Synthetic mycomelanin thin films as emergent bio-inspired interfaces controlling the fate of embryonic stem cells

Paola Manini *a, Valeria Lucci b, Valeria Lino ac, Stefania Sartini d, Francesco Rossella e, Geppino Falco *b, Cinzia Chiappe d and Marco d’Ischia a
aDepartment of Chemical Sciences, University of Napoli Federico II, Complesso Universitario Monte S. Angelo, via Cintia 4, I-80126 Napoli, Italy. E-mail: paola.manini@unina.it
bDepartment of Biology, University of Napoli Federico II, Complesso Universitario Monte S. Angelo, via Cintia 4, I-80126 Napoli, Italy. E-mail: geppino.falco@unina.it
cScuola Normale Superiore, Piazza dei Cavalieri 7, I-56126 Pisa, Italy
dDepartment of Pharmacy, University of Pisa, via Bonanno Pisano 33, I-56126 Pisa, Italy
eNEST, Scuola Normale Superiore and Istituto Nanoscienze – CNR, Piazza San Silvestro 12, I-56127 Pisa, Italy

Received 6th March 2020 , Accepted 17th April 2020

First published on 17th April 2020


The fungal pathways of melanin synthesis have so far been little considered as a source of bio-inspiration in the field of functional materials, despite the interesting properties exhibited by Ascomycetes melanins from 1,8-dihydroxynaphthalene (1,8-DHN), including the ability to shield organisms from ionizing radiation. Herein, the processing techniques and characterizations of mycomelanin thin films obtained from the solid state polymerization of 1,8-DHN is reported for the first time. Overall, the results highlighted the role of synthetic mycomelanin thin films as a prototype of next generation bioinspired interfaces featuring high structural regularity and ultrasmooth morphology, high robustness against peroxidative bleaching and adhesion under water conditions, good biocompatibility and unprecedented effects in inducing the spontaneous differentiation of embryonic stem cells prevalently towards the endodermal lineages in the absence of added factors. These data open up new avenues towards the applications of this biomaterial in the fields of tissue engineering and regenerative medicine.


Introduction

The design and implementation of functional biointerfaces for stem cell manipulation is currently an active topic in tissue engineering and regenerative medicine.1 Despite extensive investigations over the past few decades, currently, advances in this field are hindered by the incomplete understanding of the complex interactions between stem cells and their microenvironment (niche).2 Under standard cell culture conditions, there is usually a need to rely on additive molecules that orchestrate the regenerative signals and drive the differentiation pathways.3 Recent lines of evidence, however, also highlight, in addition to morphogens regulating cellular differentiation, the importance of a cellular support (a biointerface) to efficiently govern the balance between stem cell self-renewal and differentiation.4 In order to promote favorable communication between cells and their microenvironment, including the extracellular matrix (ECM) and neighboring cells and tissues, biointerfaces should fulfill a series of requisites of chemical, topographical and mechanical natures.5,6

From a chemical viewpoint, biointerfaces are usually coated or covalently functionalized with ECM proteins, peptide motifs or growth factors to overcome issues related to the failure of common materials to adequately mimic the bioactivity features of the ECM.7 Sometimes, the use of optically active groups is desirable when exploring the effect of surface chirality on cell behaviors. Emerging importance is also given to the topographical features of biointerfaces, which may encompass 2D micro/nano-patterns up to complex 3D architectures (i.e. pillars, needles and fibers) and appear to play a central role in directing cell adhesion and cell-to-cell communication.8,9

Finally, varying the physical and mechanical properties of the materials may offer insights into the contractile force and mechano-transduction mechanisms of cells, as in the case of micropost arrays used for measuring stem cell-derived cardiomyocyte contractility.10

In this context, particular attention has been devoted to the design of dynamic biointerfaces that can be used to control cell adhesion, proliferation and release, for applications in real-time cell biology, regenerative medicine, and theranostics.11 To this aim, a series of chemical groups can be used for surface functionalization or as an anchor for cell binding ligands that can respond to different types of external stimuli, including temperature, pH, bias and light.12–15

Despite the great potential of dynamic biointerfaces, their use is often associated with a series of practical complications. For example, the application of external stimuli may be invasive for living cells or may cause the release of cytotoxic molecules due to linker fragmentation. Therefore, exploitation of new biointerfaces that respond to all the requisites of cell biocompatibility and communication is very challenging but highly anticipated.

Recently, eumelanins, the dark brown pigments found in mammalian skin, hair and eyes, have emerged as versatile leads for the fabrication of innovative multifunctional dynamic biointerfaces,16 combining excellent biocompatibility17 and unique antioxidant, opto-electronic and electrical properties.18,19 A fundamental advancement towards eumelanin-based technology for surface functionalization and biointerface design has been obtained from the development of an ammonia-induced solid state polymerization (AISSP) protocol allowing the deposition of eumelanin coatings on different substrates from 5,6-dihydroxyindole (DHI) thin films.20 Due to the dry, solid state conditions of the protocol, issues related to eumelanin insolubility, high structural heterogeneity, disordered morphology and limited adhesion can be effectively overcome, allowing the buildup of 2D and 3D architectures that are fully compatible with different cell lines and stem cells.20,21

Despite the great potential of melanin-based biointerfaces associated with softness, bioavailability and biocompatibility of these polymers, several issues and practical limitations still hinder the development of robust and versatile thin films for biomedical applications. One major limitation is the marked sensitivity of eumelanin coatings to degradation processes under alkaline or oxidative conditions, a consequence of the presence of catechol/o-quinone units notoriously susceptible to oxidative ring fission, strongly affecting the performance of the biointerface.

In view of these and other issues, the rational design and synthesis of advanced robust and device-quality melanin-based biopolymers remains to date a challenging goal in materials science for the development of nanosized functional systems and biocompatible interfaces.

A promising source of inspiration towards this goal may come from non-nitrogenous catechol-free allomelanins, whose biocompatibility, antioxidant and metal chelating properties are well documented.22,23 In this context, particular attention has been paid recently to allomelanins from Aspergillus fungi.24 These pigments are derived biogenetically from 1,8-dihydroxynaphthalene (1,8-DHN) and exhibit almost complete insolubility in all solvents. Like their nitrogenous eumelanin counterparts, fungal allomelanins (hereafter referred to as mycomelanins) display broadband visible light absorption, due to their black color, and an unusually intense electron paramagnetic resonance (EPR) signal, due to their outstanding antioxidant properties.25,26

A dihydroxynaphthalene-based mycomelanin mimic has been reported as a coating agent for material-independent surface modification.27 This melanin was produced by the laccase-catalyzed polymerization of 2,7-dihydroxynaphthalene (2,7-DHN) and was deposited onto metals, polymeric materials, ceramics and mineral complexes to form functional surfaces with bactericidal and metal chelation/reduction properties, and high adhesion with proteins.

Very recently, Gianneschi et al. reported on the cellular antioxidant activity of artificial allomelanin nanoparticles obtained from the oxidative polymerization of 1,8-DHN.28 Apart from these studies, the potential of mycomelanins and related allomelanins for film preparation in biomedicine and nanotechnology has remained largely unexplored.

Herein, we report the production of unusually smooth, regular and robust mycomelanin thin films by AISSP of 1,8-DHN and disclose the unique properties of these films to induce adhesion, proliferation and differentiation of stem cells towards an endodermal lineage without added morphogens. This property may be due to the high antioxidant activity, resistance to degradation and exceptional smoothness of the allomelanin films and may guide the development of novel device-quality melanin-based biointerfaces for tissue engineering and other biomedical applications.

Results and discussion

Based on the previously developed AISSP protocol,20 widely used for melanin thin film deposition, thin films of synthetic mycomelanin from 1,8-DHN were deposited on quartz substrates by spin coating of methanol solution on a precursor followed by exposure to ammonia vapours. UV-visible analysis of the film before and after AISSP confirmed the polymeric melanin-like nature of the material, as apparent from the intense broadband absorption in both the UV and visible region of the spectrum and from the brown color of the substrate (Fig. 1).
image file: d0tb00623h-f1.tif
Fig. 1 (A) The proposed mechanism for the oxidative polymerization of 1,8-DHN. (B) UV-vis spectra of 1,8-DHN thin films on quartz substrates before (red trace) and after (black trace) AISSP; the insets show the quartz substrates before and after exposure to ammonia vapours.

The mass spectrum of the film recorded in the MALDI mode confirmed the polymeric nature of the film revealing the presence of regular patterns of peaks separated by 158 Da, corresponding to the “in-chain” dihydroxynaphthalene unit (Fig. 2). The dominant peaks were assigned to singly-charged distributions up to 20 units, confirming the lack of any detectable ammonia incorporated into the chemical structure of the polymer. Compared to the MALDI-MS data previously reported for polymers produced by aqueous polymerization, the spectra obtained from the film denoted a minor degree of distribution for each of the clusters, suggesting a more regular structure of the polymer.29


image file: d0tb00623h-f2.tif
Fig. 2 MALDI-mass spectrum of the mycomelanin thin film from 1,8-DHN.

The micro ATR spectrum registered on the film showed the presence, among others, of bands at 1700–1550 cm−1, attributable to C[double bond, length as m-dash]O and C[double bond, length as m-dash]C stretching of the conjugated quinonoid structures, and bands between 1400–1200 cm−1 suggesting the presence of C–O residues, supporting the structural hypothesis proposed for allomelanins obtained by the oxidation in solution of 1,8-DHN (Fig. 3).


image file: d0tb00623h-f3.tif
Fig. 3 Micro ATR spectrum of the mycomelanin thin film from 1,8-DHN.

The homogeneity of the mycomelanin thin film was determined by FT-IR spectroscopy imaging and atomic force microscopy (AFM).

The point-by-point scanning of the thin film carried out using FT-IR spectroscopy imaging analysis provided quite similar ATR spectra (see the ESI). This data suggested that all the regions investigated corresponded to the same compound with the only differences being in concentration, and that the sample did not contain inclusion compounds or defects, pointing out the high chemical homogeneity of the film.

Quite interesting results came from the AFM analysis (listed in Table 1). The films obtained from a nominal concentration of 100 mM 1,8-DHN were characterized by a thickness of 130 nm and an ultra-smooth surface.

Table 1 Structural parameters for 1,8-DHN mycomelanin thin films
Mycomelanin thin film from 1,8-DHN
a Average and standard deviation of six separate measurements.
Thickness (nm) 130
Roughness (rms) 0.25
Water contact anglea (°) 33.6 ± 2.1
Water contact angle image image file: d0tb00623h-u1.tif


As shown in Fig. 4, the roughness analysis, carried out for the square areas labeled I and II, revealed an unexpectedly low value of 0.25 rms for the mycomelanin film, which is quite a lot lower than that measured for the substrate (4.00 rms). This evidence confers an added value to the mycomelanin thin films with a view to design dynamic biointerfaces. Noticeably, the ultrasmooth morphology represents an important feature improving the optoelectronic properties of the biointerface. To the best of our knowledge, this is the first example of an ultra-smooth thin film obtained using spin coating deposition.


image file: d0tb00623h-f4.tif
Fig. 4 AFM analysis of the mycomelanin thin films from 1,8-DHN. (A) Colour plot of one of the AFM maps measured across sharp edges of the film; (B) average roughness profile estimated from several scans in two different regions of the map reported in panel (A): the region labeled as I lays on the mycomelanin film, while the region labeled as II lays on the substrate. (C) Three-dimensional rendering of the AFM map reported in panel (A). (D) Cross-sectional profile measured along the segment AB indicated in panel (A).

Finally, the stability of the thin films under water conditions was tested. Preliminarily water contact angle (WCA) measurements were carried out on the 1,8-DHN mycomelanin thin films providing a value of 33.6°, indicative of good surface wettability (Table 1).

Furthermore, to test the adhesiveness under water conditions, the mycomelanin-coated quartz substrates were partially immersed in water: after 24 h of immersion time, no detectable detachment of the film was observed suggesting high water resistance.

Finally, the robustness of the film was assessed by soaking the mycomelanin-coated quartz substrates in an alkaline solution of hydrogen peroxide, a standard bleaching agent for most melanin polymers; the same treatment was performed also on eumelanin-coated quartz substrates obtained via AISSP of a 5,6-dihydroxyindole (DHI) thin film. As shown in Fig. 5, while the DHI-melanin film was gradually detached from the substrate within 10 min, no detectable damage of the mycomelanin thin film was visible even after 70 min of immersion time.


image file: d0tb00623h-f5.tif
Fig. 5 Effects of alkaline hydrogen peroxide solution on DHI-melanin thin film (left) and synthetic mycomelanin thin film (right).

The high robustness of synthetic mycomelanin thin films can be attributed to the structural features of the monomer precursor, 1,8-DHN, for which the formation of catechol/o-quinone units, notoriously susceptible to oxidative ring fission, was prevented.

Experiments to assess the biocompatibility of mycomelanin thin films from 1,8-DHN were first carried out on the HEK-293 cell line. In detail, HEK-293 cells were trypsinized to obtain a single cell suspension and 2.5 × 104 cells per cm2 were plated on 100 and 200 nm mycomelanin-coated dishes, or on standard tissue culture plates. As shown in Fig. 6, the HEK-293 cells adhered to both mycomelanin-treated dishes with a similar morphology with respect to the control plates. The mycomelanin coatings also proved to be able to support the efficient growth of HEK-293 cells, with a proliferation rate at 72 h comparable to standard growth conditions.


image file: d0tb00623h-f6.tif
Fig. 6 (A) Phase contrast images of the HEK-293 cells 48 and 72 h after seeding on dishes coated with 100 nm and 200 nm mycomelanin thin films or tissue culture plates, as the control. Images were taken using a Leica DMi8 at 10× magnification. Scale bars: 250 μm. (B) Growth curves of HEK-293 cells during 72 h are shown. Cells (6 × 104) were seeded on a tissue culture plate (♦), 100 nm mycomelanin (■) and 200 nm mycomelanin (▲) coated plates and triplicate cultures counted at daily intervals. Data are expressed as mean ± SD (*p < 0.05 and ***p < 0.001).

Further experiments were carried out to investigate whether mycomelanin thin films from 1,8-DHN could also promote clonogenic cell growth in an embryonic stem cell (ESC) colony assay. ESCs are of particular interest for their sensitivity to small changes under culture conditions which can affect the morphology and behaviour of the ESC colony. ESCs were cultured for 3 days in a regular medium on cell culture plates coated with either 100 and 200 nm mycomelanin films, or gelatin as the control.

At 24 h, ESCs adhered to the plates under all conditions with different degrees of cell attachment. After 48 h, the cells proliferated forming regular dome shaped colonies, although ESCs cultured on mycomelanin coated plates were smaller compared to the control plates. Finally, at 72 h, cells on 100 nm mycomelanin coatings retained the morphology and attachment to the plate in a similar way to the control, while the cells on 200 nm mycomelanin coatings seemed to lose adhesion, floating in the medium (Fig. 7).


image file: d0tb00623h-f7.tif
Fig. 7 (A) Representative light microscopy images of ESCs of E14Tg2a.4 mice grown on cell culture low adhesion plates coated with gelatin, 100 nm mycomelanin thin film and 200 nm mycomelanin thin film. The images were acquired at 24, 48 and 72 h. Scale bars: 100 μm. (B) qRT-PCR analysis of pluripotency-specific markers. The mRNA levels were normalized for Gapdh expression and reported as a fold change with respect to the values from ESCs grown under gelatin conditions.

In addition, the expression levels of genes involved in canonical pluripotency such as Oct3/4, Nanog and Rex1 were investigated revealing no significant differences among culture conditions.

With the aim of checking if the mycomelanin thin films could also be used to direct cell differentiation into a specific lineage fate (ectoderm, mesoderm or endoderm), ESCs were cultured for five days in a regular medium without the Leukemia inhibitory factor (hereafter Lif-), a cytokine required for ESC self-renewal.

Statistical analysis was performed on all samples for three independent experiments.

The expression levels of pluripotency markers (Nanog and Oct3/4) and lineage-specific markers, such as Bry and Gsg (early endoderm markers), Cxcr4 and FoxA2 (endoderm markers), Gata2 (mesoderm marker) and NeuroD (ectoderm marker), were analyzed by RT-qPCR. Under both growing conditions, Oct3/4 and Nanog expressions were downregulated, highlighting a loss of pluripotency. Interestingly, while there were no differences in the ectoderm and mesoderm markers, Cxcr4 and FoxA2, as well as early endoderm markers, turned out to be overexpressed (Fig. 8A).


image file: d0tb00623h-f8.tif
Fig. 8 (A) ESCs plated on dishes coated with gelatin, 100 nm mycomelanin thin film and 200 nm mycomelanin thin film, cultured for five days under Lif-differentiating conditions. The mRNA expression levels of pluripotency and differentiation-associated markers were assessed by qRT-PCR and expressed as a fold change with respect to the ESCs grown under gelatin conditions. Values shown are mean ± SD, based on triplicate assays. Statistical analyses were performed using Student's t-test, where p < 0.05 was considered significant (*p < 0.05, **p < 0.01). (B) Total protein extracts prepared under the same growth conditions were separated using SDS-PAGE and subjected to western blot analysis with specific antibodies. The hybridization with vinculin assessed the uniform loading and integrity of the proteins. Loaded as follows: line 1, ESCs on gelatin coating (Lif-); line 2, ESCs on 100 nm mycomelanin coating (Lif-); line 3, ESCs on 200 nm mycomelanin coating (Lif-); n = [thin space (1/6-em)]2 independent experiments were performed with similar results.

The effectiveness of endoderm induction was further validated by analyzing the protein expressions of pluripotency and endoderm markers. In agreement with the qRT-PCR results, it was observed that the expression level of Nanog and Oct3/4 decreased more in mycomelanin than under gelatin conditions.

Similar to the upregulation of the FoxA2 mRNA expression (Fig. 8A), the FoxA2 protein expression was also increased under both culture conditions as indicated by western blot signals (Fig. 8B). These observations suggested that the mycomelanin coating could result in the differentiation prevalently towards the endodermal lineages.

Experimental

Ammonia induced solid state polymerization (AISSP): a general procedure

Suitable amounts of 1,8-DHN were dissolved in methanol and the solutions were filtered with 0.45 μm syringe filters before use. For each deposition, 100 μL of the methanol solution of 1,8-DHN was pipetted onto the upper side of the substrate just before spinning. The spin coating parameters were as follows: α = 500 rpm s−1, ω = 2000 rpm, and t = 30 s. The 1,8-DHN coated substrate was placed in a glass chamber under an ammonia atmosphere equilibrated with air. After 24 h, the polymerization was complete, and the substrate was removed from the chamber. Mycomelanin thin films of different thickness were prepared by spin coating 1,8-DHN methanolic solutions at appropriate concentrations (see the ESI).

RNA extraction, cDNA preparation and quantitative real time PCR (RT-qPCR)

Total RNA was extracted using a TRIzol reagent (Invitrogen) and treated with RNase-free DNase I (Applied Biosystems). For qRT-PCR analysis, the cDNAs were synthesized using the iScript cDNA Synthesis kit (BioRad, Hercules, CA, USA). 1 μg of total RNA was used for each cDNA synthesis. The Primer 3 software (http://primer3.ut.ee/) was used to design the oligo primers setting the annealing temperature to 59–61 °C for all primer pairs. Oligo sequences are listed in Table S1 (ESI). A real time-PCR analysis was performed using the iTaq™ Universal SYBR® Green Supermix (BioRad) in a 7500 Real-Time PCR System (Applied Biosystems) under the following conditions: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The Gapdh probe served as the control to normalize the data. The gene expression experiments were performed in triplicate on three independent experiments and a melting analysis was performed at the end of the PCR run. To calculate the relative expression levels, we used the 2−DDCT method.30

Cell culture

E14Tg2a.4 ES cells, derived from the strain 129P2/OlaHsd, were purchased from ATCC company and were cultured for two passages on gelatin-coated feeder-free plates and subsequently maintained in gelatin-coated plates in a Glasgow Minimum Essential Medium (Gibco) supplemented with 15% FBS (EuroClone), 1000 units per mL of ESGRO leukaemia inhibitory factor (LIF) (Merck Millipore), 1.0 mM sodium pyruvate (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 2.0 mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma Aldrich) and 500 U mL−1 of penicillin/streptomycin (Invitrogen). ESCs were incubated at 37 °C in 5% CO2; the medium was changed daily and cells were split every 2 to 3 days routinely. The human embryonic kidney (HEK-293) cell lines were obtained from the CEINGE Cell Culture Facility (Naples, Italy) and were grown in a DMEM medium containing 15% fetal bovine serum (EuroClone). For the growth curve, 6 × 105 cells were seeded in triplicate on 60 mm dishes coated with or without melanin and a cell count was performed after 48 h and 72 h from plating.

Protein extracts and immunoblotting

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in JS buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EGTA pH 7.8, 10% glycerol, 1% Triton, 1.5 mM MgCl2, 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF). The protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For western blot analysis, proteins were separated using SDS-PAGE, the gels were blotted onto Immobilon-P (Millipore, Bedford, MA, USA) for 2 h and the membranes were blocked in 5% non-fat dry milk in Tris-buffered saline for 2 h or overnight before the addition of the antibody for 1 h. The primary antibodies used were: anti-Vinculin (Santa Cruz, CA), anti-Nanog (Abcam), anti-Oct4 (Abcam) and anti-FoxA2 (Cell Signaling Technology). The filters were washed three times in Tris-buffered saline plus 0.05% Tween 20 before the addition of horseradish peroxidase-conjugated secondary antibodies for 45 min. Horseradish peroxidase was detected with ECL (Pierce).

Conclusions

An ultrasmooth and unusually robust biointerface with potent effects on stem cell adhesion and differentiation is disclosed herein, which is produced by AISSP of the fungal allomelanin precursor 1,8-DHN. The key features of the synthetic mycomelanin thin films include high structural regularity as determined by MALDI-MS, exceptionally low roughness determined using AFM analysis, high film homogeneity determined by FT-IR spectroscopy imaging, good wettability and strong adhesion under water conditions, exceptional resistance even to alkaline hydrogen peroxide, a standard bleaching agent for most melanin polymers. Remarkably, the mycomelanin films proved to be capable of inducing embryonic stem cell adhesion and spontaneous differentiation prevalently towards the endodermal lineages in the absence of added factors, opening up novel avenues in the fields of tissue engineering and regenerative medicine.

Conflicts of interest

The authors declare that there are no conflicts.

Acknowledgements

This work was supported in part by the Italian MIUR grants PRIN 2017YJMPZN to MdI and 2017CBHCWF_003 to PM.

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Footnote

Electronic supplementary information (ESI) available: Materials and methods, FT-IR spectroscopic imaging and AFM images. See DOI: 10.1039/d0tb00623h

This journal is © The Royal Society of Chemistry 2020