Eumelanin 3D Architectures: Electrospun PLA Fiber Templating for

Mario Barra , Irene Bonadies , Cosimo Carfagna , Antonio Cassinese , Francesca Cimino , Orlando Crescenzi , Valeria Criscuolo , d'Ischia Marco , Maria...
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Eumelanin 3D Architectures: Electrospun PLA Fiber Templating for Mammalian Pigment Microtube Fabrication Irene Bonadies,† Francesca Cimino,† Cosimo Carfagna,†,‡ and Alessandro Pezzella*,‡,§ †

Institute for Polymers, Composites and Biomaterials (IPCB), CNR, Via Campi Flegrei 34, 80078 Pozzuoli (Na), Italy Department of Chemical, Materials and Production Engineering (DICMAPI), University of Naples Federico II, P. le Tecchio 80, 80125 Napoli, Italy § Department of Chemical Sciences, University of Naples “Federico II”, Via Cintia 4, I-80126 Naples, Italy ‡

S Supporting Information *

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INTRODUCTION Eumelanin-film-coated materials have garnered considerable interest for a variety of applications requiring functional materials in different fields, including biomedicine and organic (bio)electronics.1,2 From a structural point of view, eumelanins appear as complicated disordered mixtures of different oligomeric and polymeric species arising by oxidative polymerization of the two indoles DHI and DHICA. (See SI Scheme 1.)3 Both physicochemical properties2,4 and structural features2,4 are the basis of the interest toward this pigment in material sciences.1 At the same time, the eumelanin biocompatibility5 makes this biopolymer a candidate leading choice in applications requiring functional interfaces connecting biological systems to electrical devices1,6 including: (a) devices translating optical and electronic stimuli into biosignals for engineering functional biological tissues;7 (b) substrates for advanced cell culture systems and single-cell managing,8 and (c) devices for cell sorting and differentiation.9 In this scenario, a series of recent studies have appeared reporting eumelanin-based functional devices.10,11 Key results include films for nerve tissue engineering7 and nonwoven fibers for cardiac tissue engineering.5 However, the applicability of eumelanin thin films is strongly restricted by the highly intractable nature of this human pigment arising by the oxidative metabolism of tyrosine.2,4 A series of protocols have been developed for eumelanin thin -ilm fabrication, including wet coating,10,12 laser desorption,13,14 and electrospray,15 but none of these resulted in being able to provide fully satisfying films in terms of morphological and structural/functional properties. Moreover, to the best of our knowledge, any effort has failed to go beyond eumelanin 2D thin-film fabrication, and actually no study referring to 3D or 1D eumelanin architecture assembly has been reported. Microtubular architectures have a key role in a large number of applications including cell surface interaction,16 drug delivery,17 bacterial trapping,18 optical waveguide,19 microfluidic applications,20 organic electronics,21 energy technology,22 and molecular skeleton.23 In particular, carbon-based microtubes can be key candidates24 for the long-term goal of mimicking the properties of microtubules and microtubule bundles toward functional nanomaterials for exo- and endocellular applications.25 © XXXX American Chemical Society

A number of different approaches have been developed to access carbon-based microtubes including self-assembling,17 self-rolling,26 sacrificial templating,16 and electrospinning.22 Nonetheless, each of these approaches fails to operate with eumelanins chiefly because of their insolubility in any solvent that confined the exploitation of eumelanins in fiber technology to the role of additive in substrates.27 We present the design and fabrication of eumelanin-based microtubular structures disclosing a huge potential for molding applications of this natural biopolymer beyond thin films. To achieve microtubular architectures by eumelanin, thus expanding the scope of the possible exploitation of this pigment in bioengineering, we devised an ad hoc robust and versatile protocol combining electrospinning, spin coating, solid-state polymerization28 and TUFT (TUbes by Fiber Templates) process29 in a robust and versatile procedure.

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MATERIALS AND METHODS

All commercially available reagents were used as received, and all of the solvents were analytical-grade quality. Anhydrous solvents were purchased from commercial sources and withdrawn from the container by syringe under a slight positive pressure of argon. 5,6Dihydroxindole (DHI) was prepared according to a reported procedure.30 Gel permeation chromatography (GPC) analysis of the as-received PLA pellets revealed a number-average molecular mass Mn = 94.000 and a weight-average molecular mass Mw = 139.000, with a polydispersity Mw/Mn = 1.48. Detailed procedures are reported in the Supporting Information (SI).

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RESULTS AND DISCUSSION Thanks to the availability of a solid-state protocol for eumelanin preparation,28 microscale eumelanin coating on different substrates was investigated. The protocol to realize eumelanin microtubes can be summarized as follows: (1) PLA fibers electrospinning; (2) DHI coating of PLA fibers via spin-casting; (3) eumelanin synthesis by solid-state oxidative polymerization of DHI; and (4) PLA template removal by organic solvent treatment. (See Figure 1.) The key step of the protocol is the ammonia-induced solid-state polymerization (AISSP) of the eumelanin precursors 5,6-dihydroxyindole (DHI), which is able to provide high-quality eumelanin constructs.28 Received: February 23, 2015 Revised: April 3, 2015

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DOI: 10.1021/acs.biomac.5b00239 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Key steps of the eumelanin microtube fabrication: (1) PLA electrospinning; (2) DHI coating of PLA fibers; (3) oxidative polymerization of DHI-coated fibers; (4) organic solvent PLA template removal. See SI section S3.

the different nature of the surface exposed material of the two samples. (See SI section S4.) In the last step of microtube fabrication, after the “eumelanin wall” was formed, the removal of the PLA core was achieved by its solubilization in chloroform. The washing protocol had to be optimized to prevent dissolved PLA from remaining over the fibers. A washing treatment by gently fluxing chloroform over the aluminum-foilsupported fibers allowed us to get complete PLA removal and high-quality microtubes. The morphological analysis of microtubes (Figure 3) highlights a clear formation of tubular needles featuring little polydispersity in the diameters, ranging from ∼1 μm or more, and an average wall thickness of around 200−250 nm. In Figure 3a, a microtube sample before PLA removal is reported before and after brittle fracture by stretching. The inner PLA core emerging from the exterior eumelanin coating as consequence of the different mechanical properties of PLA and eumelanin is clearly distinguishable. Although substrates were gold−palladium sputter-coated, it was impossible to avoid some electron-beam-induced deterioration of the fibers, as more clearly evident in SI section S5. As shown in Figure 3b, the well-defined hollow eumelanin tubular structure emerges after PLA removal by chloroform washing, thus revealing the dense texture of the eumelanin wall. It is worth noting here how this result was made possible by the availability of the AISSP protocol,28 which allowed us to circumvent the severe issues associated with eumelanin processing.

To gain access to microtubular structures, the templating material has to satisfy several issues such as easy processability, orthogonal solubility to DHI, stability under the AISSP conditions, and easy removability. Poly(lactic acid) (PLA) proved to be the most appropriate candidate for TUFT process31 because it can be electrospun into fibers with diameters in the micrometer/submicrometer range32 and easily removed by solvent extraction. PLA solution (5 to 10 w/v% in chloroform/dimethylformamide 90/10) was electrospun at a flow rate of 3 mL/h, room temperature, and 30% relative humidity. Not woven films were collected on an aluminum foil to allow subsequent DHI deposition and polymerization to get eumelanin coating of the PLA fibers. (See SI section S3.) The morphology of electrospun microfibers was evaluated by a field-emission scanning electron microscopy (SEM) revealing fibers with diameters in the range 400−800 nm, a random distribution on the substrate, and a quite smooth surface (Figure 2a). Electrospun fibers were subjected to DHI coating by both drop casting and spin coating from a methanol solution of DHI. After several optimization trials the spin-coating technique was preferred and a 30 mg/mL methanol solution of the indole was used. SEM micrographs of DHI coated PLA fibers reveal a neat and uniform covering of the fibers as a result of the good solubility of DHI in methanol and the stability of PLA fibers toward this solvent. Even in the presence of a packed fiber distribution, the coating resulted in a clean covering of the fibers showing very little veiling over adjacent fibers (Figure 2b). DHI-coated fibers were thus subjected to the AISSP protocol involving exposure to a NH3/O2 atmosphere under standard conditions for 2 h (see SI for details) to obtain eumelanin formation by oxidative polymerization DHI encompassing PLA fibers. After optimization of ammonia exposition time and temperature we finally got high-quality eumelanin coating even amenable of subsequent reiterations of the overall protocol since the step of DHI coating (Figure 2c). As expected, the AISSP did not modify the morphology of the fiber and produced a dark eumelanin shell featuring the typical insolubility in any solvent. The eumelanin formation after exposition to polymerization-inducing atmosphere was confirmed by IR spectroscopy. (See SI section S2.) Energy-dispersive X-ray spectroscopy (EDS) provided quantitative analysis of elemental composition of PLA fibers before and after eumelanin coating. Although EDS data cannot provide accurate elemental quantitation, the C/O ratios in uncoated (C/O = 1.6) and coated (C/O = 2.3) fibers and the detection of N (12 wt %) in this latter are quite consistent with

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CONCLUSIONS

In this work, we addressed and disclosed the potential of eumelanin pigment to be shaped into 3D tubular structures. Eumelanin tubes with submicrometer to micrometer diameter have been prepared through an ad hoc robust and versatile procedure allowing the tubular morphology to be systematically controlled by the process conditions. The inner diameters of the tubular structures replicate the diameters of the PLA template fibers and can be controlled by the size of the electrospun template fibers (ranging from 500 nm to 1 μm). The outer diameter of the tubes is dictated by the thickness of the eumelanin wall, which in turn is controlled by the coating conditions, in particular, by the concentration of DHI and the iteration of the coating and oxidation steps. (See SI sections S4−S6.) The resulting nano- and microtubes have one dimension on the microscopic scale but another dimension on the macroscopic one. The macroscopic length of the tubes realized via electrospinning as well as 3D open porous structure helps to allow their easy manipulation and integration in nano- and B

DOI: 10.1021/acs.biomac.5b00239 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 3. Sample of eumelanin tube (a) before and (b) after PLA core removal.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental methods, substrate characterization, and pictures. 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]. Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS We thank Mrs. Maria Cristina del Barone at the Transmission and Scanning Electron Microscopy Laboratories (LAMEST) of the IPCB-CNR for providing SEM images. This work was supported by a grant from Italian MIUR (PRIN 2010-2011, 010PFLRJR “PROxi” project) and was carried out in the frame of the EuMelaNet program (http://www.espcr.org/eumelanet) and the PolyMed project within the FP7-PEOPLE-2013-IRSES frame, PIRSES-GA-2013-612538.

Figure 2. SEM images of: (a) a fibrous mat of the electrospun PLA fibers and (b) DHI coated fibers before and (c) after AISSP.

microdevices, above all regarding scaffolds for biomedicine and organic (bio)electronics. The straight access to eumelanin microtubular architectures here reported reveals the unprecedented versatility of this biocompatible pigment to be handled as a moldable material, expanding the scope of eumelanin exploitation in material

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REFERENCES

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DOI: 10.1021/acs.biomac.5b00239 Biomacromolecules XXXX, XXX, XXX−XXX