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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 10617–10619 10617

Cite this: Chem. Commun., 2011, 47, 10617–10619

[60]Fullerene-based monolayers as neuroprotective biocompatible hybrid

materialswzDavide Giust,

aJose Luis Albasanz,

aMairena Martın,

aRiccardo Marega,

bArnaud Delforge

b

and Davide Bonifazi*bc

Received 3rd July 2011, Accepted 27th July 2011

DOI: 10.1039/c1cc13971a

Here we report on the surface immobilization of redox-active

[60]fullerene derivatives and the consequent neuroprotective

effects toward L-glutamate induced excitotoxicity in human

derived undifferentiated neuroblastoma cells.

Due to their unique electrochemical and photophysical

properties,1 [60]fullerene and its derivatives have been increasingly

investigated in the last few years as functional building blocks to

prepare tailored covalent2 and self-assembled3 functional

materials, some displaying great potentials in biological

applications4 such as DNA photocleavage5 and sensors

(glucose, ascorbic acid, coenzymes, proteins, or nucleic acids).6

Among others, [60]fullerene and its derivatives displayed

excellent efficiency in eliminating various reactive oxygen

species (ROS) such as superoxide radical anion (O2��),

hydroxyperoxides (ROOH), and hydroxyl radical (�OH).7 In

particular, ROS are generated in cells during oxidative stress

and play critical roles in ischemic stroke and in post-traumatic

lesions, epileptic convulsions, and in other neurodegenerative

diseases through excitotoxicity,8 such as multiple and amyotrophic

lateral sclerosis, Alzheimer’s and Parkinson’s diseases.9 In

this respect, water-soluble [60]fullerene derivatives,10 such as

hydroxylated and carboxylated conjugates,11 have shown to

efficiently scavenge free radicals in physiological solutions,

displaying neuroprotective effects,12 rapidly preventing neuronal

cells death induced by high concentrations of excitotoxins.13 On the

other hand, it has been shown that under mild irradiation

conditions (B1 mW) methano[60]fullerene-derived hexacarboxylic

acids can induce irreversible damages on cytoplasmic and

mitochondrial membranes in a dose and time-dependent

manner.14 Additionally, it is well known that freely diffusing

[60]fullerene and its derivatives can also elicit toxic responses15

in both in vitro16 and in vivo17 studies. Predictive computational

studies performed on 1207 protein entries (drug target database,

PDTD) have shown that [60]fullerene can interact with

hundreds of human proteins,18 thus potentially leading to a

series of unpredictable ‘‘off-target’’ side effects.

With the aim to undertake all these limitations deriving from

freely-diffusing [60]fullerene-based drugs, in this communication

we report on the preparation of neuroprotective hybrid materials

in which the [60]fullerene molecular cages are confined on a

surface as self-assembled19 monolayers (SAMs). Two [60]fullerene

derivatives bearing a C-unsubstituted pyrrolidine linkage (1H)

and a ferrocenyl side group (Fc, 2H) were synthesized (Fig. S1,

ESIz) and utilized for the preparation of the [60]fullerene-

bearing SAMs on Au(111)-coated mica substrates. The

presence of the Fc moiety on the one hand facilitates the SAM

characterization through X-ray Photoelectron Spectroscopy

(XPS), and on the other could be exploited for light-driven

therapeutics. Unprecedentedly, both [60]fullerene-based SAMs

provided near-complete protection (preserving most of the

characteristics from the parental differentiated20 human neuronal

cells) of the SH-SY5Y cells exposed to excitotoxic levels of

L-Glutamate (L-Glu).21

The two [60]fullerene derivatives bearing thiol anchoring groups

were synthesized through [1,3]-dipolar cycloaddition of azomethine

ylides to C60 following the well-known literature protocol22 and

further modified through amidation/deprotection reactions for

their covalent derivatization with a terminal thiol anchoring

append (Fig. S1, ESIz). Both pyrrolidino[60]fullerene derivatives

1H and 2H were thus adsorbed on Au(111)-coated mica surfaces

following the classical SAMs preparative methodology23 via wet

adsorption from CH2Cl2 solutions (see ESIz, S2), affording SAMs

[1�Au(111)] and [2�Au(111)] as depicted in Scheme 1.

With the aim to elucidate the chemical composition of

the prepared monolayers, XPS was employed as the first

characterization technique. XPS survey scans (see ESIz, S3)of both [1�Au(111)] and [2�Au(111)] monolayers revealed the

presence of peaks belonging to photoelectrons emitted by the

Au(111) surface (Au 4f at B83 eV, Au 5p and 5d in the region

from 0 and 70 eV and Au 4d at B333 eV) and by the

[60]fullerene-containing organic layers (C 1s at B284 eV, N 1

at B399 eV, O 1s at B532 eV and S 2p at B163 eV).

As expected, Fe signals (Fe 2p1/2 and 2p3/2 at B719 eV and

B707 eV, respectively) have been observed only for the

ferrocenyl-containing [2�Au(111)] monolayer.

aDepartment of Inorganic, Organic Chemistry and Biochemistry,University of Castilla-La Mancha, Avenida de Camilo Jose Cela 10,Ciudad Real, 13071, Spain

bDepartment of Chemistry, University of Namur, Rue Bruxelles 61,Namur, 5000, Belgium. E-mail: davide.bonifazi@fundp.ac.be

cDepartment of Pharmaceutical and Chemical Sciences and INSTMUdR Trieste, Universita degli Studi di Trieste, Piazzale Europa 1,Trieste, Italyw This article is part of the ChemComm ‘Molecule-based surfacechemistry’ web themed issue.z Electronic supplementary information (ESI) available. See DOI:10.1039/c1cc13971a

ChemComm Dynamic Article Links

www.rsc.org/chemcomm COMMUNICATION

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10618 Chem. Commun., 2011, 47, 10617–10619 This journal is c The Royal Society of Chemistry 2011

A high-resolution C 1s core level spectrum (see ESIz, S3-1and S3-2) allowed distinguishing between the different carbon

atom species present in the organic layer of both [1�Au(111)]and [2�Au(111)] monolayers as five main components: (i) a main

peak atB284.5 eV (78.32% and 81.60% respectively) originated

by the photoelectrons emitted from the sp2-hybridized carbon

atoms belonging to the [60]fullerene moiety; (ii) a peak

centered at B285.4 eV (6.96% and 3.54% respectively)

attributed to the sp3-hybridized carbon atoms of both the

aliphatic chain and the N-pyrrolidino moiety; (iii) a component

localized at B286.4 eV (4.09% and 1.57% respectively)

assigned to the oxidized carbon atoms in both C–O and

C–N single bonds; (iv) a peak at B287.6 eV (7.07% and

8.50% respectively) related to photoelectrons emitted from

carbon atoms by oxidized carbons belonging to carbonyl

groups and (v) a component localized at 290 eV (3.57% and

4.79% respectively) related to the carbon atoms belonging to

acidic and amidic functionalities (see ESIz, S3-3 and S3-4).

The degree of the bare Au(111) surface coverage towards

fullerenes SAMs was then assessed throughWCAmeasurements

on different portions (5 or 7) of the prepared surfaces, and

compared to the reference compound CH3(CH2)11SH (see

ESIz, S4). The contact angle between water and cleaned bare

Au(111) surface (38.9 � 2.91) suggests a low-hydrophobic

surface, while after the formation of the fullerene-containing SAMs

the WCA average values significantly increase to 77.1 � 3.01 and

84.4 � 1.91 for monolayers [1�Au(111)] and [2�Au(111)],respectively, a difference mainly ascribed to the Fc group. These

contact angles well match with the values reported in the literature

for closely-packed and organized thin film of fullerene-containing

SAMs on metallic surfaces such as Au5,24 and Hg.25

The biocompatibility and neuroprotective properties were

determined using SH-SY5Y cells (see ESIz, S5) grown for 48 h

under standard conditions (dark, humidified atmosphere with

5% CO2 at 37 1C) on both monolayer substrates [1�Au(111)]and [2�Au(111)]. Scanning electron microscopy (SEM) images

showed no evident morphological differences between cells

grown on a Petri dish, used as control, and those grown on the

[60]fullerene-containing SAMs (see ESIz, S6). In order to

investigate the monolayer neuroprotective activity, SH-SY5Y

cells grown on hybrid materials [1�Au(111)] and [2�Au(111)]were exposed for 6 h to an excitotoxic concentration (100 mM)

of L-Glu. Results from the MTT viability assay (Fig. 1) showed

how both SAMs [1�Au(111)] and [2�Au(111)] significantly

protected SH-SY5Y cells (B25% loss of cell viability) from

apoptotic events. This is a dramatic enhancement in the cell

viability as compared to the result obtained with the same cells

grown on a normal Petri dish or a cleaned Au (111) surface

exposed to the same treatment, which determined extreme loss

of viable cells (B70%, Fig. 1). We further confirmed the

biocompatibility of the fullerene-containing SAMs by growing

a population of fully-differentiated human neuronal cells. This

is of relevance because the crucial role of substrates in cell

differentiation was extensively investigated toward medical

applications, such as tissues regeneration, implants and stem cells

differentiation.26 Thus, upon treatment of the undifferentiated

SH-SY5Y cells grown on both [1�Au(111)] and [2�Au(111)]with all-trans retinoic acid (10 mM) for 7 days, followed by

administration of brain-derived neurotrophic factor (BDNF)

(50 ng mL�1) for 3 days, a fully differentiated population of

human neuron-like cells with complete neurite networks, as

shown by SEM imaging (Fig. 2b and c), was obtained. As it

clearly appears in the SEM microscopy images reported in

Fig. 2, no morphological differences were observed between

SH-SY5Y differentiated on a lysine-coated Petri dish, standard

non-neuroprotective substrate for cell attachment,27 and those

on SAMs [1�Au(111)] and [2�Au(111)]. This ultimately confirms the

biocompatibility toward neuronal cells of both fullerene-containing

SAMs, thus making them promising substrates as implanting

materials. Notably, in both biocompatibility and viability tests

no significant differences have been observed between

substrates [1�Au(111)] and [2�Au(111)], thus confirming the

biological inertness of the ferrocenyl moiety.

In conclusion, modification of Au(111) surfaces bearing

pyrrolidino-[60]fullerene derivatives led to bioactive materials

displaying full biocompatibility and neuroprotective actions in

contrasting L-Glu-induced excitotoxicity. In addition, the

Scheme 1 Schematic representation of SAMs [1�Au(111)] and

[2�Au(111)] as obtained by classical thiol adsorption on Au(111).Fig. 1 MTT viability assay performed on undifferentiated SH-SY5Y

cells grown on different substrates and exposed to a 100 mM solution

of L-Glu for 6 h. Viability of undifferentiated SH-SY5Y cells grown on

a standard Petri dish (white), a bare Au(111) surface (black),

[1�Au(111)] (light grey) and [2�Au(111)] (dark grey). **p o 0.01

significantly different from survival values of cells exposed to

L-Glutamate on a Petri dish and bare Au(111) substrates. The dotted

line represents 100% of living cells after 48 h growth on a Petri dish or

a bare Au(111) surface in the absence of L-Glutamate.

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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 10617–10619 10619

same SAMs revealed to be fully compatible materials for the

differentiation of SH-SY5Y cells into neurons, thus representing

novel nanomaterials for neuronal regeneration in damaged

tissues as in the case of ischemia. Furthermore, the possibility

of introducing lateral photoactive appends open the way to

possible devices in which a therapeutic effect, e.g. photodynamic

therapy exploiting porphyrin derivatives, can be induced

through an external stimulus such as light. These hybrid

materials represent only the initial step of an increasing effort

aimed at the design and engineering of further fullerene-based

SAMs which possess all characteristics for applications as

in vivo implants with controlled biocompatibility and bioactive

(therapeutic and/or protective) properties.

This work was supported by the EU through the MC-RTN

‘‘PRAIRIES’’ (035810), the FNRS (FRFC contract no.

2.4.550.09 and 2.4.617.07.F and MIS no. F.4.505.10.F), the

‘‘Loterie Nationale’’, the ‘TINTIN’ ARC project (contract no.

09/14-023), the University of Namur, the Fundacio La Marato

de TV3 (090331), and the Ministerio de Ciencia e Innovacion

(BFU2008-00138). AD and RM thank the FRS-FNRS for the

FRIA doctoral and post-doctoral fellowships, respectively.

Notes and references

1 L. Echegoyen and L. E. Echegoyen, Acc. Chem. Res., 1998,31, 593; D. M. Guldi andM. Prato, Acc. Chem. Res., 2000, 33, 695.

2 A.Mateo-Alonso,D. Bonifazi andM. Prato, inCarbonNanotechnology.Recent Developments in Chemistry, Physics, Materials Science andDevice Applications, ed. L. M. Dai, Elsevier, Amsterdam, 2006, pp. 155.

3 F. Diederich and M. Gomez-Lopez, Chem. Soc. Rev., 1999, 28, 263;J. F. Nierengarten, Chem. Eur. J., 2000, 6, 3667; J. F. Nierengarten,in Dendrimers V: Functional and Hyperbranched Building Blocks,Photophysical Properties, Applications in Materials and LifeSciences, Springer-Verlag Berlin, Berlin, 2003, vol. 228, pp. 87;J. F. Nierengarten, New J. Chem., 2004, 28, 1177; T. Nakanishi,Chem. Commun., 2010, 46, 3425.

4 F. Cataldo and T. da Ros,Medicinal Chemistry and PharmacologicalPotential of Fullerenes and Carbon Nanotubes, Springer, Dordrecht,2011; R. Partha and J. L. Conyers, Int. J. Nanomed., 2009, 4, 261;G. D. Nielsen, M. Roursgaard, K. A. Jensen, S. S. Poulsen andS. T. Larsen, Basic Clin. Pharmacol. Toxicol., 2008, 103, 197;M. Satoh and I. Takayanag, J. Pharmacol. Sci., 2006, 100, 513;D. Pantarotto, N. Tagmatarchis, A. Bianco andM. Prato,Mini-Rev.Med. Chem., 2004, 4, 805; S. Bosi, T. Da Ros, G. Spalluto andM. Prato, Eur. J. Med. Chem., 2003, 38, 913.

5 N. Higashi, T. Inoue and M. Niwa, Chem. Commun., 1997, 1507.6 B. S. Sherigara, W. Kutner and F. D’Souza, Electroanalysis, 2003,15, 753; F. Patolsky, G. L. Tao, E. Katz and I. Willner,J. Electroanal. Chem., 1998, 454, 9; C. Fang and X. Y. Zhou,Electroanalysis, 2001, 13, 949.

7 L. Y. Chiang, F. J. Lu and J. T. Lin, J. Chem. Soc., Chem.Commun., 1995, 1283.

8 A. Karakoti, S. Singh, J. M. Dowding, S. Seal and W. T. Self,Chem. Soc. Rev., 2010, 39, 4422.

9 X. X. Dong, Y. Wang and Z. H. Qin, Acta Pharmacol. Sin., 2009,30, 379.

10 A. Mateo-Alonso, C. Sooambar and M. Prato, Org. Biomol.Chem., 2006, 4, 1629.

11 E. Nakamura and H. Isobe, Acc. Chem. Res., 2003, 36, 807.12 T. Chen, Y. Y. Li, J. L. Zhang, B. Xu, Y. Lin, C. X. Wang,

W. C. Guan, Y. J. Wang and S. Q. Xu, J. Appl. Toxicol., 2011,31, 255; J. Tong, M. C. Zimmerman, S. M. Li, X. Yi,R. Luxenhofer, R. Jordan and A. V. Kabanov, Biomaterials,2011, 32, 3654; S. S. Ali, J. I. Hardt and L. L. Dugan,Nanomedicine, 2008, 4, 283; H. Jin, W. Q. Chen, X. W. Tang,L. Y. Chiang, C. Y. Yang, J. V. Schloss and J. Y. Wu, J. Neurosci.Res., 2000, 62, 600.

13 L. L. Dugan, D. M. Turetsky, C. Du, D. Lobner, M. Wheeler,C. R. Almli, C. K. F. Shen, T. Y. Luh, D. W. Choi and T. S. Lin,Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 9434; A. M. Y. Lin,C. H. Yang, Y. F. Ueng, T. Y. Luh, T. Y. Liu, Y. P. Lay andL. T. Ho, Neurochem. Int., 2004, 44, 99; X. Q. Cai, H. Q. Jia,Z. B. Liu, B. Hou, C. Luo, Z. H. Feng, W. X. Li and J. K. Liu,J. Neurosci. Res., 2008, 86, 3622.

14 X. L. Yang, L. Chen, X. Qiao and C. H. Fan, Int. J. Toxicol., 2007,26, 197.

15 K. Aschberger, H. J. Johnston, V. Stone, R. J. Aitken, C. L. Tran,S. M. Hankin, S. A. K. Peters and F. M. Christensen, Regul.Toxicol. Pharmacol., 2010, 58, 455; H. J. Johnston,G. R. Hutchison, F. M. Christensen, K. Aschberger andV. Stone, Toxicol. Sci., 2010, 114, 162.

16 B. Han and M. N. Karim, Scanning, 2008, 30, 213; H. Yamawakiand N. Iwai, Am. J. Physiol.: Cell Physiol., 2006, 290, C1495;G. Jia, H. F. Wang, L. Yan, X. Wang, R. J. Pei, T. Yan,Y. L. Zhao and X. B. Guo, Environ. Sci. Technol., 2005,39, 1378; H. Kato, N. Shinohara, A. Nakamura, M. Horie,K. Fujita, K. Takahashi, H. Iwahashi, S. Endoh andS. Kinugasa, Mol. BioSyst., 2010, 6, 1238; L. W. Zhang,J. Z. Yang, A. R. Barron and N. A. Monteiro-Riviere, Toxicol.Lett., 2009, 191, 149.

17 K. T. Kim, M. H. Jang, J. Y. Kim and S. D. Kim, Sci. TotalEnviron., 2010, 408, 5606; T. M. Blickley and P. McClellan-Green,Environ. Toxicol. Chem., 2008, 27, 1964; C. Y. Usenko,S. L. Harper and R. L. Tanguay, Carbon, 2007, 45, 1891;S. Q. Zhu, E. Oberdorster and M. L. Haasch, Mar. Environ.Res., 2006, 62, S5; S. Yamago, H. Tokuyama, E. Nakamura,K. Kikuchi, S. Kananishi, K. Sueki, H. Nakahara, S. Enomotoand F. Ambe, Chem. Biol., 1995, 2, 385.

18 M. Calvaresi and F. Zerbetto, ACS Nano, 2010, 4, 2283.19 C. A. Mirkin and W. B. Caldwell, Tetrahedron, 1996, 52, 5113;

D. Bonifazi, O. Enger and F. Diederich, Chem. Soc. Rev., 2007,36, 390; L. Sanchez, R. Otero, J. M. Gallego, R. Miranda andN. Martin, Chem. Rev., 2009, 109, 2081.

20 J. L. Biedler, S. Suzanne Roffler-Tarlov, M. Schachner andL. S. Freedman, Cancer Res., 1978, 38, 3751.

21 Z.-W. Sun, L. Zhang, S.-J. Zhu, W.-C. Chen and B. Mei, Neurosci.Bull., 2010, 26, 8.

22 M. Maggini, G. Scorrano and M. Prato, J. Am. Chem. Soc., 1993,115, 9798; K. Kordatos, T. Da Ros, S. Bosi, E. Vazquez,M. Bergamin, C. Cusan, F. Pellarini, V. Tomberli, B. Baiti,D. Pantarotto, V. Georgakilas, G. Spalluto and M. Prato,J. Org. Chem., 2001, 66, 4915.

23 L. Valli and D. M. Guldi, in Fullerenes: from Synthesis toOptoelectronic Properties, ed. D. M. Guldi and N. Martin, KluwerAcademic Publ., Dordrecht, 2002, vol. 4, p. 327.

24 Y. Shirai, L. Cheng, B. Chen and J. M. Tour, J. Am. Chem. Soc.,2006, 128, 13479; Y. S. Shon, K. F. Kelly, N. J. Halas andT. R. Lee, Langmuir, 1999, 15, 5329; O. Enger, F. Nuesch,M. Fibbioli, L. Echegoyen, E. Pretsch and F. Diederich,J. Mater. Chem., 2000, 10, 2231; W. B. Caldwell, K. Chen,C. A. Mirkin and S. J. Babinec, Langmuir, 1993, 9, 1945.

25 F. Y. Song, S. Zhang, D. Bonifazi, O. Enger, F. Diederich andL. Echegoyen, Langmuir, 2005, 21, 9246.

26 S. D. Sheridan, S. Gil, M. Wilgo and A. Pitt, in Stem Cell Culture,Elsevier Academic Press Inc., San Diego, 2008, vol. 86, p. 29.

27 M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen,V. Cena, C. Gallego and J. X. Comella, J. Neurochem., 2000,75, 991.

Fig. 2 (a) Phase contrast image of neuron-like cells grown on a

normal cell culture Petri dish by differentiation of SH-SY5Y.

(b, c) SEM images of differentiated neuron-like SH-SY5Y cells grown

on SAMs [1�Au(111)] and [2�Au(111)], respectively.

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