatility of preparation. Beside this, their chemistry is complex and it yields structures with different molecular weight and composition ranging from oligomers, to polymers as well as nanoparticles (NP). The comprehension of the correlation of the different compositions and morphologies to the optical properties of melanin is still incomplete and challenging, even if it is fundamental also from a technological point of view. In this minireview we focus on scientific papers, mostly recent ones, that indeed examine the link between composition and structural feature and photophysical and photochemical properties proposing this approach as a general one for future research.

Local Light-Controlled Generation of Calcium Carbonate and Barium Carbonate Biomorphs via Photochemical Stimulation.

Photochemical activation is proposed as a general method for controlling the crystallization of sparingly soluble carbonates in space and time. The photogeneration of carbonate in an alkaline environment is achieved upon photo-decarboxylation of an organic precursor by using a conventional 365 nm UV LED. Local irradiation was conducted focusing the LED light on a 300 µm radius spot on a closed glass crystallization cell. The precursor solution was optimized to avoid the precipitation of the photoreaction organic byproducts and prevent photo-induced pH changes to achieve the formation of calcium carbonate only in the corresponding irradiated area. The crystallization was monitored in real-time by time-lapse imaging. The method is also shown to work in gels. Similarly, it was also shown to photo-activate locally the formation of barium carbonate biomorphs. In the last case, the morphology of these biomimetic structures was tuned by changing the irradiation intensity.

A Selective Ratiometric Fluorescent Probe for No-Wash Detection of PVC Microplastic.

Microplastics (MP) are micrometric plastic particles present in drinking water, food and the environment that constitute an emerging pollutant and pose a menace to human health. Novel methods for the fast detection of these new contaminants are needed. Fluorescence-based detection exploits the use of specific probes to label the MP particles. This method can be environmentally friendly, low-cost, easily scalable but also very sensitive and specific. Here, we present the synthesis and application of a new probe based on perylene-diimide (PDI), which can be prepared in a few minutes by a one-pot reaction using a conventional microwave oven and can be used for the direct detection of MP in water without any further treatment of the sample. The green fluorescence is strongly quenched in water at neutral pH because of the formation dimers. The ability of the probe to label MP was tested for polyvinyl chloride (PVC), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), poly methyl methacrylate (PMMA) and polytetrafluoroethylene (PTFE). The probe showed considerable selectivity to PVC MP, which presented an intense red emission after staining. Interestingly, the fluorescence of the MP after labeling could be detected, under excitation with a blue diode, with a conventional CMOS color camera. Good selectivity was achieved analyzing the red to green fluorescence intensity ratio. UV-Vis absorption, steady-state and time-resolved fluorescence spectroscopy, fluorescence anisotropy, fluorescence wide-field and confocal laser scanning microscopy allowed elucidating the mechanism of the staining in detail.

Extending photocatalysis to the visible and NIR: the molecular strategy.

Photocatalysis exploits light to perform important processes as solar fuel production by water splitting, and CO2 reduction or water and air decontamination. Therefore, photocatalysis contributes to the satisfaction of the increasing needs for clean energy, environmental remediation and, most recently, sanification. Most of the efficient semiconductor nanoparticles (NP), developed as photocatalysts, work in the ultraviolet (UV) spectral region and they are not able to exploit either visible (Vis) or near infrared (NIR) radiation. This limitation makes them unable to fully exploit the broad band solar radiaton or to be applied in indoor conditions. Recently, different approaches have been developed to extend the spectral activity of semiconductor NP, like for example band-gap engineering, integration with upconversion NP and plasmonic enhancement involving also hot-electron injection. Nevertheless, the use of organic molecules and metal complexes, for enhancing the photoactivity in the Vis and NIR, was one of the first strategies proposed for sensitization and it is still one of the most efficient. In this minireview we highlight and critically discuss the most recent and relevant achievements in the field of photocatalysis obtained by exploiting dye sensitization either via dynamic or static quenching.

Bio-Applications of Multifunctional Melanin Nanoparticles: From Nanomedicine to Nanocosmetics.

Bioinspired nanomaterials are ideal components for nanomedicine, by virtue of their expected biocompatibility or even complete lack of toxicity. Natural and artificial melanin-based nanoparticles (MNP), including polydopamine nanoparticles (PDA NP), excel for their extraordinary combination of additional optical, electronic, chemical, photophysical, and photochemical properties. Thanks to these features, melanin plays an important multifunctional role in the design of new platforms for nanomedicine where this material works not only as a mechanical support or scaffold, but as an active component for imaging, even multimodal, and simple or synergistic therapy. The number of examples of bio-applications of MNP increased dramatically in the last decade. Here, we review the most recent ones, focusing on the multiplicity of functions that melanin performs in theranostics platforms with increasing complexity. For the sake of clarity, we start analyzing briefly the main properties of melanin and its derivative as well as main natural sources and synthetic methods, moving to imaging application from mono-modal (fluorescence, photoacoustic, and magnetic resonance) to multi-modal, and then to mono-therapy (drug delivery, anti-oxidant, photothermal, and photodynamic), and finally to theranostics and synergistic therapies, including gene- and immuno- in combination to photothermal and photodynamic. Nanomedicine aims not only at the treatment of diseases, but also to their prevention, and melanin in nature performs a protective action, in the form of nanopigment, against UV-Vis radiations and oxidants. With these functions being at the border between nanomedicine and cosmetics nanotechnology, recently examples of applications of artificial MNP in cosmetics are increasing, paving the road to the birth of the new science of nanocosmetics. In the last part of this review, we summarize and discuss these important recent results that establish evidence of the interconnection between nanomedicine and cosmetics nanotechnology.