Insulin loaded iron magnetic nanoparticle-graphene oxide composites: Synthesis, characterization and application for in vivo delivery of insulin

Abstract

One of the focal subjects in insulin delivery is the development of insulin formulations that protect the native insulin from degradation under acidic pH in the stomach. In this work we show, for the first time, that a graphene oxide (GO) based matrix can ensure the stability of insulin at low pH. GO and GO modified with 2-nitrodopamine coated magnetic particle (GO-MPdop) matrices loaded with insulin were prepared and the pH triggered release of the insulin was studied. The loading of insulin on the GO nanomaterials proved to be extremely high at pH < 5.4 with a loading capacity of 100 +/- 3% on GO and 88 +/- 3% on GO-MPdop. The insulin-containing GO matrices were stable at acidic pH, while insulin was released when exposed to basic solutions (pH = 9.2). Using Xenopus laevis oocytes as a model we showed that the meiotic resumption rate of GO and GO-MPdop remained unaltered when pre-treated in acidic conditions, while pre-incubated insulin (without GO nanomaterials) has lost almost entirely its maturation effect. These results suggest that GO based nanomatrices are promising systems for the protection of insulin.

Full text

Insulin loaded iron magnetic nanoparticle–
graphene oxide composites: synthesis,
characterization and application for in vivo delivery
of insulin
Kostiantyn Turcheniuk,
ab
Manakamana Khanal,
a
Anastasiia Motorina,
ac
Palaniappan Subramanian,
a
Alexandre Barras,
a
Vladimir Zaitsev,
c
Victor Kuncser,
d
Aurel Leca,
d
Alain Martoriati,
e
Katia Cailliau,
e
Jean-Francois Bodart,
e
Rabah Boukherroub
a
and Sabine Szunerits*
a
One of the focal subjects in insulin delivery is the development of insulin formulations that protect the native
insulin from degradation under acidic pH in the stomach. In this work we show, for the first time, that a
graphene oxide (GO) based matrix can ensure the stability of insulin at low pH. GO and GO modified
with 2-nitrodopamine coated magnetic particle (GO–MP
dop
) matrices loaded with insulin were prepared
and the pH triggered release of the insulin was studied. The loading of insulin on the GO nanomaterials
proved to be extremely high at pH < 5.4 with a loading capacity of 100 3% on GO and 88 3% on
GO–MP
dop
. The insulin-containing GO matrices were stable at acidic pH, while insulin was released
when exposed to basic solutions (pH ¼9.2). Using Xenopus laevis oocytes as a model we showed that
the meiotic resumption rate of GO and GO–MP
dop
remained unaltered when pre-treated in acidic
conditions, while pre-incubated insulin (without GO nanomaterials) has lost almost entirely its
maturation effect. These results suggest that GO based nanomatrices are promising systems for the
protection of insulin.
1. Introduction
The use of nanomaterials as carriers of glycans,
1,2
drugs,
3,4
genes,
5,6
and other biologically active compounds
7
has become a
widely investigated research eld. Most recently, graphene, a
two-dimensional nanomaterial, has been intensively explored as
an alternative nanocarrier for biological materials due to its
large surface area, rich surface chemistry and its potential for
crossing the plasma membrane and promoting the cellular
uptake of molecules.
8,9
The interest in using graphene and gra-
phene oxide (GO) for loading and release of chemical and bio-
logical molecules is in addition linked to the different ways the
molecule can be linked to the graphene matrix: hydrogen
bonding, hydrophobic, p–pstacking and electrostatic interac-
tions can act as anchors that are sensitive to external stimuli
(pH, temperature, chemical substances, electrical eld, etc.),
enabling controlled release.
10–14
Since the pioneering work of Dai
and colleagues
13,15
on the use of PEGylated (PEG ¼polyethylene
glycol) GO as a nanocarrier to load anticancer drugs via non-
covalent physisorption and study its cellular uptake, several
papers have been devoted to improving the loading efficiency
and release of anticancer drugs such as doxorubicin (DOX)
16
or
to the preparation of multi-functionalized graphene nano-
materials.
17–20
Besides graphene and GO, graphene/iron oxide
nanoparticles composite materials have shown great promise as
drug carriers
4,20,21
and for the immobilization and enrichment of
biomolecules.
22
The magnetic particles modied graphene
sheets were synthesized by in situ oxidation of Fe
2+
salts to Fe
3+
and deposited as Fe
3
O
4
particles onto GO, being at the same
time reduced to reduced graphene oxide (rGO).
23
Other
approaches exploited the strong complexation of the carboxylate
anions of GO with FeCl
3
and FeCl
2
, before precipitating Fe
3
O
4
nanoparticles onto GO by treatment with sodium hydroxide.
4,20
It is worth mentioning that in the earlier reports the
magnetic particles were not chemically protected and therefore,
they were prone to corrosion upon immersion in cell culture
media. Here, we report a different strategy for the preparation of
GO–magnetic nanoparticles composite. It is based on the ex situ
synthesis of chemically stabilized magnetic particles with
a
Institut de Recherche Interdisciplinaire (IRI, USR CNRS 3078), Universit´
e Lille 1, Parc
de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France.
E-mail: Sabine.Szunerits@iri.univ-lille1.fr
b
Department of Fine Organic Synthesis, Institute of Bioorganic Chemistry and
Petrochemistry NAS of Ukraine, 1 Murmanska Str., 02660, Kiev, Ukraine
c
Taras Shevchenko University, 60 Vladimirskaya str., Kiev, Ukraine
d
National Institute of Materials Physics, Atomistilor 105 bis, 077125 Magurele,
Romania
e
EA 4479, IFR 147, Universit´
e Lille 1, 59658 Villeneuve d’Ascq, France
Cite this: RSC Adv.,2014,4, 865
Received 31st October 2013
Accepted 11th November 2013
DOI: 10.1039/c3ra46307a
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2-nitrodopamine, followed by their insertion onto the GO
matrix (Fig. 1). This approach prevents any subsequent reduc-
tion of water soluble GO to water insoluble rGO and ensures the
formation of a chemically stable GO–magnetic nanoparticles
interface. This GO matrix is well suited for the uptake of
biomolecules such as insulin.
Insulin, a polypeptide composed of 51 amino acid residues
and secreted by the pancreas, plays an important role in the
control of blood glucose. Diabetic people suffer from low levels of
insulin production and/or from abnormal resistance to the
insulin hormone. Current treatment methods involve regular
injections of insulin, which can be both painful and inconve-
nient. In order to overcome these hurdles, the oral route is
considered as one of the most convenient means of drug uptake.
However, oral administration of hydrophilic macromolecules
such as insulin encounters (or faces) major problems such as
hydrolysis in the low pH of gastric medium, splitting by
proteinases in the stomach and weak penetration through the
membrane of epithelial cells of theintestine.
24,25
One of the most
promising strategies to achieve oral insulin uptake is the use of
microsphere systems, which act both as protease inhibitors by
protecting the encapsulated insulin from enzymatic degradation
and as permeation enhancers by effectively crossing the epithe-
lial layer aer oral administration.
26–31
Behavior, toxicity and
biocompatibility of nanomaterials in vivo is associated to size,
surface of coating and administration routes.
32
Nevertheless, oral
administration appears as an appealing strategy. Indeed, a recent
study underlined the biocompatibility of PEGylated GO deriva-
tives aer oral administration since the injected material
exhibited a long-term retention but no toxicity.
33
2. Experimental part
2.1. Materials
Graphite powder (<20 microns), hydrogen peroxide (H
2
O
2
),
sulfuric acid (H
2
SO
4
), dimethylsulfoxide (DMSO), acetonitrile
(CH
3
CN), ammonium hydroxide (NH
4
OH), iron(II) chloride tet-
rahydrate (FeCl
2
$4H
2
O), iron(III) chloride hexahydrate
(FeCl
3
$6H
2
O), dopamine hydrochloride, sodium nitrite, insulin
(from bovine pancreas, code 10516), dispase, and collagenase
were purchased from Sigma-Aldrich and used as received. 3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) was obtained from invitrogen.
2.2. Preparation of graphene oxide (GO)
Graphene oxide (GO) was synthesized from graphite powder by
a modied Hummers method.
34
5 mg of the synthesized GO was
Fig. 1 (A) Synthetic route of 2-nitrodopamine and functionalization of magnetic nanoparticles (MP) with 2-nitrodopamine. (B) Insulin loading on
GO and GO–MP
dop
. (A).
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dispersed in 1 mL of water and exfoliated through ultra-
sonication for 3 h. This aqueous suspension of GO was used as a
stock solution in subsequent experiments.
2.3. Synthesis of 2-nitrodopamine
2-Nitrodopamine was synthesized according to ref. 35. Dopa-
mine hydrochloride (1.90 g, 10 mmol) and sodium nitrite (1.52 g,
22 mmol) were dissolved in water (25 mL) and cooled to 0 C.
Sulfuric acid (17.4 mmol in 10 mL of water) was added slowly to
the mixture, and a yellow precipitate was formed. Aer stirring at
room temperature overnight, the precipitate was ltered and
recrystallized from water to give a product as a hemisulfate salt.
Yield 1.9 g (77%).
1
H NMR (DMSO-d
6
, 300 MHz, ppm): 3.10
(br s, 4H, CH
2
CH
2
), 6.85 (s, 1H), 7.47 (s, 1H).
2.4. Preparation of 2-nitrodopamine modied magnetic
particles (MP
dop
)
Magnetic particles (MP) were prepared as reported previously.
36
FeCl
2
$4H
2
O (0.34 g, 1.7 mmol) and FeCl
3
$6H
2
O (0.95 g, 3.5
mmol) were dissolved in deareated water (20 mL) and subse-
quently added to a nitrogen-protected three-necked ask under
sonication. The resulting mixture was heated at 50 C for
30 min. Then concentrated ammonium hydroxide (2 mL) was
added dropwise and kept at constant temperature (50 C) for
30 min. The system was nally cooled to room temperature and
the solid product was isolated via a non-uniform magnetic eld
generated by a Nd–Fe–B permanent magnet. The resulting
Fe
3
O
4
particles were washed six times with Milli-Q water to
remove unreacted chemicals and then stored in water.
A water dispersion of bare MP (10 mg mL
1
, 1 mL) was mixed
with 2-nitrodopamine (7 mg) and sonicated for 1 h at room
temperature. The nitrodopamine modied MP (MP
dop
) were
isolated by means of magnet and puried through six consec-
utive wash/precipitation cycles with water to ensure complete
removal of unreacted dopamine. The precipitate was dried in an
oven at 50 C.
2.5. Preparation of GO–MP
dop
nanohybrid
2 mL of GO in water (2 mg mL
1
) was sonicated for 1 h before
2mLofMP
dop
(1 mg mL
1
) were added and further sonicated
for 2 h at 30 C under N
2
.Inarst step, the resulting precipitate
was isolated by centrifugation at 13.500 rpm (20 min) and
puried through two consecutive wash/centrifugation cycles at
13.500 rpm (20 min) with water. Further purication was ach-
ieved by magnetic separation to separate the magnetic GO–
MP
dop
hybrid from the non-magnetic phase. This procedure
yielded z5mgofGO–MP
dop
hybrid.
2.6. Loading of insulin onto GO and GO–MP
dop
hybrid
GO or GO–MP
dop
nanohybrid (150 mgmL
1
) was sonicated with
the desired concentration of insulin for 2 h and then stirred for
22 h at room temperature. All samples were centrifuged at
13.500 rpm for 30 min. The concentration of insulin in the
supernatant was determined using a standard insulin concen-
tration curve generated with a UV/Vis spectrophotometer at
275 nm from a series of insulin solutions of different
concentrations.
2.7. Release of insulin from GO and GO–MP
dop
hybrid
The release behavior of insulin from the GO–MP
dop
hybrid and
GO was investigated at 37 C under stirring at 40 10 rpm by
varying the pH. At predetermined time-points, samples were
centrifuged at 13.500 rpm for 30 min and the supernatant was
analyzed using a UV/Vis spectrometer at 275 nm. The precipi-
tate was redispersed in 1 mL of fresh PBS and release studies
were continued.
2.8. Cell viability/cytotoxicity studies (MTT test) on HEK
cells
HEK cells were seeded in 96 wells plate at a density of 3 10
4
cells per well at 37 C. Aer 24 h of culture, the medium in the
wells was replaced with fresh medium, containing the GO–
MP
dop
insulin hybrid in varying concentrations. Aer incuba-
tion of the HEK cells for 24 hours, the medium was replaced and
10 mL of MTT (12 mM in sterile PBS) was added in each well and
incubated for 4 h at 37 C. Then medium was carefully removed
and formed formazan crystals were solubilized with DMSO
(50 mL). The absorbance of each well was read on a microplate
reader (PHERAstar FS, BMG LABTECH) at 540 nm. Each
condition was replicated for four times and wells without GO–
MP
dop
insulin hybrid were taken as negative control.
2.9. Biological assays: cytotoxicity and M-phase entry in
Xenopus oocytes
Adult Xenopus females were purchased from University of Ren-
nes I, France. Aer anesthetizing Xenopus females by immersion
in MS222 solution (tricaine methane sulfonate, 1 g L
1
), ovarian
lobes were surgically removed and kept in ND96 physiological
medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl
2
, 1 mM MgCl
2
,5
mM HEPES–NaOH, pH 7.5). For follicular cells removal from
oocytes, fragments of ovarian lobes were treated by dispase (3 h,
0.4 mg L
1
), rinsed and bathed in collagenase (1 h, 0.4 mg L
1
)to
end defolliculation. Fully-grown stage VI oocytes were selected
according to their morphology.
37
The latter oocytes are arrested
at the G2/M border of the rst meiotic division and resume
meiosis in response to hormonal stimulation in vitro upon
progesterone or insulin addition in the medium.
38,39
Oocytes
were stored at 14 C in ND96 medium until use.
In the case of GO and GO–MP
dop
, the oocytes were pre-incu-
bated for 30 min with the GO matrix before hormonal stimulation
by insulin. In the case of GO–insulin and GO–MP
dop
–insulin, the
matrix was directly added to the oocytes. The pH media were
adjusted using HCl/NaOH solutions at pH 1, 2, 5.3, 7.4 and 9.2.
Kinetic of Germinal Vesicle Breakdown (GVBD) was scored by the
appearance of a white spot (WS) at the animal pole of the cell,
which attests of the M-phase entry and meiosis resumption.
2.10. Instrumentation
2.10.1. Fourier transformed infrared (FTIR) spectroscopy.
Fourier transform infrared (FT-IR) spectra were recorded using
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a Perkin-Elmer Spectrum One FT-IR spectrometer with a reso-
lution of 4 cm
1
. Dried powder (1 mg) was mixed with KBr
powder (100 mg) in an agate mortar. The mixture was pressed
into a pellet under 10 tons load for 2–4 min and the spectrum
was recorded immediately. Sixteen accumulative scans were
collected. The signal from a pure KBr pellet was subtracted as a
background.
2.10.2. X-ray photoelectron spectroscopy (XPS). X-ray
photoelectron spectroscopy (XPS) measurements were per-
formed with an ESCALAB 220 XL spectrometer from Vacuum
Generators featuring a monochromatic Al KaX-ray source
(1486.6 eV) and a spherical energy analyzer operated in the CAE
(constant analyzer energy) mode (CAE ¼100 eV for survey
spectra and CAE ¼40 eV for high-resolution spectra), using the
electromagnetic lens mode. No ood gun source was needed
due to the conducting character of the substrates. The angle
between the incident X-rays and the analyzer is 58. The
detection angle of the photoelectrons is 30.
2.10.3. Particle size measurements. Homogeneous
suspensions of nanoparticles (20 mgmL
1
) in water were
prepared by ultrasonication. The particle size of the nano-
particles suspension was measured at 25 C using a Zetasizer
Nano ZS (Malvern Instruments S.A., Worcestershire, UK) in 173
scattering geometry and the zeta potential was measured using
the electrophoretic mode.
2.10.4. UV/Vis measurements. Absorption spectra were
recorded using a Perkin Elmer Lambda UV/Vis 950 spectro-
photometer in plastic cuvettes with an optical path of 10 mm.
The wavelength range was 400–1100 nm or 400–700 nm.
2.10.5. Magnetic measurements. Temperature and eld
dependent magnetic measurements have been performed by
SQUID magnetometry (MPMS XL magnetometer from Quantum
Design) under the high sensitivity reciprocal space option, RSO.
In addition, the Fe phase composition and local magnetic
interactions were analyzed by the powerful method of the
57
Fe
M¨
ossbauer spectroscopy. M¨
ossbauer spectra were collected at
different temperatures between 5 K and 240 K, in transmission
geometry, by inserting the sample into a close cycle He cryostat.
AM
¨
ossbauer drive system operating in constant acceleration
mode combined with conventional electronics and a
57
Co(Rh
matrix) source of about 30 mCi activity were employed.
2.10.6. Transmission electron microscopy (TEM). TEM
measurements were performed in a FEI Tecnai G2 20 equipped
with EDS micro-analysis, Gatan energy lter (EELS), electron
precision and tomography.
2.10.7. Thermogravimetric analysis (TG). Thermogravi-
metric analysis measurements were made in Al
2
O
3
crucibles in
an atmosphere of nitrogen at a heating rate of 10 C min
1
using a TA Instruments Q50 thermogravimetric analyzer.
3. Results and discussion
3.1. Graphene oxide (GO)-2-nitrodopamine modied iron
oxide nanoparticles (MP
dop
): synthesis and characterization
The synthesis of the GO–MP
dop
hybrid matrix and the chemical
structure of the capping ligand employed are illustrated in
Fig. 1. We synthesized Fe
3
O
4
NPs using the co-precipitation
reaction of Fe
2+
and Fe
3+
in alkaline media as reported previ-
ously by us.
36
The magnetic particles were functionalized using
2-nitrodopamine as capping agent. The introduction of an
electron withdrawing nitro group onto the catechol nucleus is
known to result in a catecholate anchor far superior to dopa-
mine.
40–42
The higher oxidation potential of the 2-nitrodop-
amine ligand implies that nanoparticles surface degradation is
diminished, insuring irreversible binding of ligand and good
stability of the resulting nanostructures. These properties are
crucial when using such particles for follow-up reactions and in
biomedical applications.
43
This procedure results in MP
dop
with
a mean diameter of 15 5 nm obtained from the analysis of
several thousands of nanoparticles by transmission electron
microscopy (TEM) images (Fig. 2A).
The chemical composition of the 2-nitrodopamine modied
particles (MP
dop
) was examined using FTIR spectroscopy
(Fig. 3A). The MP
dop
nanostructures exhibit bands at 1291 and
1500 cm
1
corresponding to C–O and C]C vibrations of the
catechol system, bands at 1233 and 1548 cm
1
due to symmetric
and asymmetric vibrations of NO
2
group, and bands at 3367 and
1619 cm
1
due to the stretching and bending modes of primary
amines. The bands at 2854 and 2920 cm
1
are due to CH
stretching vibrations of the dopamine ligand. The MP
dop
particles were incorporated onto graphene oxide (GO) nano-
sheets by sonicating GO and MP
dop
(mass ratio 2/1) for 2 h
(Fig. 1). MP
dop
nanoparticles adsorption on GO is believed to be
Fig. 2 TEM images of (A) MP
dop
and histogram of particle size distri-
bution; (B) GO–MP
dop
.
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driven by p–pstacking interactions between the 2-nitrodop-
amine ligand on the magnetic particles and sp
2
rings on GO.
Electrostatic interactions between the acid groups of GO and
the amine groups of the 2-nitrodopamine ligand are unlikely as
the pK
a
of 2-nitrodopamine is near pH 6.5, lower when
compared to dopamine ligands with a pK
a
>9.
40,44
TEM
measurements of GO–MP
dop
nanohybrid reveal the presence of
spherical particles with 15 5 nm in diameter as in the case of
free magnetic particles (Fig. 2B). The hydrodynamic size of the
hybrid is estimated to be 294 68 nm (polydispersity index ¼
0.649 0.113) with a surface charge of 50.4 1 mV.
We have shown, recently, thatdopamine and its derivatives are
excellent reducing agents of GO.
45,46
In case of an irreversible
binding of the 2-nitrodopamine capping agent to the magnetic
particles, reduction of GO is unlikely to occur, as the 1,2-diols of
the catechol linker are not oxidizable to their corresponding
quinine structure. The FTIR spectra of GO before and aer
loading with MP
dop
particles are shown in Fig. 3A. In the case of
GO, the bands at 1734 and 1624 cm
1
correspond to v(C]O) of
–COOH and the skeletal vibration of unoxidized graphite
domains, respectively. Loading 2-nitrodopamine modied
magnetic particles on GO resulted in a comparable FTIR spec-
trum as for GO with additional bands at 1291 and 1500 cm
1
corresponding to C–O and C]C vibrations of the catechol
system. To gain further information on the chemical composition
of the resulting GO–MP
dop
nanomaterials, X-ray photoelectron
spectroscopy (XPS) analysis was performed. The C1s core level
XPS spectrum of GO nanosheets is displayed in Fig. 3B. It can be
deconvoluted into four components with binding energies at
about 283.8, 284.7, 286.7 and 287.9 eV assigned to sp
2
-hybridized
carbon, C–H/C–C, C–O and C]O species, respectively. Deposi-
tion of MP
dop
onto GO did not alter the C1s spectrum signicantly
showing contributions at 284.7, 286.7 and 287.9 eV due to C–C/C–
H, C–O/C–N and C]O moieties, respectively. The presence of GO
rather than rGO is in line with the irreversible binding of the 2-
nitrodopamine ligand to the magnetic particles. The success of
the incorporation of MP
dop
particles is furthermore conrmed by
the presence of 14.2 mass% of iron.
The UV/Vis absorption spectra of GO, 2-nitrodopamine and
GO–MP
dop
hybrid are depicted in Fig. 3C. GO dispersed in water
exhibits a maximum absorption at 228 nm, attributed to the
p–p*transition resulting from C]C bonds of the aromatic
skeleton, and a broad shoulder at 297 nm due to the n–p*
transition of C]O bonds from carboxylic acid functions. The
UV/Vis spectrum of the free 2-nitrodopamine ligand exhibits a
prominent peak at 352 nm. In the case of the GO–MP
dop
hybrid,
a broad peak between 350 and 390 nm with a maximum at 370
nm was observed due to the presence of MP
dop
.
Thermogravimetric analysis was carried out to understand
better the binding nature of MP
dop
and insulin to GO. Fig. 3D
indicates gradual decomposition of the hybrid with two stages
of weight loss at 150 C and 320 C indicating the decomposi-
tion of functional groups from insulin and graphene oxide.
Large weight loss at 850 C is accounted for by breakdown of
coordination bond between nitrodopamine and Fe
3
O
4
nanoparticles.
4,23,47
The magnetic properties of the MP
dop
and once onto the GO
matrix were in addition determined. Indeed, it has been shown
by Finotelli et al.,
31
that insulin could be released from alginate/
chitosan beads containing magnetic nanoparticles by the use of
a magnetic eld. While not investigated here, the magnetic
properties were nevertheless determined. The hysteresis loop of
the MP
dop
sample obtained at 300 K in a eld range of 20 kOe
(above the pseudo-saturation) is shown in Fig. 4A. In the inset of
the same gure is presented the magnetization at increasing
temperature obtained in a eld of 80 Oe aer cooling the
sample in zero eld. The well known zero eld cooling (ZFC)
procedure gives rise to a magnetization curve specic to nano-
particulate systems, with a maximum at a blocking temperature
of about 250 K. It means that above such a temperature (e.g. at
300 K), the MP
dop
behave superparamagnetically, in agreement
with the zero coercive eld shown by the hysteresis loop. The
specic saturation magnetization of MP
dop
at 300 K is about 60
emu g
1
,
43
being about 10% lower than that of naked (uncoated)
magnetic nanoparticles
34
at the same temperature (these values
are lower than the specic spontaneous magnetization of bulk
magnetite, due to both thermal effects related to the reported
magnetization at 300 K as well as due to an expected more
defective structure related to size effects). However, the lower
saturation magnetization in the MP
dop
as compared to naked
MP has to be related to a diminished relative weight of the
magnetic ions in the sample, due to the presence of the addi-
tional 2-nitrodopamine surfactant.
Fig. 3 (A) FTIR spectra of MP
dop
, GO and GO–MP
dop
; (B) C1s core level
XPS spectra of GO (a) and GO–MP
dop
(b); (C) UV/Vis spectra of GO in
water (blue), 2-nitrodopamine (black) and GO–MP
dop
(red), (D) TGA (in
nitrogen, scanning rate of 10 C min
1
)ofGO–MP
dop
–insulin.
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The hysteresis loop of the GO–MP
dop
sample obtained in
similar conditions as for sample MP
dop
is shown in Fig. 4B. In
the down inset of the same gure is also presented the ZFC
magnetization curve in 80 Oe, which is clearly different from
that of the MP
dop
sample. It is worth mentioning the increase of
the magnetization at lower temperature (e.g. from 150 K down
to 10 K), suggesting the presence of a ferromagnetic-like inter-
action among the nanoparticles as compared to the antiferro-
magnetic dipolar type usually observed in non-diluted
nanoparticulate systems. Such interactions seem to be present
also at higher temperatures, leading to a consistent shiof the
blocking temperature well above 290 K. If the nature of such
unusual interactions requires additional studies of the GO–
MP
dop
mixtures, the consistent difference of the ZFC curve of
the GO–MP
dop
sample as compared to the ZFC curve of the
MP
dop
sample clearly proves the formation of the GO–MP
dop
hybrid with specic properties induced by the strong interac-
tions of the nanoparticles via the GO support. In the upper inset
of the same Fig. 4B is shown the hysteresis loop of the GO
substrate, collected in similar conditions as for the GO–MP
dop
hybrid. It is observed that in the maximum eld of 20 kOe, the
magnetization of GO (0.008 emu g
1
) is 1000 times lower than
that of the hybrid sample (about 8 emu g
1
) and therefore can
be clearly neglected. Hence, the saturation magnetization of the
GO–MP
dop
hybrid is just 13% from the saturation magnetiza-
tion of MP
dop
sample, inferring an equivalent (13 mass%) of
loading magnetic material in the analyzed sample. This is in
accordance with XPS analysis where a 14.3 mass% of iron was
determined.
3.2. Insulin loading and release
The kind of interactions of GO, MP
dop
and insulin is of utter-
most importance not only for the construction of a stable GO–
MP
dop
hybrid but also for insulin loading and release strategies.
As discussed above, 2-nitrodopamine ligands are used as
capping agent for the formed magnetic particles. The linkage
insuring an irreversible binding of the ligand. Indeed, no
degradation of the MP
dop
particles size and chemical compo-
sition was observed upon immersion for 4 h into aqueous
solutions of low pH (pH ¼1), as might be observed under
biological conditions. The interaction of the dopamine ligand
to the iron oxide nanoparticles is not disrupted in the lower pH
range. Interaction of the dopamine-capped MP particles with
the GO matrix is mostly over p–pstacking interactions between
the hexagonal cells of graphene and the aromatic ring structure
of dopamine. As the diol functions of the used catechol are not
available, the formation of ortho-quinol structures is inhibited
in this case and a further covalent binding not feasible.
45
The loading capacity of insulin onto GO–MP
dop
can be
evaluated by measuring the concentration of insulin using UV/
Vis spectra at 275 nm in solution before and aer insulin
loading. The difference corresponds to insulin loaded onto the
GO–MP
dop
matrix (Fig. 5A). Indeed, due to the high UV/Vis
absorbance of GO occurring in the same spectral area as
insulin, a direct determination of the insulin concentration on
the GO–MP
dop
matrix is not possible. The insulin loading
capacity of the GO–MP
dop
nanohybrid was calculated according
to eqn (1):
Loading capacity ¼ c0csup
cGOMPdop !100% (1)
where c
0
is the initial concentration of insulin added to GO, c
sup
is the concentration of insulin in the supernatant aer reaction
determined by UV/Vis and c
GO–MP
dop
is the concentration of GO–
MP
dop
(150 mgmL
1
).
Many studies have shown that aromatic molecules including
chemotherapy drugs such as doxorubicin can be loaded onto
the surface of graphene via p–pstacking interactions.
12,15,21
In
the case of insulin, a polypeptide composed of 51 amino acid
residues, electrostatic forces will additionally affect insulin
loading as the isoelectronic point (pI) of insulin is reported to be
5.4.
27
The inuence of the pH on the loading of a xed
concentration of insulin onto GO–MP
dop
is displayed in Fig. 5B.
Using a loading time of 24 h, increased insulin loading was
observed at pH < 5.5 in accordance with an insulin pI ¼5.4.
27
Below pH 5.4, insulin is positively charged and interacts more
strongly with the negatively charged GO–MP
dop
matrix. This
interaction is weakened at pH > 5 and at pH 7 the loading
Fig. 4 Magnetic properties of MP
dop
particles (A) and of GO–MP
dop
(B). Main graphs show the corresponding hysteresis loops in 20 kOe at 300 K,
down insets show the dependence of the magnetization versus temperature after zero field cooling and subsequent measuring at increasing
temperatures in a field of 80 Oe and upper inset in (B) shows the hysteresis in 20 kOe of a reference GO sample.
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capacity of insulin is 3 times lower as only p–pstacking inter-
actions and/or hydrophilic interactions will prevail between
insulin and GO–MP
dop
. The loading capacity of GO–MP
dop
is as
high as 88 3%, although some areas of the multifunctional
GO has been previously occupied with magnetic particles.
Indeed, this results in a decrease in the loading capacity when
compared to GO (100 3%), but is still remarkably high when
compared to other nanostructures.
47,48
For mesoporous silica
insulin loading of 15% was reported,
47
while poly(lactide-
ethylene glycol) nanoparticles showed a maximal insulin
loading of 58.8%.
48
To understand better which of the materials, GO or MP
dop
is
more effectively loading insulin, the loading capacitance of
MP
dop
was in addition determined. No detectable amounts of
insulin could be determined by the colorometic assay, indi-
cating that all the insulin reacts with the GO matrix rather than
the magnetic particles.
Insulin release from GO and GO–MP
dop
hybrid at pH 5 was
analyzed by incubating the matrices at 37 Catdifferent pH
while shaking. Fig. 6 shows the cumulative release of insulin
from GO (Fig. 6A) and GO–MP
dop
(Fig. 6B) matrices as a func-
tion of pH. At pH ¼2, even though a small release of insulin is
observed, probably due to weakly bound insulin, GO and GO–
MP
dop
appear to have a high insulin retention capacity.
Comparable behavior was observed on core–shell poly(ethylene
glycol)polyhedral oligosilsesquioxane nanoparticles.
30
Following a pH change to 5, insulin release is initiated and
sustained for the rst 90 min. The amount of insulin released is
highly pH dependent with about 28 3% of insulin released at
pH 9 for GO–MP
dop
and 40 3% for GO. The insulin release
from the GO–MP
dop
hybrid turns to be less successful than from
GO alone, which likely accounts for some levels of interaction
between insulin and nanoparticles within the hybrid. The
release at pH 9 is most likely due to electrostatic repulsion
between negatively charged insulin and negatively charged GO
and GO–MP
dop
. The release is fast and low when compared to
mesoporous silica nanoparticles with a maximal release of 77%
at pH 8.5 aer 10 h.
47
It is comparable to poly(lactide-ethylene
glycol) nanoparticles with a release of 59% aer 10 days,
48
or
alginate/chitosan microcapsules with a release of 18% in the
rst hours and about 45% aer 3 days.
31
The high insulin retention capacity at low pH, comparable to
that of gastric pH suggests that insulin is well protected on GO
and GO–MP
dop
hybrid, while at intestinal pH (pH 6–7) insulin is
activated and released. We thus investigated, if GO and GO–
MP
dop
hybrids could be used as potential carriers for an insulin
drug delivery system.
3.3. Cell viability assay of GO and GO–MP
dop
Two different cell viability assays were performed to obtain
information about the cytotoxicity of the GO–MP
dop
hybrid. The
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diephenyltetrazolium
bromide) assay is a simple colorimetric assay to measure cell
cytotoxicity, proliferation or viability and used in this work. As
seen in Fig. 7A under the investigated concentration range of
GO–MP
dop
and GO–MP
dop
–insulin no cytotoxicity to HEK cells
is observed.
In Xenopus laevis large number of oocytes are easily obtained
at all stages of maturation, making this organism an excellent
model for studying the role of insulin and insulin growth
factors on the development of the organism.
49,50
Fully grown
Fig. 5 Insulin loading on GO and GO–MP
dop
: (A) UV/Vis spectra of
free insulin at different concentrations and the corresponding cali-
bration curve (inset); (B) insulin loading capacity of GO (blue) and
GO–MP
dop
(red) as a function of pH.
Fig. 6 Insulin release from GO (A) and GO–MP
dop
(B) for different pH
and at different time points (error bars are based on triplicate
measurements).
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Xenopus laevis oocytes are physiologically arrested at the
prophase of the rst meiotic divisions. These oocytes must
resume meiosis and proceed to the metaphase of meiosis II
before fertilization is possible. The process which enables
fertilization and drive the oocyte from prophase of rst meiotic
division to a block in metaphase of second meiotic division,
Fig. 7 (A) Cytotoxicity of GO–MP
dop
(grey) and GO–MP
dop
insulin (blue) to HEK cell lines; (B) schematic illustration of insulin induced process of
meiotic resumption of fully grown Xenopus laevis oocytes: Xenopus oocytes (stage VI) before (a) and after treatment with insulin (10 mgmL
1
)at
pH ¼9.2 (b). A typical white spot, attesting for the germinal vesicle breakdown (GVBD) transition from the G2 to the M phase of the cell cycle, is
seen; (C) meiotic resumption rate of Xenopus oocytes incubated for 24 h with GO (black) and GO–MP
dop
(red) at different concentrations and
after injection of insulin (c¼50 mgmL
1
); (D) meiotic resumption rate as a function of pH.
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termed maturation, is triggered in vivo by a preovulatory
gonadotropin surge followed by follicular production of
progesterone.
39,51–53
In addition to progesterone and other
hormones, both insulin and insulin-like growth factor-1 (IGF-1)
can induce meiotic resumption and oocyte maturation.
36,39
Fig. 7A shows photographs of Xenopus laevis oocytes (stage VI)
before and aer treatment with insulin (10 mgmL
1
) at pH 9.2.
A typical white spot, attesting for the germinal vesicle break-
down (GVBD), is observed under the binocular at the animal
pole of the oocytes. We use the monitoring of oocyte meiotic
resumption in this study for testing their viability and respon-
siveness towards insulin aer being released from GO and GO–
MD
dop
nanostructures. To ensure that GO and GO–MD
dop
nanostructures without insulin have no cytotoxic effect on
Xenopus oocytes, the fully-grown stage VI oocytes were exposed
for 24 h to increasingly high concentrations of GO and GO–
MP
dop
at pH 7.4. Fig. 7B shows the meiotic resumption rate
upon insulin induction and indicates that exposure to GO and
GO–MP
dop
even at high concentrations is not toxic for oocytes,
showing a comparable meiotic resumption rate when the Xen-
opus laevis oocytes were not pre-incubated with the nano-
structures. The meiotic resumption rate of Xenopus laevis
oocytes upon injection of insulin (50 mgmL
1
)atdifferent pH
was investigated to insure that insulin release at higher pH
would have an important inuence on oocytes. As seen from
Fig. 7C, no signicant changes in meiotic resumption rates were
observed when insulin induction was performed at pH above 5.
However, at pH ¼2, the meiotic resumption rate is signicantly
decreased, indicating deviations from native insulin most likely
linked to conformational changes that have occurred in the
polypeptide chains of insulin.
54
3.4. Meiotic resumption rates of Xenopus laevis oocytes
upon addition of GO–insulin and GO–MP
dop
–insulin
The dose effect of insulin at pH 5 and 9.2 on the meiotic
resumption rate of oocytes was investigated. As seen in Fig. 8A,
at pH ¼9.2, the minimal insulin concentration resulting in
high rates of meiotic resumption is around 1.2 mgmL
1
. Below
this concentration level, no meiotic resumption was observed.
At pH 5, this concentration limit was shied to higher insulin
concentrations. A comparable concentration range was thus
chosen for the insulin loaded GO and GO–MP
dop
nano-
structures. Fig. 8B compares the meiotic resumption rates of a
variety of different experimental set-ups. GO (5.6 mgmL
1
) and
insulin (5.6 mgmL
1
) were used as negative and positive
controls in this comparative experiment. GO–insulin and GO–
MP
dop
–insulin nanostructures showed a dose-dependence
response: while at a concentration of 0.8 mgmL
1
, both
matrices exhibited only low meiotic resumption rates at pH 9.2,
concentrations higher than 5.6 mgmL
1
resulted in high levels
of meiotic resumption as for free insulin. While this behaviour
is expected, a surprisingly different meiotic resumption
behaviour was observed once insulin, GO–insulin and GO–
MP
dop
–insulin were pre-incubated for 5 h at pH ¼2. For insulin,
the meiotic resumption rate was highly decreased in line with
the observation in Fig. 7C. However, acid pre-treated
GO–insulin and GO–MP
dop
–insulin nanostructures did not
show any altered meiotic resumption characteristics. This in
vitro experiment proves that the insulin incorporated onto the
nanostructures is not affected by the low pH, and the GO
“protects”insulin from acidic degradation. The GO–insulin and
GO–MP
dop
–insulin nanostructures might be thus considered as
novel insulin formulations next to microcapsules, polymers and
others.
26,31,48,54,55
The appealing character of GO–insulin and
GO–MP
dop
–insulin nanostructures is that the nanocomposites
are easy to prepare and can be produced on a larger scale. The
incorporation of magnetic particles does not alter the meiotic
resumption prole of Xenopus laevis oocytes, used as model
system here. The attractiveness of the incorporation of the
Fig. 8 Meiotic resumption response curves of Xenopus oocytes: (A)
influence of the insulin concentration (0–10 mgmL
1
) and the solution
pH; progesterone (10 mgmL
1
) was used as positive control. (B)
Influence of the concentration of GO–insulin and GO–MP
dop
–insulin
on the meiotic resumption rate at pH 9.2; GO was used as negative
control and insulin (5.6 mgmL
1
) as positive control; meiotic
resumption rate of pre-incubated (pH ¼2; 6 h) insulin, GO–insulin and
GO–MP
dop
–insulin.
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magnetic particles is that insulin controlled release can be
enhanced in the presence of a magnetic eld, as previously
demonstrated by Finotelli et al. using alginate/chitosan beads
containing magnetic particles.
31
4. Conclusions
In this study, we have demonstrated that graphene oxide
matrices can be easily loaded with different carriers. In our case
2-nitrodopamine coated magnetic particles and/or insulin was
incorporated onto the GO nanosheets. The insulin loading
capacity on the GO nanomaterials was pH-dependent, but
proved to be extremely high at pH lower than 5.4 with 100 3%
and 88 3% loading on GO and GO–MP
dop
, respectively.
Insulin-loaded on GO matrices was stable at acidic pH, but was
released when exposed to basic solutions (pH ¼9.2). Insulin
retained its native structure when released from the matrix. In
addition, the insulin loaded on GO and GO–MP
dop
were strongly
resistant to acidic pH, as for that encountered in the gastric
environment. These results open new avenues for further
investigations of the potential application of insulin loaded on
GO matrices for treatment of patients with insulin deciency.
Acknowledgements
A.B, R.B and S.S. gratefully acknowledge nancial support from
the Centre National de Recherche Scientique (CNRS), the
Universit´
e Lille 1, the Nord Pas de Calais region, and the Institut
Universitaire de France (IUF). Support from the European
Union through a FP7-PEOPLE-IRSES (PHOTORELEASE) is
acknowledged. Support from the Romanian project PNII IDEI
75/2011 is gratefully acknowledged.
References
1 A. Barras, F. A. Martin, O. Bande, J. S. Baumann, J.-M. Ghigo,
R. Boukherroub, C. Beloin, A. Siriwardena and S. Szunerits,
Nanoscale, 2013, 5, 2307.
2 M. Durka, K. Buffet, J. Iehl, M. Holler, J.-F. Nierengarten,
J. taganna, J. Bouckaert and S. P. Vicent, Chem. Commun.,
2011, 47, 1321.
3 J. J. Shi, A. R. Votruba, O. C. Farokhzad and R. Langer, Nano
Lett., 2010, 10, 3223.
4 X. Yang, X. Zhang, Y. Ma, Y. Huan, Y. Wang and Y. Chen,
J. Mater. Chem., 2009, 19, 2710.
5 X. Yang, G. Niu, X. Cao, Y. Wen, R. Xiang, H. Duan and
Y. Chen, J. Mater. Chem., 2012, 22, 6649.
6 L. Zhang, Z. Lu, Q. H. Zhao, J. Hunag, H. Shen and
Z. J. Zhang, Small, 2011, 7, 460.
7 K. E. Sapsford, W. R. Algar, L. Berti, K. Boeneman Gemmill,
B. J. Casey, E. Oh, M. H. Stewart and I. L. Medintz, Chem.
Rev., 2013, 113, 1904.
8 H. Y. Mao, S. Laurent, W. chen, O. Akhavan, M. Imani,
A. A. Ashkarran and M. Mahmoudi, Chem. Rev., 2013, 113,
3407–3424.
9 K. Yang, L. Feng, X. Shi and Z. Liu, Chem. Soc. Rev., 2013, 42,
530.
10 Y. Pan, H. Bao, N. G. Sahoo, T. Wu and L. Li, Adv. Funct.
Mater., 2011, 21, 2754.
11 U. Dembereldorj, M. Kim, S. Kim, E.-O. Ganbold, S. Y. Lee
and S.-W. Joo, J. Mater. Chem., 2012, 22, 22845.
12 W. Zhang, Z. Guo, D. Huang, Z. Liu, X. Guo and H. Zhong,
Biomaterials, 2011, 32, 8555.
13 Z. Liu, J. T. Robinson, X. M. Sun and H. J. Dai, J. Am. Chem.
Soc., 2008, 130, 10876.
14 J. Hong, N. J. Shah, A. C. Drake, P. C. DeMuth, J. B. Lee,
J. Chen and P. T. Hammond, ACS Nano, 2012, 6, 81.
15 X. M. Sun, Z. Liu, K. Welsher, et al.,Nano Res., 2008, 1, 203.
16 X. Yang, X. Zhang, Z. Liu, Y. Ma, Y. Huang and Y. Chen,
J. Phys. Chem. C, 2008, 112, 17554.
17 W. Miao, G. Shim, S. Lee, S. Lee, Y. S. Choe and Y.-K. Oh,
Biomaterials, 2013, 34, 3402.
18 L. Zhang, J. Xia, Q. Zhao, L. H. Liu and Z. Zhang, Small, 2010,
6, 537.
19 Y. Yang, Y.-M. Zhang, Y. Chen, D. Zhao, J.-T. Chen and
Y. Liu, Chem.–Eur. J., 2012, 18, 4208.
20 X. Yang, Y. Wang, X. Huang, Y. Ma, Y. Huang, R. Yang,
H. Duan and Y. Chen, J. Mater. Chem., 2011, 21, 3448.
21 X. Ma, H. Tao, K. Yang, L. Feng, L. Chen, X. Shi, Y. Li, L. Guo
and Z. Liu, Nano Res., 2012, 5, 199.
22 G. Cheng, Y.-L. Liu, Z.-G. Wang, J.-L. Zhang, D.-H. Sun and
J.-Z. Ni, J. Mater. Chem., 2012, 22, 21998.
23 Y. Xue, H. Y. Chen, D. Yu, S. Wang, M. Yardeni, Q. Dai,
M. Guo, Y. Liu, F. Lu, J. Qu and L. Dai, Chem. Commun.,
2011, 47, 11689.
24 M. Goldberg and I. Gomez-Orellana, Nat. Rev. Drug Discovery,
2003, 2, 289.
25 N. A. Peppas and N. J. Kavimandan, Eur. J. Pharm. Sci., 2006,
29, 183.
26 F. Wang, Y. Chen and H. A. E. Benson, Open Drug Delivery J.,
2008, 2,1.
27 F. Cui, K. Shi, L. Zhang, A. Tao and Y. Kawashima,
J. Controlled Release, 2006, 114, 242.
28 G. P. Carino and E. Mathiowitz, Adv. Drug Delivery Rev., 1999,
35, 249.
29 D. P. Huynh, M. K. Nguyen, B. S. Pi, M. S. Kom, S. Y. Chae,
K. C. Lee, B. S. Kim, S. W. Kim and D. S. Lee, Biomaterials,
2008, 29, 2527.
30 K.-O. Kim, B.-S. Kim and I.-S. Kim, J. Biomater.
Nanobiotechnol., 2011, 2, 201.
31 P. V. Finotelli, D. Da Silva, M. Sola-Penna, A. Malta Rossi,
M. Farina, L. R. Andrada, A. Y. Takeuchi and M. Rocha-
Leao, Colloids Surf., B, 2010, 81, 206.
32 J. Liu, L. Cui and D. Losic, Acta Biomater., 2013, 9, 9243–9257.
33 K. Yang, H. Gong, X. Shi, J. Wan, Y. Zhang and Z. I. Liu,
Biomaterials, 2013, 34, 2787.
34 O. Fellahi, M. R. Das, Y. Coffinier, S. Szunerits, T. Hadjersi,
M. Maamache and R. Boukherroub, Nanoscale, 2011, 3, 4662.
35 M. Rodenstein, S. Z¨
urcher, S. G. Tosatti and N. D. Spencer,
Langmuir, 2010, 26, 16211.
36 M. Mazur, A. Barras, V. Kuncer, A. Galatanu, V. Zaitzev,
P. Woisel, J. Lyskawa, W. Laure, A. Siriwardena,
R. Boukherroub and S. Szunerits, Nanoscale, 2013, 5, 2692.
37 J. N. Dumont, J. Morphol., 1972, 136, 153.
874 |RSC Adv.,2014,4,865–875 This journal is © The Royal Society of Chemistry 2014
RSC Advances Paper
Published on 11 November 2013. Downloaded by Univ Lille 1 on 03/12/2013 09:37:51.
View Article Online

38 F. Baert, J. F. Bodart, B. Bocquet-Muchembled, A. Lescuyer-
Rousseau and J. P. Vilain, J. Biol. Chem., 2003, 278, 49714.
39 M. El-Etr, S. Schorderet-Slatkine and E. E. Baulieu, Science,
1979, 205, 1397.
40 E. Amstad, A. U. Gehring, H. Fisher, V. V. Nagaiyanallur,
G. H¨
ahner, M. Textor and E. Reimhult, J. Phys. Chem. C,
2011, 115, 683.
41 E. Amstad, T. Gillich, I. Bilecka, M. Textor and E. Reimhult,
Nano Lett., 2009, 9, 4042.
42 E. Amstad, M. Textor and E. Reimhult, Nanoscale, 2011, 3,
2819.
43 S. J. Clarke, C. A. Hollmann, Z. Zhang, S. Suffern,
S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik and
J. L. Nadeau, Nat. Mater., 2006, 5, 409.
44 A. K. L. Yuen, G. A. Hutton, A. F. Masters and T. Maschmeyer,
Dalton Trans., 2012, 41, 2545.
45 I. Kaminska, M. R. Das, Y. Coffinier, J. Niedziolka-Jonsson,
J. Sobczak, P. Woisel, J. Lyskawa, M. Opallo,
R. Boukherroub and S. Szunerits, ACS Appl. Mater.
Interfaces, 2012, 4, 1016.
46 I. Kaminska, W. Qi, A. Barras, J. Sobczak, J. Niedziolka-
Jonsson, P. Woisel, J. Lyskawa, W. Laure, M. Opallo, M. Li,
R. Boukherroub and S. Szunerits,Chem.–Eur. J., 2013, 19,8673.
47 L. Sun, X. Zhang, C. Zhen, Z. s. Wu and C. Li, J. Phys. Chem. B,
2013, 117, 3852.
48 L. Tomar, C. Tyagi, M. Kumar, P. Kumar, H. Singh,
Y. E. Choonara and V. Pillay, Int. J. Nanomed., 2013, 8, 505.
49 L. Scavo, A. R. Shuldiner, J. Serrano, R. Dashner, J. Roth and
F. De Pablo, Proc. Natl. Acad. Sci. U. S. A., 1991, 88, 6214.
50 I. B. Dawid, T. D. Sargent and F. Rosa, Curr. Top. Dev. Biol.,
1990, 24,2.
51 L. Zhu, N. Ohan, Y. Agazie, C. Cummings, S. Farah and
X. J. Li, Endocrinology, 1997, 139, 949.
52 Y. Mazui and H. J. Clarke, Int. Rev. Cytol., 1979, 57, 185.
53 L. D. Smith, Development, 1989, 107, 685.
54 K. Yoshida, K. Sato and J.-I. Anzai, J. Mater. Chem., 2010, 20,
1546.
55 R. A. Shimkunas, E. Robinson, R. Lam, S. Lu, X. H. Xu,
X.-Q. Zhang, H. Huang, E. Osawa and D. Ho, Biomaterials,
2009, 30, 5720.
This journal is © The Royal Society of Chemistry 2014 RSC Adv.,2014,4,865–875 | 875
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