Electrochimica Acta 201 (2016) 220–227

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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta

Highly Water-soluble and Surface Charge-tunable Fluorescent

Fullerene Nanoparticles: Facile Fabrication and Cellular Imaging
Rongbin Xiea , Zifei Wanga , Hongtao Yua , Zetan Fana , Fanglong Yuana , Yunchao Lia ,
Xiaohong Lia , Louzhen Fana,* , Hong Fanb,**
a
Department of Chemistry, Beijing Normal University, Beijing 100875, China
b
Shanxi Provincial People’s Hospital, Taiyuan, Shanxi 030012, China

A R T I C L E I N F O A B S T R A C T

Article history:
Received 1 February 2016 Water-soluble and surface charge-tunable amine-functionalized polyhydroxylated fullerene nano-
Received in revised form 22 March 2016 particles with a strong green emission were synthesized by grinding and hydrothermal treatment. The
Accepted 31 March 2016 quantum yield of the nanoparticles was as high as 17%, which is the highest value recorded for fluorescent
Available online 1 April 2016 fullerene materials. The amine-functionalized polyhydroxylated fullerene nanoparticles with high
surface charge were found to easily penetrate into breast cancer cells, HeLa cells and cardiac progenitor
Keywords: stem cells, opening up great opportunities for their bio-medical applications.
Fullerene ã 2016 Elsevier Ltd. All rights reserved.
Water-soluble
Fluorescence
Charge-tunable
Stem Cells

1. Introduction them as bioimaging agents. The nanoparticles exhibited bright

Fluorescent carbon nanomaterials (FCNs) with tunable emis-
sion are considered to be next generation green nanomaterials and
are promising alternatives to toxic fluorescent semiconductor
nanocrystals. FCNs have drawn increasing attention in recent years
owing to their exceptional advantages such as stable photo-
luminescence (PL), excellent biocompatibility and low cytotoxicity
[1–4]. Among carbon nanomaterials, fullerene (C60) has attracted
an increasing interest in various fields including material chemis-
try and biomedicine because of their unique structural and
physicochemical properties [5–9]. However, the extremely low
fluorescence efficiencies and poor water solubility for these C60
nanomaterials limited their biomedical applications [10]. Jeong
et al. synthesized high water-soluble and color-tunable C60
nanoparticles by conjugating tetraethylene glycol (TEG) with
lithium hydroxide (LiOH) as a catalyst. However, the quantum yield
(QY) of the nanoparticles was only 0.86%, which was inadequate
widely used in practical applications [11]. In our previous study, we
also prepared water-soluble and strongly fluorescent C60 nano-
particles by electrochemical method and successfully utilized
* Corresponding author. Fax: +86 10 5880 2075.
** Corresponding author.
E-mail addresses: lzfan@bnu.edu.cn (L. Fan), fanhong661016@163.com (H. Fan).
blue fluorescence, and the QY was about 5–6% [12]. Therefore, it is
highly important but remains challenging to synthesize strongly
fluorescent and highly water-soluble C60 nanomaterials.
Fullerenol (polyhydroxylated fullerene, PHF), a hydroxylated
derivative of C60, is significantly more hydrophilic than C60.
Depending on the number of hydroxyl groups, the aqueous
solubility of PHF may achieve 58.9 mg mL
1, nearly ten orders of
magnitude greater than the aqueous solubility of C60 [13–15]. Due
to the hydrophilic nature, high proton conductivity and excellent
medicinal properties of PHF, it has been explored for biomedical
applications such as in the delivery of drugs or as probes for
imaging [11,12,16,17]. Unfortunately, The QY of PHF in water is very
low, which greatly limits its potential applications in the biological
imaging.
Herein, we successfully prepared water-soluble and surface
charge-tunable amine-functionalized fullerenol nanoparticles
(PHF-NH2 NPs) under grinding conditions in a porcelain mortar
at room temperature and then with hydrothermal treatment in a
Teflon-lined stainless steel autoclave at high temperature (150  C).
The methods of poly-hydroxylation and amine-functionalization
in this work are by far the simplest and high-yielding, providing
the possibility of large-scale production. Due to the amine-
functionalization, the QY of the green-emitting PHF-NH2 NPs is
approximately 17%, which is the highest value recorded for

http://dx.doi.org/10.1016/j.electacta.2016.03.198
0013-4686/ ã 2016 Elsevier Ltd. All rights reserved.
 R. Xie et al. / Electrochimica Acta 201 (2016) 220–227
fluorescent fullerene materials [11,12], raising the prospect of the
fullerene for biomedical applications. Our experiment has
demonstrated that PHF-NH2 NPs with high surface charge were
easily internalized in cancer cells by virtue of their high QY, stable
PL and low cytotoxicity. Most importantly, they could penetrate
into stem cells, opening up great opportunities for exploring their
native development and tissue regeneration.
2. Experimental
2.1. Materials
C60 fullerene (99.9%) and guanidine carbonate were purchased
from J&K. NaOH, KOH and HCl (68%) were purchased from Beijing
Chemical Works. All solvents and reagents were used without
further purification.
2.2. Preparation of PHF NPs
PHF NPs were prepared from C60 powder by mechanical
grinding method. Briefly, excessive KOH powder was mixed and
grind with C60 powder in a porcelain mortar for 15 min. A small
amount of toluene was added in the process of grinding to keep the
mixture moist. After grinding, the color of the mixture turned
yellow-brown. Then deionized water was added, followed by
stirring the mixture for 10 min to dissolve the sludge completely.
The obtained dark-brown solution was then centrifuged at
12000 rpm for 10 min to remove the insoluble residual. Pure
PHF NPs were obtained by dialysis in a dialysis bag (retain
molecular weight 3500 Da) for one day to fully remove the KOH
and other impurities.
2.3. Preparation of PHF-NH2 NPs
PHF-NH2 NPs with strong fluorescence were obtained by
hydrothermal treatment. PHF NPs and guanidine carbonate (at
concentrations of 5, 2.5, 1.5, 1, 0.5, and 0.25 mg/mL, respectively)
were dispersed in 20 mL deionized water, and heated hydrother-
mally in a Teflon-lined stainless steel autoclave at 150  C for 8 h.
After that, the obtained colloidal solution was further dialyzed in a
dialysis bag (retain molecular weight 3500 Da) for one day to fully
remove the guanidine carbonate and other impurities.
2.4. Characterization
Atomic force microscopy (AFM) images were taken using a
SPM-9600 atomic force microscope by using tapping mode with
the rotated Si tips. The tips specification: 40 N/m, 300 kHz, radius
8 nm, no coating. Transmission electron microscope (TEM) and
high-resolution transmission electron microscopy (HRTEM)
observations were performed on a JEOL JEM-2010F electron
microscope operating at 200 kV. The sample solution was drop-
cast from solution onto a carbon-coated TEM grid and the solvent
was evaporated at room temperature. X-ray powder diffraction
(XRD) patterns were obtained with a Rigaku D/max-2500 using
Cu-Ka radiation. Absorption and fluorescence spectra were
recorded on a Hitachi 3100 spectrophotometer and a Hitachi
7000 fluorescence spectrophotometer at room temperature,
respectively. Fourier transform infrared (FTIR) spectra were
recorded with a Bio-Rad FTIR spectrometer FTS165. The Raman
spectra were taken with Laser Confocal Micro-Raman Spectros-
copy (Lab-RAM Aramis). X-ray photoelectron spectroscopy (XPS)
spectra were collected using a Kratos Axis Ultra DLD X-ray
photoelectron spectrometer. The zeta potential measurements
were performed on a Zeta-Plus Zeta Potential Analyzer (Broo-
khaven, 100–240 V, 50/60 Hz).
221
Scheme 1. Schematic representation for the preparation of green fluorescent PHF-
NH2 NPs.
2.5. Cellular experiments
Approximately 5  104 Breast Cancer cells (MCF-7), HeLa cells
and Cardiac progenitor stem cells (CPCs) were seeded in a 96 well
plate and cultured in Dulbecco's Modified Eagle Medium (DMEM)
with 10% (v/v) fetal bovine serum (Hyclone, USA), 0.1 mg mL
1
penicillin G sodium and 0.1 mg mL
1 streptomycin sulfate
(Amresco, USA) in an incubator (37  C, 5% CO2) for 24 h, then
cultivated in the same culture medium solution containing
PHF-NH2 NPs at concentrations of 0, 10, 20, 50, 100, 200 and
500 mg mL
1 (200 mL per well). After incubation, the cells were
washed three times and then fixed with 4% paraformaldehyde for
20 min at room temperature.
3. Results and discussion
3.1. Morphology and conformation of PHF-NH2 NPs
The preparation of PHF-NH2 NPs involves two steps as shown in
Scheme 1. PHF NPs were synthesized by grinding C60 and KOH with
a small amount of toluene. The strong alkali reacts with C60 to
break p-p carbon bond and combine with oxygen in various forms
of carbon-oxygen conjugate, the toluene plays as a role of the
dispersing agent which makes C60 react with oxygen quickly and
fully in the present of alkali. Subsequently, the PHF-NH2 NPs were
prepared by hydrothermal treatment of PHF NPs solution with
guanidine carbonate. The guanidine carbonate contains rich amino
groups which provide the amino source of PHF-NH2 NPs.
Pure NPs were obtained by a set of post-processing procedures
of dissolution, precipitation and dialysis. TEM and AFM were used
to determine the morphology of the NPs. TEM images (Fig. 1a and
b) show that the PHF NPs are well-dispersed and have a relatively
narrow size distribution with an average diameter of 1.5–3.0 nm.
From the HRTEM images, PHF NPs displayed a typically spherical
agglomeration nanoparticle shape. As demonstrated in previous
research, the diameter of PHF NPs was higher than theoretical
(0.7 nm), corresponding to the aggregation of C60 [18]. In the AFM
images (Fig. 1c and d) reveal a typical topographic height below
1.4 nm. From typical TEM and HRTEM images (Fig. 2a and b) of
PHF-NH2 NPs, it can be seen that the PHF-NH2 NPs are spherical
and well-dispersed with average sizes of 1.0–2.0 nm. The AFM
images (Fig. 2c and d) demonstrate the average thickness is mostly
222 R. Xie et al. / Electrochimica Acta 201 (2016) 220–227

Fig. 1. (a) TEM image of the PHF NPs. The inset of (a) is the HRTEM image of the PHF NPs. (b) Diameter distribution of the PHF NPs. (c) AFM image of the PHF NPs deposited on
freshly cleaved mica substrates. (d) Height profile along the line A-B in (c).

below 1 nm. PHF-NH2 NPs are smaller and thinner than PHF NPs. It
was likely that the functionalization of PHF NPs further broke
symmetry of C60, and aggregated C60 became more disperse and
uniform.
In order to verify the structures of the NPs, XRD and Raman
spectroscopy were performed to explore the structural changes in
the process of preparation. As shown in Fig. 3a, both PHF and
PHF-NH2 NPs have two XRD characteristic peaks ((11 0) and
(11 2)), similar to those of pristine C60, indicating that the PHF NPs
and PHF-NH2 NPs have the formation of hexagonal close packing
type (HCP) crystal structure of pristine C60. The Raman spectra
(Fig. 3b) reveal a band at 1467 cm
1, which correspond to the cage
structure of C60 [19] , which further prove that the PHF NPs and
PHF-NH2 NPs have retained the skeleton structure of C60.
XPS measurements are performed to determine the composi-
tion of the as-prepared PHF-NH2 NPs. The full spectra presented in
Fig. 4a show three typical peaks: C 1s (284 eV), N 1s (400 eV), and O
1s (532 eV). The resultant PHF-NH2 NPs have a higher O/C atomic
ratio (40%) than that of PHF (25%), because of the oxidation of C¼C
during treatment with guanidine carbonate. A pronounced N 1s
peak is observed for the resultant PHF-NH2 NPs, whereas no N
signal can be detected on the PHF NPs. In the high-resolution
spectra, the N 1s peaks of the PHF-NH2 at 399.6 eV and 401.8 eV are
respectively attributed to amide and protonated amine N atoms
(Fig. 4b). The C 1s band of the PHF-NH2 NPs can be deconvoluted Fig. 2. (a) TEM image of the PHF-NH2 NPs. The inset of (a) is the HRTEM image of the
PHF-NH2 NPs. (b) Diameter distribution of the PHF-NH2 NPs. (c) AFM image of the
into four peaks (Fig. 4c), corresponding to C¼C (284.9 eV), C

N PHF-NH2 NPs deposited on freshly cleaved mica substrates. (d) Height profile along
(286.4 eV), C

O (286.0 eV), and O

C

O (288.2 eV). No C

N peak the line A-B in (c).
 R. Xie et al. / Electrochimica Acta 201 (2016) 220–227 223

Fig. 3. (a) XRD patterns and (b) Raman spectrum of the C60, PHF and PHF-NH2 NPs.

was detected on the PHF NPs, which further confirms the
successful incorporation of N into the PHF-NH2 NPs. The FTIR
spectrum of the PHF-NH2 NPs (Fig. 4d) further confirms the
presence of amide groups as evidenced by the N-H bending
vibration at 1410 cm
1 [20], excepting the characteristic bands at
3400, 1610, 1380, and 1045 cm
1 assigned to O

H, n(C¼C),
ds(C

O

H), and n(C

O) absorption. These characteristic peaks
are invariably reported as diagnostic absorption from PHF NPs.
[13,15,21,22]
3.2. Photophysical properties of PHF-NH2 NPs
The most distinctive feature of the PHF-NH2 NPs which sets
them apart from other previously reported PHF NPs is their specific
PL behavior. From the UV–vis spectrum of the PHF and PHF-NH2
NPs aqueous solution (Fig. 5a), both PHF and PHF-NH2 NPs showed
the typical absorption peaks at 230 due to the p–p* transition of
C¼C and other two peaks at 260 and 350 nm assigned to n-p*
absorption band [23,24]. Besides, PHF-NH2 NPs exhibit another
new absorption peak at 460 nm, indicating the further broken
symmetry of the C60. To further explore the optical properties of
the PHF-NH2 NPs, a detailed PL investigation is carried out at
different excitation wavelengths. As shown in Fig. 5b, at the
excitation wavelength of 460 nm, the PL spectrum shows the
strongest peak at 515 nm. The maximum emission peak shows a
Stokes shift of 55 nm. The fluorescent emission peak shifted with
different excitation wavelengths ranging from 320 to 560 nm,
indicating that the PL origin is markedly similar to that of the
common excitation wavelength dependent PL of other FCNs
[4,20,25]. The QY of PHF-NH2 NPs is approximately 17% (Fig. 6a),
as accurately measured using rhodamine 6 G as a standard, which
is the highest value yet recorded for fullerene nanoparticles in
water [11,12,26].
In order to explicate the roles of alkali and guanidine carbonate
during this process, control experiment was conducted. The
further hydroxylated PHF (PHF-OH) NPs was produced by
hydrothermal treating PHF NPs in KOH solution. Because of the
intersystem crossing from singular state to the excited triplet state
and the forbidden electronic transitions resulted from the high
symmetry of Ih, no fluorescence could be observed in isolate C60

Fig. 4. Fig. 4 (a) XPS spectra, (b) N 1s and (c) C 1s XPS spectra of the PHF and PHF-NH2 NPs. (d) FTIR spectra of the PHF and PHF-NH2 NPs.
224 R. Xie et al. / Electrochimica Acta 201 (2016) 220–227

Fig. 5. (a) UV–vis absorption spectra of the PHF-NH2 and PHF NPs aqueous solution. (b) PL spectra of the PHF-NH2 NPs aqueous solution at different excitation wavelengths.
The inset of (b) is the photograph of the PHF-NH2 solution under UV light. (c) UV–vis absorption of the PHF-OH aqueous solution. (d) PL spectra of the PHF-OH aqueous
solution at different excitation wavelengths. The inset of (d) is the photograph of the PHF-OH solution under UV light.

and PHF NPs. However, the PHF-OH NPs could emit green PL
centered at 505 nm with the corresponding absorption band at
340 nm (Fig. 5c and d), and the QY of PHF-OH is about 5% (Fig. 6a),
which is most likely due to the increase of hemiketal moieties
produced by the further hydroxylation of PHF. The proposed PL
mechanism based on the hemiketal moieties is supported by the
observed pH-dependent PL [23]. Under alkaline conditions, the
PHF-OH emits green PL, whereas, under acidic conditions, the PL is
nearly completely quenched (Fig. 6b). PL intensity of PHF-OH NPs
can be readily recovered when pH is switched repeatedly between
13.0 and 1.0, which is related to the interconversion of hemiketal
and ketone according with the previous report. Under acidic
conditions, most of hemiketal moieties convert into the structure
of ketone. Thus, the PL of PHF-OH NPs is quenched. However, under
alkaline conditions, the hemiketal moieties are restored, thereby
leading to the recovery of PL.
PHF-NH2 NPs obtained by hydrothermally treated PHF NPs with
guanidine carbonate emit stronger green PL than PHF-OH NPs, and
the QY of PHF-NH2 NPs could reach as high as 17%, which are
attributed to the amine-functionalization. Amine-functionaliza-
tion further breaks the electronic symmetry of the PHF-OH NPs.
Such a structure may involve a further electronic symmetry
breaking of the C60 molecules engendered by the special charge
distribution in the neutral core/anionic shell C60 nanoparticles,
which would partially allow the intrinsically forbidden transitions
[27,28].

Fig. 6. (a) Photoluminescence intensity and absorbance of the PHF-NH2, PHF-OH aqueous solution and Rhodamine 6 G ethanol solution (FF  95%). (b) pH-dependent PL
spectra of the PHF-OH aqueous solution when pH is switched between 13 and 1.
 R. Xie et al. / Electrochimica Acta 201 (2016) 220–227 225

Table 1 microscope. As shown in Fig. 7, after incubation with PHF-NH2 NPs

Zeta potential and N atomic concentration of PHF NPs and PHF-NH2 NPs.
for 24 h, the MCF-7 cells emit green fluorescence, predominantly in
NPs Guanidine Carbonate (mg/mL) Z-potential (mV) N1s (%) the cytoplasm. Moreover, the intracellular fluorescence image
PHF 0
34.96 0 grew brighter as the surface charge of PHF-NH2 NPs increased. The
PHF-NH2 0.25
32.96 1.52 possible pathway for fullerene derivatives entering the cells may
PHF-NH2 0.5
28.03 2.81 be adsorption on proteins and receptor-mediated endocytosis
PHF-NH2 1.0
24.09 3.23
since PHF NPs barely penetrate across the membrane directly
PHF-NH2 1.5
18.57 3.95
PHF-NH2 2.5
13.45 5.34
[29,30]. Chithrani et al. have demonstrated that serum proteins
PHF-NH2 5.0
5.63 7.14 could adsorb fullerene derivatives and dictated the uptake of the
nanoparticles. Thus, we speculated that a part of PHF-NH2
nanoparticles are adsorbed by the nonspecific serum proteins,
which induce these nanoparticles to enter cells via the mechanism
of receptor-mediated endocytosis. [31] With the higher surface
charge, the more PHF-NH2 NPs are adsorbed by the nonspecific
3.3. Tunable surface charge of PHF-NH2 NPs serum proteins. Therefore PHF-NH2 NPs with high surface charge
could easily penetrate into MCF-7 cells. So we obtained the
PHF-NH2 NPs with different surface charge were obtained by optimized PHF-NH2 NPs and we also incubated another kind of
hydrothermal treatment of PHF with different concentrations of cancer cells HeLa cells with PHF-NH2 NPs. As shown in Fig. 8a and
guanidine carbonate. As shown in Table 1, zeta potential measure- b, PHF-NH2 NPs are able to label both the cell membrane and the
ments display that both PHF and PHF-NH2 NPs are negatively cytoplasm of HeLa cells.
charged. With the increase of guanidine carbonate concentrations,
the amount of amine groups in PHF-NH2 NPs increased. As the 3.5. Stem cells bioimaging of PHF-NH2 NPs
amine could bound with the H+ from ionization of water to form
positive charged
NH3+, the zeta potential of PHF-NH2 NPs Stem cells are central to the understanding of native develop-
increased. Thus we obtained tunable surface charge of PHF-NH2 ment and tissue regeneration, which give rise to tissue progenitor
NPs by varying degrees of amine-functionalization. cells and in turn differentiate into tissue-forming cells [32–34].
Because of the particularity of stem cells, their labeling poses a
3.4. Cancer cells bioimaging of charge-tunable PHF-NH2 NPs considerable challenge and currently no technique is available for
labeling stem cells directly and efficiently [35]. Although almost all
The water soluble, strongly fluorescent PHF-NH2 NPs have nanomaterials can penetrate the cancer cells effortlessly for
prompted us to explore their potential for cellular imaging. The imaging, only unique graphene quantum dots (GQDs) with specific
uptake and bioimaging experiments of PHF-NH2 NPs with different structure can enter the stem cells as labeling agents [36,37].
surface charge were performed by the confocal fluorescence According to previous reports, the fluorescent C60 nanoparticles

Fig. 7. Confocal fluorescence microscopy images of MCF-7 cells with different surface charge of PHF-NH2 NPs: (a)
5.63 mV. (b)
13.45 mV. (c)
18.57 mV. (d)
24.09 mV. (e)

28.03 mV. (f)
32.96 mV.
226 R. Xie et al. / Electrochimica Acta 201 (2016) 220–227
Fig. 8. Confocal fluorescence microscopy images of HeLa cells (a), CPCs (c) with the fluorescent PHF-NH2 NPs and CPCs (e) with the fluorescent PHF-OH NPs incorporated at
the excitation wavelength of 405 nm and corresponding images under bright field (b, d, f). The surface charge value of PHF-NH2 and PHF-OH NPs:
28.53 mV,
29.31 mV.
could difficultly penetrate into stem cells [37]. The high QY and
biological compatibility of PHF-NH2 NPs are compelling and have
prompted us to explore their potential use in stem cells. Cardiac
progenitor stem cells (CPCs) were incubated with PHF-NH2 NPs for
24 h. As shown in Fig. 8c and d, the PL spots could be observed in
the cytoplastic area at the CPCs with the excitation wavelength of
405 nm. We did also incubate CPCs with PHF-OH NPs (Fig. 8e and
f), the surface charge values of PHF-NH2 NPs and PHF-OH NPs are

28.53 mV and
29.31 mV, respectively. The PL in CPCs incubated
with PHF-OH NPs was much weaker than that in CPCs incubated
with PHF-NH2 NPs. This due to without amine-functionalization,
the QY of PHF-OH NPs is lower than PHF-NH2 NPs. The easy
internalization of PHF-NH2 NPs in stem cells may be due to the
certain unique interactions between stem cells and PHF-NH2 NPs
with the novel carbon cage structure and special groups, which is
an interesting topic worthy of further investigation. All above
results indicate that PHF-NH2 NPs provide a great potential for
biomedical applications.
4. Conclusions
We have successfully prepared highly water-soluble and
surface charge-tunable fluorescent PHF-NH2 NPs by grinding
and hydrothermal treatment. The green-emitting PHF-NH2 NPs
with a 17% QY is demonstrated for the amine-functionalization,
and the QY is the highest value recorded for fluorescent
fullerene materials. PHF-NH2 NPs with high surface charge were
found to easily penetrate into cancer cells. Moreover, such
PHF-NH2 NPs have demonstrated direct and easy penetration
into stem cells without affecting their viability, proliferation or
differentiation capacity. With excellent optical properties, good
water-solubility, low cytotoxicity and tunable surface charge,
these new PHF-NH2 NPs may offer new opportunities for the
development of fluorescent labeling agents and thus extend
their widespread application in biomedicine. Detailed follow-up
work is underway in our laboratory and will be reported in due
course.
Acknowledgements
This work is supported by NSFC (21573019), the Major Research
Plan of NSFC (21233003), Fundamental Research Funds for the
Central Universities.
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