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Eumelanin Coated PLA Electrospun Micro Fibers as Bio-inspired Cradle for SH-SY5Y Neuroblastoma Cells Growth and Maturation Ines Fasolino, Irene Bonadies, Luigi Ambrosio, Maria Grazia Raucci, Cosimo Carfagna, Federica Maria Caso, Francesca Cimino, and Alessandro Pezzella ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13257 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on November 4, 2017

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ACS Applied Materials & Interfaces

Eumelanin Coated PLA Electrospun Micro Fibers as Bio-inspired Cradle for SH-SY5Y Neuroblastoma Cells Growth and Maturation Ines Fasolino†, Irene Bonadies*††, Luigi Ambrosio†, Maria Grazia Raucci*†, Cosimo Carfagna††,‡, Federica M Caso∥, Francesca Cimino††, Alessandro Pezzella§,††

†

Institute of Polymers, Composites and Biomaterials (IPCB) – CNR, Viale J.F. Kennedy 54,

Mostra D’Oltremare Pad 20, 80125 Naples, Italy ††

Institute of 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, 80126

Naples, Italy National Interuniversity Consortium of Materials Science and Technology (INSTM), Via G. Giusti, 9, 50121 Florence, Italy ∥

Nanofaber Spin-off at Italian National Agency for New Technologies, Energy and Sustainable

Economic Development (ENEA), Casaccia Research Centre, Via Anguillarese 301, 00123 Rome, Italy

ACS Paragon Plus Environment

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KEYWORDS:

eumelanin,

neuronal

cell

growth,

neurodegenerative

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applications,

electrospinning, tissue engineered scaffold

ABSTRACT Within the framework of neurodegenerative disorder therapies, the fabrication of 3D eumelanin architectures represents a novel strategy to realize tissue-engineering scaffolds for neuronal cell growth and control by providing both mechanical support and biological signals. Here, an appropriate procedure combining electrospinning, spin coating and solid-state polymerization process is established to realize the scaffolds. For biological analysis, a human derived cell line SH-SY5Y from neuroblastoma is used. Cell maturation on eumelanin microfibers, random and aligned, is evaluated by using confocal analysis and specific markers of differentiating neurons (βIII tubulin and GAP-43 expression). Cell morphology is tested by SEM analysis and immunofluorescence techniques. As results, eumelanin coated microfibers prove capable to support biological response in terms of cell survival, adhesion and spreading and to promote cell differentiation toward a more mature neuronal phenotype as confirmed by GAP-43 expression over the culture.


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INTRODUCTION The regeneration of neurite network constitutes a strategy for the treatment of neurodegenerative disorders characterized by a loss and dysfunction of trophic factors such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF).1 Neurotrophic factors play a critical role in neuronal regeneration, but their clinical use is limited by their inability to cross the blood brain barrier.1 Moreover, the use of primary mammalian neurons derived from embryonic central nervous system tissue is limited by the impossibility of propagation after cells terminal differentiation into mature neurons. To overcome these limitations in reproduction of neuronal in vitro systems, researchers suggest the use of transformed neuronal-like cell lines such as the popular SH-SY5Y neuroblastoma cell line.2 The ability of SH-SY5Y to differentiate into cells possessing a more mature neuron-like phenotype has afforded numerous advantages in the field of neuroscience research. In addition, since SH-SY5Y cells are human-derived, they express a number of human-specific proteins and protein isoforms that could reproduce more inherently the human neuronal in vivo microenvironment. From a morphological point of view, the undifferentiated SH-SY5Y cells are characterized by neuroblast-like truncated processes, the presence of clusters and the tendency to form a cell mass.3 However the most important feature of undifferentiated SH-SY5Y cells is their analogy to immature catecholaminergic neurons.4, 5 In fact, following treatment with differentiation-inducing stimuli, SH-SY5Y cells can be driven toward a wide variety of adult neuronal phenotypes including cholinergic, adrenergic, or dopaminergic and exhibit numerous but randomly distributed processes of mature cells.5 Tissue engineering advancement requires design and fabrication of bioactive and biocompatible structures whose chemical and morphological characteristics are devised to affect cell functions


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and fate.6 In particular, for neurodegenerative applications, much effort has been devoted to the design of 2D/3D microenvironments that combining diverse cues and factors are able to can manipulate biological response in terms of proliferation and differentiation of different cell lines. .7, 8, 9, 10 More often natural materials proved a valuable tool for the modulation of the surface properties, such as adhesivity or hydrophobicity, since they are able to confer additional biofunctionality or influence protein adsorption and ultimately biological response.11 Eumelanin-based blends as well as eumelanin coatings are emerging as versatile tools for the fabrication of innovative multifunctional bio-interfaces.12, 13, 14 Eumelanin biopolymers are the most relevant mammal exposed pigment and are responsible for the brown to black coloration of skin hair and iris. Eumelanins are formed by the oxidative polymerization 5,6-dyhydroxyndoles (DHI and DHICA) arising by the oxidative metabolism of the amino acid tyrosine15 and allows interfaces to take advantage of the enhanced chemical-physical properties of the poly hydroxyindole backbone, providing, at the same time, adhesion

16, 17

bio-compatibility

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electrical conductivity 19 and UV-vis absorption 20. In a series of studies addressing a systematic investigation of eumelanin coatings, we recently succeed in the fabrication of biocompatible interfaces for stem cell growth and induced differentiation.21 Key tools for the eumelanin interface fabrication were provided by the design of a solid-state eumelanin fabrication protocol allowing access to nearly any possible morphology. Indeed eumelanin microtubes were recently fabricated combining PLA electrospinning technology and DHI solid-state polymerization.22 Thanks to the versatile electrospinning technology, a cutting edge tool for producing significant in length polymer fibers with diameters in the micro- nanoscale range 23 at laboratory as well as


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industrial scale24, it is possible to obtain three-dimensional scaffolds for advanced applications in tissue engineering.24, 25, 26 Among different materials, Polylactide (PLA) electrospun fibers are widely used for fabricating scaffolds to be exploited in tissue engineering

27, 28

as well as in experimental models aimed to

study cell behavior.29, 30 A number of studies disclosed the valuable application of biocompatible and biodegradable PLA fibers, also integrated with active components, as efficient tools for neural tissue regeneration 25, 31, 32 and neural stem cell differentiation control.28, 33 Here we present a chief advancement of these studies demonstrating the actual exploitation of eumelanin coated PLA microfibers as a tool for controlling and directing the growth and maturation of SH-SY5Y cells, a neuronal-like cell line.

RESULTS AND DISCUSSION Neuronal-like SH-SY5Y cells are widely investigated to assess their in vitro response in several models for neurodegenerative disorders. Two-dimensional (2D) surfaces are an useful tool to study the behaviour of different cell populations in an isolated system, but they are not able to provide information on correlation between neuronal anatomical and functional connectivity.34 Furthermore, conventional 2D culture systems show limitations also because they cannot allow the survival of dissociated neurons for several weeks as previously reported35. 3D matrices such as hydrogels, electrospun fibers and spheroid-based systems or porous materials are widely investigated to assess their in vitro effect on cell response because they mimic more realistically 3D in vivo microenvironments.36 Also, a three-dimensional architecture preserves cell-cell interaction and influences biological response by using specific chemical signals as well as morphological features.37 Here, we have investigated the effect of scaffold consisting of


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eumelanin coated PLA electrospun microfibers (EU@PLA) (Figure S1) prepared with a welldefined protocol (Scheme 1 SI) on SH-SY5Y cell in vitro response. Eumelanin film stability during in vitro and in vivo tests is reported in literature.

18, 21

Indeed

SEM inspection (Figure S2) of EU@PLA samples before and after 21 days of in vitro cell culture, confirmed the actual stability of the eumelanin coatings under the explored conditions: no detachment phenomena were noticeable, both fiber surface and diameter are unaffected by the cell culture medium. Biocompatibility of the EU@PLA microfibers was assessed following the growth profile of SHSY5Y cultures in the basal medium over random and aligned microfiber substrates (Figure 1).

Day 1 Day 3

150 Percent of reduction (%)

*

°

Day 7

#

p≤0.05, p≤0.01 and p≤0.001vs control

Day 14 Day 21

100

* #

50

#

°

°

#

°

@ PL EU lig A

do m an R

ne d

EU

on

@

tr o

PL

l

A

A

0

C

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Figure 1. Quantitative analysis of cell proliferation by using Alamar blue assay after 1, 3, 7, 14 and 21 day of cell culture in presence of a conventional 2D plate surface (control), random


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eumelanin coated microfibers and aligned eumelanin microfibers. Data represent mean ± SEM (standard error of mean) of 3 experiments. *p < 0.05 and °p < 0.01 and #p < 0.01 versus control.

As 2D flat surface (control), random and aligned eumelanin microfibers increased cell proliferation in a time depended manner until day 7 of cell culture. Notably, random eumelanin fiber substrates promote cell proliferation until day 21 of culture time and over and aligned microfibers support cell growth with better proliferation values than random microfibers at day 14. Conversely, at day 21 of cell culture aligned microfibers induced a significant decrease in cell proliferation as 2D flat surface. It is known that dissociated neurons exhibit inability to survive for several weeks on 2D conventional culture systems.38 It may be speculated that SHSY5Y cells recognize aligned EU@PLA microfibers as a 2D patterning.38 Indeed, SEM inspection of the cultures at day 7 confirmed (Figure 2 and S3) cell proliferation and disclosed their good adhesion onto the random EU@PLA microfibers. This result, consistently with previous studies disclosing the high biocompatibility of AISSP fabricated eumelanin coatings 39, 21

towards different cell lines, may be ascribed to the chemical microenvironment experienced by

the cells in contact with the eumelanin layer which is characterized by the presence of polar groups 40, 41 (i.e. phenolic, carboxylic, carbonyl, etc.) as well as aromatic ring systems and by the enhanced water wettability respect to the mat of neat PLA (Figure S4).


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Figure 2. SEM analysis of fibrous scaffolds seeded with SHY5Y cells after 7 days of cell culture: random EU@PLA fibers at magnification of 1000x (A), 2000x (B), 4000x (C); aligned EU@PLA fibers at magnification of 2500x (D), 6000x (E).

Figure 2 reveals the marked tendency of the cell membranes to embed the micro-fibrous scaffold, witnessing not only a strong interaction between cell surface and the eumelanin surface but also a very efficient cell – cell and cell – ECM adhesion. Indeed, the presence of eumelanin coating allowed to obtain better values of cell substrate integration than those obtained by functionalization of PLA fibers with peptides such as RGD sequences to facilitate recognition by integrin.9, 10, 42 In order to further investigate EU@PLA microfiber biocompatibility, immunofluorescence analysis was performed by using cell tracker kit. A large amount of EU@PLA microfibrous surface was covered with well-spread and more substrate-adherent cells. No morphological differences were detected comparing cells cultured for 7 days on random EU@PLA microfibers


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with cells cultured on aligned EU@PLA microfibers (Figure 3). Furthermore, the combined evaluation of data from immunofluorescent cell tracker method and SEM analysis (Figure S5), confirmed a good biological response also in terms of cell proliferation in presence of random and aligned EU@PLA microfibers.

Figure 3. Effect of random eumelanin microfibers (A) and aligned eumelanin microfibers (B) on cell morphology, cell density and cell spreading after 7 days of exposure by using confocal microscopy.

In a further deepening of the study, neuronal maturation of SH-SY5Y cells was followed by confocal analysis detection of Beta tubulin class III expression, over the culture time. Beta tubulin class III is a protein primarily expressed in neurons and involved in neurogenesis, axon guidance and maintenance. Here, SH-SY5Y cells clearly expressed Beta tubulin class III at day 7 of cell culture on random microfibers (Figure 4, A-B). Furthermore, the actively proliferating SH-SY5Y showed polygonal morphology with a short filopodia at day 7 and some of them had characteristics of growing axons as noticeable from their termination by a growth cone.


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Conversely, aligned EU@PLA microfibers inhibited Beta tubulin class III expression at day 7 of exposure and increased outwardly cell proliferation rate (Figure 4, C). According to previous data on cell proliferation, qualitative analysis on Beta tubulin class III confirmed that SH-SY5Y cells cultured on random EU@PLA microfibers, at day 21, were able to maintain an excellent degree of viability and tend to form aggregates thus increasing cell-cell interaction and ameliorating their maturation (Figure 5, A). In fact, cell clusters were observed and most of cells migrated into ganglion-like aggregates (Figure S6).

Figure 4. Representative images of βIII tubulin (green) SHY5Y cells on random eumelanin microfibers (A-B) and of βIII tubulin SHY5Y cells on aligned eumelanin microfibers (C) after 7 days of cell culture by using immunofluorescence analysis. Nucleus is stained with DAPI (blue).


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Figure 5. Immunocytochemistry of SHY5Y cells. The SHY5Y cells were grown on random (A) and aligned (B) eumelanin microfibers for 21 days. Cells were stained with neuron specific marker βIII tubulin (green). Nucleus is stained with DAPI (blue). The histogram in Figure C represents βIII tubulin fluorescence intensity calculated on qualitative images using ImageJ software.

Otherwise on aligned EU@PLA microfibers at day 21 of cell culture a significant decrease in cell proliferation was observed (Figure 5, B). This effect may be due to a different signal derived by aligned fiber settlement. Probably, the random disposition of microfibers provides a more homogeneous environment compared to aligned fibers thus promoting a better cellular response in terms of cell proliferation and morphology. Qualitative results on βIII tubulin expression at day 21 were confirmed by quantitative image analysis as reported in Figure 5 C. Finally, the potential of EU@PLA to induce cell maturation was assessed under basal conditions up to 21 days, a period reported to lead to greatest levels of viable MSC cell adhesion over polar substrates.40 The neuronal adult form was evident by the SEM analysis revealing the actual formation of dendrite structures and their propagation over the EU@PLA microfibers at day 21 (Figure 6). At this time point, cells cultured on random EU@PLA microfibers exhibited network of neuritic processes (Figure 6, A). By contrast these morphological features were not evident in cells plated on aligned EU@PLA microfibers (Figure 6, B). These results on cell morphology supported cell response in terms of proliferation observed in Figure 1. Notably, a relevant difference

between

random

and

aligned

EU@PLA

microfibers

was

detected

by

immunofluorescence analysis on GAP43 expression (Figure 7). GAP43 growth associated protein 43, well known as B-50-PP46, is expressed at high levels in neuronal growth cones


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during development and axonal regeneration.43 In fact, this protein is considered a crucial component of an effective regenerative response in the nervous system. In the present study, immunopositive GAP43 was more concentrated in cells cultured on random EU@PLA microfibers compared to plate control and cells on aligned EU@PLA microfibers at day 21 (Figure 7, A-C). Qualitative results on GAP-43 expression at day 21 were confirmed by quantitative image analysis as reported in Figure 7 D.

Figure 6. SEM images of SHY5Y cells cultured on random (A) and aligned (B) eumelanin microfibers for 21 days.


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Figure 7. Immunofluorescent staining of the neuronal marker GAP-43. SHY5Y cells were maintained in basal culture medium for 21 days in the presence a conventional 2D flat surface (A), 3D random eumelanin microfibers (B) and 3D aligned eumelanin microfibers (C). Random eumelanin microfiber exposure (B) induced accumulation of GAP-43 immunoreactivity (green). Nucleus is stained with DAPI (blue). The histogram in Figure 7 D represents GAP-43 fluorescence intensity calculated on qualitative images using ImageJ software.

Notably, no relevant morphological differences were detected by comparison with mature cells arising by a standard retinoic acid (RA) induced differentiation in a control experiment (Figure S7). Comparing to differentiation protocols based on cell stimulation by chemical factors (e.g. RA), results here disclosed present longer reaction time, but a remarkable gain concerning the culture media composition and cellular environment and the possibility to capitalize on the substrate chemistry in order to direct stem cell in the same culture towards different maturation.


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CONCLUSIONS An eumelanin coated mat based on electrospun PLA microfibers was fabricated and tested as an effective tool for the control of SH-SY5Y growth in vitro. Several studies in vitro on neuronal regeneration have addressed that functional surface chemistries can control SH-SY5Y cell fate, here we reported unprecedented eumelanin functionalized microfibers capable to support cell maturation towards adult neurons without added differentiating factors in the culture media. Moreover, eliminating the need of differentiating factors in the media, eumelanin coatings expand the scope of substrate driven cell culture growth and maturation, opening to culture substrates (2D and 3D) capable to exhibit regio-controlled cell maturation stimuli for tissue engineering. These findings have a wide impact in the developing field of design and fabrication of eumelanin based bio-interfaces and further in cell biology, as prospecting the ability to control biological response in terms of proliferation, adhesion and maturation, via a natural pigment featuring peculiar chemical physical properties such as UV-vis absorption and hybrid ionic electronic charge transport.

EXPERIMENTAL SECTION Materials and Methods Polylactic acid (PLA) (Ingeo™ 4032D) was supplied by NatureWorks LLC; all solvents were reagent grade. 5,6-Dihydroxindole (DHI) was prepared according to a reported procedure

44

as

detailed in SI.

Fibers fabrication and characterization


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The PLA solution (10% w/v in chloroform/dimethylformamide 90/10) was electrospun with an electrospinning setup NF103 MECC Co., Ltd. (Fukuoka, Japan) at a flow rate of 3 mL h-1, room temperature and 30% relative humidity. The voltage and distance between the needle tip and the collector were 30 kV and 25 cm, respectively. Not woven films were collected on an aluminum foil, whereas aligned fibers were collected by using static and separated conductive inclined electrodes (gap method).45 Eumelanin coating was obtained via spin coating starting from a DHI solution and a subsequent annealing at 30 °C for 30’ under a nitrogen atmosphere; the DHI coating efficiency was calculated in term of eumelanin amount (wt. %) for different samples by thermogravimetric analysis in nitrogen atmosphere at 10°C/min as detailed in SI (Figure S8). The morphology of electrospun microfibers and cells was evaluated by a field-emission scanning electron microscopy (FESEM, QUANTA200, FEI, The Netherlands) at an accelerating voltage of 30 kV after gold–palladium sputter coating. We used Directionality, which is an ImageJ plugin, to infer the preferred orientation of fibers (Figure S9).

In vitro cell culture SH-SY5Y cell line derived from a bone marrow biopsy taken from a four-year-old female with neuroblastoma was purchased from Sigma-Aldrich (Italy). SH-SY5Y, often used as in vitro models of neuronal function and differentiation, were cultured in a medium consisting of 220 ml of Eagle's Minimum Essential Medium (EMEM), 220 ml of Dulbecco's Modified Eagle Medium (DMEM),10 vol.% fetal bovine serum (FBS), antibiotic solution (streptomycin 100 µg/ml and penicillin 100U/ml, Sigma Chem. Co) and 2 mM L-glutamine. Cells were maintained in an incubator at 37 °C, 95% relative humidity and 5% CO2 atmosphere. Cell viability (> 80%) was evaluated by trypan blue staining. 5x104 cells were seeded onto the eumelanin coated microfibers


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and were maintained in culture for 21 days, with the cell culture medium being changed every 3 days. The biological response on the eumelanin microfibers was investigated. For this aim, the eumelanin substrates were sterilized by UV-irradiation for 1 h.

Cell proliferation The preliminary biological studies were performed in order to evaluate the effect of eumelanin coated microfibers on the cell behavior in terms of proliferation. To achieve this goal SH-SY5Y cells (at density of 5x104/well) were seeded onto eumelanin microfibers using a 48-well plate. Cells in plate were used as control. The cell culture medium was removed after 1, 3, 7, 14 and 21 days of culture time and the in vitro cell proliferation was checked by the Alamar blue assay (AbDSerotec, Milano, Italy). AlamarBlueTM reagent was added directly to each well, the plates were incubated at 37°C for 4hrs to allow cells to convert resazurin to resorufin and the optical density was immediately measured with a spectrophotometer (Sunrise, TECAN, Männedorf, Zurich, Switzerland) at wavelengths of 570 and 600 nm. The Alamar blue assay allows to control the cell proliferation by measuring the metabolic activity of live cells. Wells without any cells were used to correct any background interference from the redox indicator.

Cell adhesion and morphology The interaction of SH-SY5Y cells onto eumelanin substrates in terms of morphology and cell spreading was evaluated by SEM analysis. For this aim 5x104 cells were seeded onto eumelanin coated microfibers incubated for 7 and 21 days at 37°C. After these time points, cells seeded onto microfibers were fixed with a solution of 4% paraformaldehyde for 24hrs at 4oC. Cellloaded samples were washed and dehydrated using a series of increasing ethanol concentration,


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prior to the analysis. After that, samples were fixed by means of a double adhesive tape to aluminum stubs applying a tension of 20 kV. The stubs were sputter-coated with gold to a thickness of around 20 nm (Emitech Sk500). Cell adhesion and morphology were also evaluated by confocal laser scanning microscopy (LSM 510, CarlZeiss). For this purpose, cells were treated with a cell tracker green CMFDA (5µM) in phenol red-free medium in 75 cm2 cell culture flask and incubated for 30 min at 37°C. Later, several washing steps with PBS were performed to remove the non-attached cells, while the attached ones were incubated for 1 h in complete medium. After that, 20,000 cells were seeded onto each sample and incubated for 24hrs at 37°C and 5%CO2. Moreover, in order to investigate the formation of neuronal processes, cells were fixed, permeabilised as described earlier and incubated with the Anti-beta III Tubulin antibody (βIII tubulin is a microtubule element of the tubulin family found almost exclusively in neurons) at 1:200 dilution overnight at 4 °C. After washing the samples thrice with PBS for 15 min, incubation for 1-2 hours with goat anti-rabbit IgG –FITC secondary antibody (Santa Cruz) in the dilution of 1:500 was performed. The cells were again washed with PBS thrice to remove any excess staining then incubated for 10 minutes at 37 °C with DAPI (for nuclei detection). The samples were again washed with PBS thrice and mounted over glass slides. The glass slides were observed using confocal laser scanning microscopy (LSM 510, Carl Zeiss). The mean fluorescence intensity of βIII tubulin in stained cells was calculated by using Image J software.

Cell maturation To observe SH-SY5Y neuronal maturation, immunofluorescent staining was performed using GAP-43 antibody (bs-0154R) that is a specific neuronal marker. Expression of GAP-43 was analyzed using 20,000 cells cultured for 21 days onto eumelanin coated microfibers in basal


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culture medium. Then, cells were fixed and permeabilised as described earlier.30.Later, neuronal specific marker protein Gap-43 FITC conjugated (1:100 dilution) was added and incubated overnight at 4 °C. After washing the samples thrice with PBS for 15 min, incubation for 10 minutes at 37 °C with DAPI (for nuclei detection) was performed. The cells were again washed with PBS thrice to remove any excess staining. The samples were then removed and mounted over glass slides. The glass slides were observed using confocal laser scanning microscopy (LSM 510, Carl Zeiss). The mean fluorescence intensity of GAP-43 in stained cells was calculated by using Image J software.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Preparation scheme, SEM, photos of surface, confocal microscopy images (PDF) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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