Neuro-Regenerative Choline-Functionalized Injectable Graphene Oxide Hydrogel Repairs Focal Brain Injury

Article abstract of DOI:10.1021/acschemneuro.8b00514

Brain damage is associated with spatial imbalance of cholinergic system, which makes severe impact in recovery of damaged neurons of brain. Therefore, maintenance of cholinergic system is extremely important. Here, we fabricated an injectable hydrogel with acetylcholine-functionalized graphene oxide and poly(acrylic acid). Results revealed that this hydrogel is non-cytotoxic, promotes neurite outgrowth, stabilizes microtubule networks, and enhances the expression of some key neural markers in rat cortical primary neurons. Further, this hydrogel exhibits significant potential in neuro-regeneration and also promotes fast recovery of the sham injured mice brain. Moreover, we found significant enhancement of reactive astrocytes in the hippocampal dentate gyrus region of the sham injured brain, indicating its excellent potential in neural repair of the damaged brain. Finally, above results clearly indicate that this neuro-regenerative hydrogel is highly capable of maintaining the cholinergic balance through local release of acetylcholine in the injured brain, which is crucial for brain repair.

Full text of DOI:10.1021/acschemneuro.8b00514

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Article
Neuro-Regenerative Choline Functionalized Injectable
Graphene Oxide Hydrogel Repairs Focal Brain Injury
Krishnangsu Pradhan, Gaurav Das, Juhee Khan, Varsha
Gupta, Surajit Barman, Anindyasundar Adak, and Surajit Ghosh
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00514 • Publication Date (Web): 14 Nov 2018
Downloaded from http://pubs.acs.org on November 15, 2018
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Neuro-Regenerative Choline Functionalized
Injectable Graphene Oxide Hydrogel Repairs
Focal Brain Injury
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Krishnangsu Pradhan,1# Gaurav Das,1,2# Juhee Khan,1 Varsha Gupta,1 Surajit Barman,1
Anindyasundar Adak,1 and Surajit Ghosh*1,2
1. Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4 Raja
S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Fax: +91-33-2473-
5197/0284; Tel: +91-33-2499-5872
2. Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical
Biology Campus, 4 Raja S. C. Mullick Road, Kolkata 700 032, India.
# These two authors contributed equally
Abstract: Brain damage is associated with spatial imbalance of cholinergic system, which makes
severe impact in recovery of damaged neurons of brain. Therefore, maintenance of cholinergic
system is extremely important. Here, we fabricated an injectable hydrogel with acetylcholine
functionalized graphene oxide and polyacrylic acid. Results revealed that this hydrogel is non-
cytotoxic, promotes neurite outgrowth, stabilizes microtubule networks, and enhances the
expression of some key neural markers in rat cortical primary neurons. Further, this hydrogel
exhibits significant potential in neuro-regeneration and also promotes fast recovery of the sham
injured mice brain. Moreover, we found significant enhancement of reactive astrocytes in the
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hippocampal DG region of the sham injured brain, indicating its excellent potential in neural repair
of the damaged brain. Finally, above results clearly indicate that this neuro-regenerative hydrogel
is highly capable of maintaining the cholinergic balance through local release of acetylcholine in
the injured brain, which is crucial for brain repair.
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Keywords:
Acetylcholine, Graphene oxide, Injectable hydrogel, Neurite outgrowth, Brain injury,
Neuroprotection.
1. Introduction:
Brain is an epicentre of all major complex biochemical activities that has mesmerized scientists
for eras.1,2 This enigmatic machinery is a highly sophisticated and extremely complex network
structure consisting of millions of neurons and glial cells that interconnect to give rise to the
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intellects . It acts as an essential integrated system, which controls all the physiological, motor
and cognitive functions. Slight perturbations or damage to this complex array of system leads to
severe complications of its essential functions.4 The major concern with this complex machinery
is its limited regenerative capabilities, and due to this fact adopting novel approaches to understand
the repair mechanism is extremely important.5 Recently, various attempts such as stem cell based
regeneration have been made towards this extremely complex issue.6 However, none of those
approaches have translated into any clinical success.7 Therefore, to address this complex issue
many novel strategies like fabrication of potential biomimetic scaffolds such as electrospun fibers,
hydrogels.8 and various chemically functionalized carbon based materials have been conceived.9
Graphene-based materials (GO) 10,11,12 have attracted huge attention for the fabrication of various
biomaterials due to their excellent electrical, chemical, thermal and charge carrier properties along
with negligible electrical noise, low toxicity and high biocompatibility.13,14 Interestingly, unique
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electrical properties 15 of graphene oxide have been utilized for studying neural growth as well as
neural stimulations.16 Acetylcholine plays crucial role as it maintains cortical activation,17 synaptic
function,18 neuroprotection,19,20 neurogenesis,21 cortical differentiation22,23, neuronal plasticity24
and cortical reorganization25,26. During brain injury, cholinergic system is severely damaged. It
has been shown that cholinergic system plays a crucial role in cortical plasticity and functional
recovery after brain injury.27 Therefore, maintaining the choline balance in injured brain tissue is
crucial for the successful repair of damaged brain.28 So far a few attempts have been made towards
maintenance of choline balance in damaged brain.29 Herein, we have adopted a simple but potential
approach to maintain the choline balance in the damaged brain using covalently conjugated
choline-graphene oxide nanomaterials.
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2. Results and Discussion
2.1 Synthesis of CFGO based hydrogel
For the functionalization of the graphene oxide, 2-hydroxy-N′,N′,N′-trimethylethanaminium was
prepared from 2-aminoethanol and conjugated with GO (Figure S1, S2). Then, we prepared a novel
injectable hydrogel30,31 using this choline-graphene oxide nanomaterial with polyacrylic acid
(CFGO) (Figure 1A).
2.2 FT-IR spectroscopy for characterization of CFGO
GO and CFGO were characterized by FT-IR spectroscopy. For that purpose, GO and CFGO
samples were lyophilized to get the powder. The data was recorded using FT-IR JASCO FT-IR
4200 model. The FT-IR spectrum of GO reveals peak at 882 cm-1 signifying the presence of epoxy
group in the GO surface. The peak at 1115 cm-1 for GO signifies C-OH stretching frequency of -
COOH group supporting the presence of -COOH group in GO surface.32 The peak at 1720 cm-1
due to carbonyl stretching of the carboxyl group also supported the presence of -COOH group in
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GO surface. But in case of CFGO, the characteristic peak at 1735 cm-1 due to ester bond supports
covalent functionalization on GO and formation of CFGO.32 Peaks at the region 3000 cm-1 to 3700
cm-1 for both the cases are due to O-H stretching (both free and H bonded) and amide N-H bond
(secondary) stretching (Figure S3).
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2.3 UV-Vis spectroscopy for characterization of CFGO
UV spectroscopy was used to characterize GO and CFGO. The UV spectroscopy results revealed
that the characteristic absorption peaks appear at 234 and 300 nm, which signify the formation of
GO. But for CFGO a similar spectral analysis results in the reduction in intensity of the peak at
234 nm, which supports the functionalization of the GO surface and formation of CFGO (Figure
S4).
2.4 X-ray photoelectron spectroscopy (XPS) for characterization of CFGO
The formation of CFGO was further confirmed by XPS measurements. In the wide scan spectrum
of CFGO, carbon (C 1s at 284.6 eV), nitrogen (N 1s at 402.2 eV) and oxygen (O 1s at 532.0 eV)
were appeared (Figure S5). This result indicates that acetylcholine mimicking functional group is
successfully covalently attached onto the GO sheets. Further evidence for the formation of CFGO
was obtained through high-resolution XPS spectra. The high-resolution XPS spectra of C 1s for
CFGO (Figure S6A) revealed peaks at 284.6 eV (C-C), 286.7 eV (C-O, C-N+) and 288.7 eV (O-
C=O). Atomic percentage of the each group of the CFGO was also calculated. The result showed
that C-C was 56.2%, C-O, C-N+ was 29.5% and O-C=O was 14.3% (Figure S6B). The high
resolution XPS spectra of N 1s showed peak at 402.4 eV for C-N+ peak (Figure S7). These
informations also support the successful functionalization of acetylcholine on the GO surface.
These XPS results also suggest that the CFGO nanocomposite was successfully prepared.
2.5 Transmission Electron Microscopy (TEM) for characterization of CFGO hydrogel
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Choline functionalized graphene oxide (CFGO) hydrogel was characterized by transmission
electron microscopy (TEM). TEM image of the CFGO hydrogel shows the presence of a 3D
network structure comprising of self-assembled large nanosheets (Figure 1B).
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2.6 Rheological study for CFGO hydrogel
Viscoelastic behaviour of the CFGO based hydrogel was analyzed by rheological analyses in terms
of storage modulus (G′) and loss modulus (G″). Hydrogels are generally characterized by a "solid
like" behavior and the storage modulus is much higher than the loss component. Frequency sweep
experiment of the hydrogel reveals that both G′ and G″ remain independent in the frequency range
from 0.1 to 100 rad/s (G′ > G″) (Figure 2A). This result also supports the solid like rheological
behaviour of the CFGO hydrogel. To get better information about the yield stress of the CFGO
based hydrogel, the amplitude sweep experiment was performed. The maximum yield stress of the
CFGO hydrogel was ~6 KPa (Figure 2B). To analyze the thixotropic behaviour of CFGO, step
strain experiment was performed. When the strain is applied above the linear viscoelastic (LVE)
region, the hydrogel loses its solid like behaviour (G′ < G″). But the sol form of the hydrogel
quickly goes back to the solid like state (G′ > G″) when the strain is applied to the LVE region of
the CFGO hydrogels (Figure 2C). This thixotropic property demonstrates that CFGO hydrogel has
a great character to recover the gel like property, which is crucial for injectable applications of the
hydrogel.
2.7 Injectable nature of CFGO hydrogel
Hydrogel gains a lot of attention in the medicinal field due to its injectable nature. Injectable
hydrogels can easily be delivered to the target region in vivo via a simple syringe without giving
the pain of surgical treatment to the patient. Therefore, we checked the practical applicability of
this type of hydrogel i.e whether they are injectable or not. The injectable nature of the hydrogel
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was checked with a syringe. The images revealed that hydrogel can be injected through a syringe
and removed from the syringe as a sol (Figure S8). After some time, the sol is converted to the gel
automatically.
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2.8 Biocompatibility and neurite outgrowth of PC12 derived neurons on CFGO hydrogel
The growth of the PC12 derived neurons on the biomimetic CFGO hydrogel was assessed by
plating cells on the surface of an overnight UV sterilized hydrogel coated on the surface of the
glass slides. Following this, the cells were cultured for 7 days on these gels and were found to
retain healthy morphology while promoting neurite outgrowth. For a control setup, cells were
grown on CFGO uncoated glass coverslips. Before proceeding any further, it was important to
know the biocompatibility of CFGO, the PC12 cells were cultured and differentiated into neurons
in the presence of NGF. Then, the neurons were treated with CFGO at various concentrations upto
200 µg/mL. MTT assay was performed to check the cell viability, CFGO did not exert any
cytotoxic effects (Figure 2D). This high cell viability of the PC12 derived neurons even after the
treatment with CFGO indicated that CFGO possess excellent biocompatibility for neuron culture.
The various neuronal morphological characteristics (no. of branches, mean neurite length, cell
body area and maximum neurite length) as indicated in the figure were quantified using the
CellSens software. It was found that the number of branches, the area of the cell body, the
maximum and mean length of neurites of the PC12 derived neurons grown on CFGO were
significantly higher than those grown on GO coated or (control) uncoated coverslips (Figure 3A-
E). The health of the neurons which were further confirmed through day wise increase in
expression of neuronal marker GAP 43 (Figure S9). All the above results indicate that our choline
functionalized (CFGO) hydrogel promoted growth, proliferation and health of the neurons.
Microtubules are one of the key cytoskeletal filaments of the cells responsible for the various cargo
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transports. These microtubules are particularly very important in context of neurons as without
this filaments the neurons would not be able to maintain its exaggerated shape. Along with the
maintenance of the dynamicity of the neuronal architecture, they regulate the transport of cargo
proteins along its long axon and dendrites. It is due to this reason that the microtubule arrays are
tightly organized in axons, dendrites, growth cones and migratory neurons with respect to their
intrinsic polarity which is important for both their assembly and transport mechanisms.33 These
inexplicably important roles of microtubules in neurons prompted us to check whether our choline
functionalized hydrogel was able to confer stability to the microtubule networks in the PC12
derived neurons or not. The 7 day old differentiated neurons grown on CFGO coated coverslips
were stained with anti-alpha tubulin antibody and analysed under the microscope. Microscopic
images revealed that in comparison to neurons grown on uncoated coverslips, the neurons grown
on CFGO hydrogel were not only healthy but also showed microtubule stabilization (Figure 3F, G
and Figure S10, S11).
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2.9 Actin filament stabilisation of PC12 derived neurons on CFGO hydrogel
Since CFGO based hydrogel promoted microtubule stabilization, we checked its effect on another
important cytoskeletal filament, actin. Actin is a highly conserved protein that takes part in many
protein-protein interactions and carries the inherent ability to transition between monomeric (G-
actin) and filamentous (F-actin) states controlled by nucleotide hydrolysis and a group of actin-
binding proteins. Thus actin participates and regulates a host of cellular functions like cell motility,
maintenance of cell shape and polarisation of transcription regulation. Actin filaments of the
neurons cultured on the CFGO hydrogel coated glass cover slides were stained with Alexa Fluor
488 phalloidin. Phalloidin is a rigid bicyclic heptapeptide of phallotoxin class isolated from the
Amanita phalloides “death cap" mushroom which selectively labels F-actin and can be used for
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imaging actin filaments. Microscopic images revealed that CFGO hydrogel promoted healthy
network of actin filaments and hence this CFGO hydrogel can be regarded as a cytoskeleton
stabilizer (Figure S12). This exhibits the multifunctional nature of CFGO hydrogel whereby along
with promotion of neurites, they also confer stability to their cytoskeleton framework.
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2.10 The growth and culture of primary rat cortical neurons on CFGO hydrogel
In our earlier observations, it was noted that CFGO hydrogel promoted the growth and attachment
of PC12 derived neurons. Therefore, we decided to check how the primary rat cortical neurons
would behave when plated on CFGO hydrogel coated coverslips. For this purpose, primary cortical
neurons were isolated from the cortex of Sprague Dawley embryonic day 18 rats and plated on the
CFGO hydrogel coated coverslips. It was observed that the rat cortical neurons attached and grew
on this coverslips for the next 14 days. Their growth on the CFGO surfaces over the next 14 days
were marked by extensive neurite outgrowths and numerous dendritic projections, mostly
resembling morphological features observed in cortical neurons in situ (Figure S13). Since CFGO
hydrogel has already shown microtubule stability, we stained the primary cortical neurons with
MAP2 (Microtubule Associated protein 2), a neuron specific cytoskeletal protein, highly enriched
in dendrites with a significant role in microtubule assembly that promotes stabilization of the
dendritic shape during neurogenesis. The MAP2 stained images of the primary cortical neurons
cultured on CFGO hydrogel indicated the ability of the gel to promote the neuronal morphology
of the cortical neurons along with additional stability to its microtubule network (Figure 4A, B).
Next, we checked the biocompatibility of the CFGO by performing the live dead cell assay using
calcein AM/ Propidium iodide on the primary cortical neurons cultured on CFGO. Microscopic
images revealed that even after 14 days of culture the neurons retained almost 100% cell viability,
indicating that CFGO possesses good biocompatibility for cortical neuron culture (Figure 4C, D).
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In our previous studies, we observed that along with PC12 derived neurons, CFGO also promoted
the growth and differentiation of primary rat cortical neurons. This leads us to check the expression
of three key neuronal markers - Growth Associated Protein 43 (GAP 43), Beta Tubulin-III (Tuj 1)
and Microtubule Associated Protein 2 (MAP2) in the primary cortical neurons cultured on the
CFGO coated plates. GAP 43 is also known as the “growth” or “plasticity” protein as it is highly
expressed in developing or regenerating neurons during axon growth. Beta Tubulin-III (Tuj 1) is
a class III member of the beta tubulin protein family, specifically localized to neurons having
implications in neurogenesis, axon guidance and maintenance. MAP 2 as already discussed
indicates microtubule stability and dendritic growth in neurons. To investigate the expression of
these key neuronal proteins, those were isolated from the primary cortical neurons and cultured on
CFGO hydrogel for 14 days and subsequently immunoblot analysis was performed with the
respective primary antibodies. It was observed that the expression of all three neuronal markers
(GAP-43, MAP2 and Tuj1) were higher in the neurons cultured on CFGO hydrogel as compared
to those cultured on uncoated surfaces (Figure 4E). These results indicate that the CFGO hydrogel
promotes neuronal outgrowth and sprouting through the up regulation of the key neuronal markers
(Figure 4F).
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2.11 Effect of CFGO on focal cryogenic injury at the parietal cortex of C57BL/6J mice brain
Both components of our CFGO hydrogel, graphene oxide and choline, have neuroprotective roles.
Out of them especially, choline is known to exert neurogenesis in an injured brain and has been
evaluated for its potential of neuro-recovery in the treatment of stroke as well as Traumatic Brain
injury (TBI) in various animal models and human clinical trials.34,35 Based on all the observations
made so far with PC12 derived and primary rat cortical neurons, CFGO seems to also have neuro-
protective activity, which should be further validated in animal models. Therefore, in our next
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study we tried to understand the neuroprotective role of CFGO in cryogenic injury model (CIM)
of mice. Unilateral cryogenic lesions is a type of focal injury made on the exposed skull bone of
the parietal cortex with a copper rod having a tip diameter of 2.5 mm cooled in liquid nitrogen.
For our study, we used C57BL/6J male mice and divided them into 3 groups each having of 5 mice
(n=5) (Unlesioned control, Control Sham injury and sham injury to be treated with CFGO).
Thereafter, we performed a focal injury in the parietal cortex following the cryoinjury technique.
In the third group of mice, CFGO was applied over the cryolesion and the mice brains were isolated
following the 5th day and 7th day of treatment through transcardial perfusion. Similarly, lesioned
and unlesioned brains were isolated from the sham injury and unlesioned control mice groups
respectively. The brains were then fixed in 4% formalin and images were taken. On careful
examination, a distinct focal injury was observed in the mice brain which persisted in the sham
injured mice upto 7 days while absence of any such cryolesion in the CFGO treated brains
indicated complete recovery. For the histopathological studies, 5 µm thick coronal sections of the
brain were taken and stained with cresyl violet. Cresyl violet acetate stains Nissl substances in the
cytoplasm of formalin fixed neurons and are commonly used to identify neuronal structures in
brain and spinal cord tissue. Cresyl violet stains indicated cortical loss in the brains of sham injured
mice taken at three different time points (1d, 5d and 7d) while no such cortical loss was observed
in the CFGO treated mice brains treated for 5 and 7 days, indicating its potential for neuro-
recovery. The unlesioned control mice brain also showed no such cortical cell loss. No cell loss
was observed in the ipsilateral dentate gyrus region in any of the mice brain sections indicating the
focal nature of our injury. All the above observations indicate the neuroprotective nature of CFGO
hydrogel in injured mice brains (Figure 5A-F). Cryogenic injury in the brain leads to a cascade of
events that result in severe neuroinflammation and neuronal cell death. In response to such injury,
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the astrocytes become reactive exhibiting hypertrophy and hyperplasia. These bunches of reactive
astrocytes have shown to play essential roles in preservation of neural tissue and reduction of
neuroinflammation after a focal injury by forming a glial scar. Reactive astrocytes are marked by
an elevated expression of GFAP (Glial Fibrillary Acidic Protein), a cell specific marker for
astrocytes. In our study, we tried to evaluate the protective role of these reactive astrocytes by
performing immunostaining of GFAP in the coronal sections of the fixed brains. A number of
hypertrophoid astrocytes were observed in the cortical region of the CFGO treated brain sections
as could be seen from the enlarged image which promotes the protective nature of our hydrogel
(Figure 5G-I). Moreover it is well documented that neurogenesis and cell proliferation increases
in the hippocampal DG (dentate gyrus) region after injury which being a potential source of
neurons for repair. Hence, we calculated the number of astrocytes in the DG region from the 7 day
CFGO treated and Sham injured mice brain sections, it was found that there is a higher expression
of GFAP in the CFGO treated mice indicating speedy recovery (Figure 5K). This result further
validates the potential role of our CFGO gel in neuro-recovery following injury.
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3. Conclusions
The brain although an incredible organ comes with its own weaknesses for example it struggles to
repair itself after sustaining damage from injury or stroke. These limited regenerative potential of the
brain prompted us to design an acetylcholine functionalized graphene oxide hydrogel due to the ionic
interaction of positive charge of the quaternary amine with the negative charge of the polyacrylic acid.
Acetylcholine is known to be an essential neurotransmitter. Previously, various neurotransmitters have
shown potential in regulating neuronal development and enhancing neurite outgrowth. The studies with
PC12 derived neurons revealed that CFGO hydrogel is biocompatible with neurons, can be used for
prolong cultures up to 7 days with no additive cyto-toxicity and promotes neurite branching vigorously.
This experiment first gave us the idea of the importance of its neurotransmitter mimicking functional
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group and how it potentiates its neurogenic attributes. Next, we also cultured primary rat cortical
neurons on CFGO and it was observed up to 14 days of culture the neurons showed almost 100 %
biocompatibility. Along with that, CFGO also leads to an increase in the expression of some key
neuronal markers like GAP43, Tuj1 and MAP2, thereby proving that CFGO promotes growth of the
primary cortical neurons. CFGO was additionally found to stabilize key cytoskeletal filaments
microtubules and actin of the neurons which helps in structure maintenance and intracellular/axonal
transport. Along with this, our neuro-regenerative gel showed injectable property and therefore we
treated it with a sham injured mice brain at the site of cryolesion. To our surprise, our hydrogel showed
almost full recovery of the injury within 5 and 7 days of treatment. This was more clearly observed
through cresyl violet staining of the brain slices. At the DG region of the brain slices, a larger number
of reactive astrocytes in the treated mice brain further indicated its neuro-regenerative potential. This
result is supported by measuring the GFAP expression which signifies the reactive astrocytes in
the hippocampal DG region of the injured brain. Overall, these findings support that acetylcholine
functionalized graphene oxide based hydrogel may be considered as an excellent platform for
neuronal outgrowth and neuron repair in the mice model.
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4. Experimental section
4.1 Chemicals
Graphite powder (<60 μm) was bought from Loba Chemie. Trifluoroacetic acid (TFA) was
purchased from Spectrochem. Potassium permanganate and sodium hydroxide were purchased
from Fisher Scientific. Sodium nitrate, sulphuric acid, hydrogen peroxide (30%) and potassium
carbonate were purchased from Merck. Dry methanol was purchased from Acros Organic. 2-
aminoethanol was purchased from Sigma Aldrich. Methyl iodide was purchased from
Spectrochem. N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 3-(4, 5-
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dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), β-Mercaptoethanol, kanamycin
sulfate, nerve growth factor (NGF) 7S, formaldehyde solution (36%), 2, 2, 2-Tribromoethanol,
HEPES buffer, sodium bicarbonate, and low glucose RPMI (Roswell Park Memorial Institute)
1640 medium were purchased from Sigma Aldrich. Fetal bovine serum (FBS), horse serum, PBS,
pH 7.4, GlutaMAX™ Supplement, B27 supplement, neurobasal medium, MAP2 Monoclonal
Antibody (M13), Hank's Balanced Salt Solution (HBSS), Minimum Essential Medium (MEM)
and Penicillin-Streptomycin solution were purchased from Invitrogen. Primary antibodies such as
anti-Beta III Tubulin, anti-GAP 43, anti-α-Tubulin (Clone DM1A) and secondary antibodies goat
anti-Mouse IgG (H+L) Cy3 conjugate, goat anti-mouse and anti-rabbit IgG antibody (H+L) HRP
conjugates were purchased from Merck Millipore. Alexa Fluor 488 Phalloidin was purchased from
Thermo Fisher Scientific.PVDF membrane of 0.45-micron size, BSA, Triton-X 100 and Tween
20 were purchased from Merck Millipore. Glycine, Tris-base, sodium dodecyl sulphate (SDS),
cresyl violet stain, ammonium persulphate, Tetramethylethylenediamine (TEMED) and
bromophenol blue were purchased from Himedia. Surgicals (scalpel, hemostats, scissors, forcep,
suture needle and thread) and copper rod (3.4 mm) were procured locally. propidium iodide
solution and Calcein AM were purchased from BD Biosciences. GFAP antibody (GF5) was
purchased from Abcam. UV data was recorded on UV-1800 ENG 240V Shimadzu Model and FT-
IR data was recorded on JASCO FT-IR 4200 model.
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4.2 Preparation of 2-hydroxy -N,N′,N′-trimethylethanaminium from 2-aminoethanol
250 L of 2-aminoethanol and 20 mL of dry methanol were mixed under nitrogen atmosphere in
a two necked round bottom flask. 2 gm of anhydrous Potassium carbonate (K2CO3) was added into
the round bottom flask. Next, methyl iodide (500 L) was added drop wise into the solution while
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stirring and the solution was stirred for overnight at room temperature followed by evaporation of
solvent to get the product.
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4.3 Preparation of CFGO from GO
The graphene oxide was prepared from the commercially available graphite powder (<60 μm)
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following the modified Hummers method.36 Then, the graphene oxide was dissolved in Milli-Q
water by ultra-sonication. The sonication was performed for an hour and concentration of the
solution was maintained at 4 mg/mL. After that, the solution was treated with 3M aqueous NaOH
solution in a round bottom flask. The solution was sonicated for additional 3h. Then, the alkaline
solution was neutralized (pH 7.0) using dilute HCl solution. The solution was filtered through
Whatman 40 filter paper and the residue was washed with Milli-Q water several times to get pure
graphene oxide (GO-COOH) residue. For the functionalization of the graphene oxide, the
preformed 2-hydroxy -N′,N′,N′-trimethylethanaminium (100 mg) was added into the 10 mL GO-
COOH solution. The concentration of the graphene oxide solution was maintained at 1 mg/mL.
Then the solution was sonicated for 5 min. To the mixture of the solution, N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) was added to reach 5 mM
concentration and again sonicated for 30 min. Then, EDC was added into the reaction mixture to
reach 20 mM concentration. This reaction mixture was left for stirring overnight at room
temperature. After the stirring, 50 μL of β-Mercaptoethanol was added to the reaction to terminate
it. After completion of the reaction, the reaction mixture was centrifuged for 10 min at 12000 rpm
and washing was performed with milli-Q water for 4-5 times. Finally, the residue was lyophilized
and dried CFGO was stored at room temperature.
4.4 UV-Vis Spectroscopy
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For this experiment, solution of GO and acetylcholine functionalized GO (CFGO) were prepared
in Milli-Q water. Then, UV spectra of these solutions were measured maintaining the
concentration of the solution at 2 mg/mL.
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4.5 FT-IR Spectroscopy
FT-IR spectra of GO and CFGO was measured using KBr pellet. GO and CFGO solutions were
lyophilized to get powder compound. Then, these powder compounds were mixed with KBr to
prepare the KBr pellet. FT-IR data was recorded in a Perkin-Elmer Spectrum 100 FT-IR
spectrometer through 48 times scan using the above KBr pellets with speed 0.2 cm/s at a resolution
of 1.6 cm-1. The LiTaO3 detector was used for plotting the data. For each samples, background
correction was performed to eliminate interference from air (or any other parameters).
4.6 X-ray photoelectron spectroscopy (XPS)
For this analysis freshly prepared CFGO was used. The sample was drop casted on a glass cover
slide. Then, the glass slide was put in a vacuum chamber to remove the solvents. All the XPS
analyses were performed using an Al X-ray source operated at 150W on an Axis Ultra X-ray
photoelectron spectrometer. The experiments were performed at an electron takeoff angle of 90°.
General survey scan was performed over the binding energy range of 200-800 eV and high-
resolution spectra was recorded in the regions of C1s, N1s.
4.7 Preparation of CFGO Based Hydrogel
For the preparation of CFGO based hydrogel, a stock solution of poly-acrylic acid was prepared
by dissolving 4 mg in 1 mL of water and another stock solution of CFGO was also prepared by
dissolving 8 mg of CFGO in 1 mL milli-Q water. Then, in the ten sets vial we mixed poly acrylic
acid with CFGO in different proportion. But, when 0.2 mL of poly acrylic acid solution was added
into the solution containing 0.8 mL of CFGO solution, hydrogel was formed easily. This is the
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optimized concentration for the hydrogel formation. To form the hydrogel, mixture was heated
and occasionally sonicated for 15 min. Then the mixture was kept at room temperature for 30 min
to form the CFGO based hydrogel.
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4.8 Rheology
TA Instrument, USA rheometer was used to carry out rheology experiments with a cone-plate
(diameter 40 mm and angle 4°). All the experiments were performed at a constant temperature (25
°C). The temperature was maintained using a Peltier. For these studies, fresh samples were used.
The amplitude and frequency sweep analyses were performed with a fixed gap value. Thixotropic
experiments were performed by applying successive deformation and recovery steps. The steps
were performed repeatedly at a fixed frequency of 1 rad/s.
4.9 Transmission Electron Microscopy (TEM)
For this experiment, a 20 L CFGO hydrogel was dissolved in milli-Q water (80 L) by sonication.
Then, 10 μL of suspensions solution was placed on a carbon coated copper grid. After 1min, excess
solution was removed by filter paper and the grid was stained with 2% uranyl acetate in water.
After 1 min of staining, excess uranyl acetate solution was removed with filter paper from the grid.
Then, the grids were dried under vacuum. Images were captured using a TECNAI G2 SPIRIT
BIOTWIN CZECH REPUBLIC 120 KV electron microscope operating at 80 kV.
4.10 Cell Culture
Rat pheochromocytoma cells (PC12) was a kind gift from Dr. Suvendra Nath Bhattacharyya. The
cells were cultured in RPMI medium supplemented with 10% heat inactivated horse serum (HS),
5% heat inactivated FBS, 100 units/mL penicillin and 100 μg/mL streptomycin in a humidified
atmosphere with 5% CO2 at 37 ºC. For differentiation into neurons, PC12 cells were treated with
nerve growth factor (NGF) (100 ng/mL) in RPMI medium supplemented with 1% HS for 7 days.
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4.11 Cell Viability Assay
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To assess the biocompatibility of our hydrogel, undifferentiated PC12 cells were plated on 96-well
plates and then differentiated for 7 days. Then, the cell viability was checked by MTT assay in
PC12 derived neurons. Around 5000 cells were seeded per well (total medium volume of 100 L).
After 24 h, post-seeding, the medium was replaced with solutions of different concentration of
hydrogel compounds to each well (five wells for each concentration). The MTT assays were
performed. Cells without treatment was used as the control.
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4.12 Neurite Outgrowth Assay37
To assess neurite outgrowth on CFGO hydrogel surfaces, coverslips were coated with CFGO
hydrogel and another set of coverslips were coated with only GO. Then, the PC12 cells were plated
on these CFGO and GO surfaces. The cells were then differentiated with NGF over the next 7 days
and their neurite outgrowth was monitored. As control experiment, PC12 cells were plated on
uncoated coverslips. Consequently, after 7 days the cells were fixed with 4% formaldehyde and
analyzed under microscope (Olympus) in DIC mode. The cells were mainly characterized using
four parameters namely number of branches, mean neurite length, cell body area and maximum
neurite length. Evaluation of these parameters was carried out using the analysis and calculation
mode of CellSens software. The quantitative data has been presented as the mean ± SD. Tests of
data significance was performed using one-way ANOVA.
4.13 Microtubule Stability through Immunostaining
In order to check whether our hydrogel confers stability to microtubules, a very important
component of neuronal cytoskeleton, the PC12 cells were plated on CFGO coated coverslips and
cultured over the next 7 days with NGF in order to induce them into neurons. Similarly, for control
experiments the cells were plated on uncoated coverslips and cultured on them. After 7 days of
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culture, the cells first fixed with 4% formaldehyde for 30 min at room temperature and
permeabilized using 0.2% Triton X-100 for another 15 min. The permeabilized neurons were
incubated with mouse anti-α tubulin overnight at 4 °C. The neurons were then washed with PBS
and further incubated with fluorescence-labeled secondary goat anti-mouse antibody for 2 h at 37
°C. The neuronal nuclei were finally stained with Hoechst dye H33258 (0.05 mg/mL) The
microtubule stability was assessed through microscopic imaging using Olympus microscope
equipped with Andor iXON3 897 EMCCD.
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4.14 F-actin Staining of PC12 derived neurons cultured on CFGO hydrogel
For visualization of F-actin filaments, PC12 cells first cultured on CFGO coated surfaces and then
differentiated into neurons using NGF for 5 days. Thereafter the cells were fixed with 4 %
formaldehyde and were stained with 165 nM Alexa Fluor 488 Phalloidin for 20 minutes at room
temperature. Then it was washed twice with PBS, nucleus stained with Hoechst dye H33258 (0.05
mg/mL) and observed under Olympus microscope equipped with Andor iXON3 897 EMCCD
camera.
4.15 Primary Culture of Rat Cortical Neuron
The culture of primary cortical neurons in this work has been performed following a previously
described method.38,39 Briefly, timed-pregnant Sprague-Dawley rats were taken and brains were
isolated from E18 embryos. Then the brain cortices were micro dissected, processed and filtered.
Next, in order to assess the growth response of the cortical neurons on CFGO hydrogel surface,
cortical neurons with an initial cell density of 1 × 105 cells/mL were cultured on CFGO coated
surfaces in Neurobasal media supplemented with B27, Pen/Strep, and GlutaMAX for the next 14
days. Each experiment was performed in triplicate and control experiments were performed
simultaneously.
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4.16 Immunochemistry staining of the cortical neurons
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The neurons cultured on the CFGO hydrogels were identified by staining them with MAP2, a
neuronal marker. In brief, the cultured neurons were fixed with 4% formaldehyde, permeabilized
with 0.2%Triton X-100 and thereafter incubated with mouse anti-MAP2 overnight at 4 °C. After
washing with PBS, the neurons were further incubated with fluorescence-labeled secondary goat
anti-mouse for 1 h at 37 °C and nuclear staining was performed using Hoechst dye H33258 (0.05
mg/mL).
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4.17 Live/dead Cell Assay
For this experiment, freshly prepared CFGO hydrogel was coated on a cover glass slides using
spin coater. Then, the primary cortical neurons were plated on the CFGO hydrogel coated glass
surfaces. The neurons were cultured for 14 days. After 14 days of culture, the neurons were treated
with a solution containing both 2 M calcein-AM and 4 M propidium iodide (PI) in the dark.
After 30 min, neurons were washed with PBS buffer to remove the excess dye. Finally, live/dead
cell status of the cell was revealed through microscopy of the stained cells using Olympus
microscope equipped with Andor iXON3 897EMCCD camera.
4.18 Western Blot Analysis
Whole cell extracts isolated from PC12 cells and rat primary cortical neurons cultured on CFGO
hydrogel were fractionated by SDS-PAGE and transferred to a 0.45 µm polyvinylidene difluoride
(PVDF) membranes using a transfer apparatus following the manufacturer’s protocol (Biorad).
After that the membranes were blocked using 5% non-fat milk in TBST for 1 h and sequentially
probed with primary antibodies against GAP 43, Beta-III tubulin (Tuj 1) and MAP2. Membranes
were washed thrice for 10 min with TBST and incubated with horseradish peroxidase-conjugated
anti-mouse or anti-rabbit secondary antibodies for 2 h. Blots were again washed thrice with TBST
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and developed with the ECL system using Luminata Forte Western HRP Substrate. -Tubulin was
used as a protein loading control. All quantitative densitometry analysis of the blots obtained have
been performed using ImageJ software and relative expressions represented have been normalized
to the alpha-tubulin.
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4.19 Animal Models
In our laboratory, we have used male pathogen-free C57BL/6 mice (8-11 weeks) for the
development of Cryogenic injury model. All animal experiments have been conducted following
the laws and regulations of the regulatory authorities of our Institutional Animal Ethics Committee.
4.20 Cryogenic Injury Model (CIM) and Histological Studies
The cryogenic injury model has been developed following previously described method.40 Briefly,
the mice was divided into three groups (five mice in each group n=5). The animals were
anaesthetized using 2.5 % Avertin injected intraperitoneally at a dosage of 10 mg/mL body weight
of the mice and were placed on an elevated platform for the surgery. The hair was shaved and was
rubbed with ethanol from the head region to disinfect the area. A midline sagittal incision was
made to expose the skull of the mice. The bregma and lambda were used as a landmark to mark
the left parietal area of the brain for the injury. A cylindrical copper rod of diameter 3.4 mm was
dipped in liquid nitrogen prior to the surgery and the rod was placed on the exposed left parietal
skull for about 60 seconds to make an injury. Wound was closed using suture needle after the
injury. The experimental mice was applied with the gel over the injury prior the wound closure
whereas the sham operated mice were left untreated. All the operated mice were kept for recovery
and were sacrificed by transcardial perfusion using 4 % formaldehyde at two different time points
post injury (5d and 7d). The brains from traumatized mice were isolated and kept overnight in
formaldehyde for post fixation and then it was processed for histological sections. Coronal brain
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sections of 5 μm thickness were obtained and were stained with Cresyl Violet and GFAP antibody
for histological and immunohistological studies respectively. The reactive astrocyte of the hilus
region of DG were counted using Image J software program (NIH, Bethesda, MD, USA). Data is
represented as Mean  S.D.
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4.21 Data Analysis
To analysis various microscopic images, image J software was used. Origin 8.5 pro was used to
calculate various spectroscopic data and bar diagram. For statistical analysis, tow tailed Student’s
t-test and one-way ANOVA were performed. In various experiment, statistical values were for
*P≤0.03, **P≤0.05.
ASSOCIATED CONTENT
Supporting Information
This materials are available free of charge via the Internet at http://pubs.acs.org
The Supporting figures of synthetic scheme for preparation of 2-hydroxy -N′,N′,N′-
trimethylethanaminium, mass spectra of 2-hydroxy -N,N,N-trimethylethanaminium, FT-IR
spectra of GO and CFGO, UV spectra of GO and CFGO, XPS spectra of CFGO, illustration of the
injectable nature of the CFGO based hydrogel, expression of GAP 43 protein isolated from PC12
derived neurons at different days cultured on the CFGO based hydrogel, microscopic images
displaying F-actin staining, microscopic images of PC12 derived neurons for neurite outgrowth in
different channels, microscopic images of primary cortical neurons in different channels.
■ AUTHOR INFORMATION
Corresponding Author
*Fax: +91-33-2473-5197/0284. Phone: +91-33-2499-5872.
Email: sghosh@iicb.res.in.
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ORCID ID
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Gaurav Das: 0000-0002-8432-5384
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Surajit Ghosh: 0000-0002-8203-8613
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Abbreviations
Graphene oxide, GO. Acetylcholine functionalized GO, CFGO.
Author contributions
K.P. carried out synthesis, purification and characterization of CFGO hydrogel. K.P. performed
various in vitro assays such as FT-IR, UV and XPS. G.D performed all the cell based assays such
biocompatibility, neurite outgrowth, intracellular microtubule imaging, F-actin staining and
western blot. J.K., G.D. and VG executed the rat primary cortical neuron culture experiment and
generated the cryo-injury mice model. G.D. and J.K. performed the immunocytochemistry and
histological studies. S.B. and A.S.A supported K.P. and G.D. for performing various experiments
and edited the manuscript. S.G. conceived the idea supervised the project and wrote the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
KP, SB, ASA thank UGC fellowship. GD thanks ICMR fellowship. VG, JK thank DST fellowship.
We thank Dr. Subhadip Bodhak, CGCRI for performing the XPS experiment. SG kindly
acknowledges SERB India for providing financial support (EMR/2015/002230) and CSIR-IICB
for financial support (HCP-0012), infrastructure and animal facility.
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Figure 1. Synthesis and charecterization of CFGO based hydrogel. (A) Synthetic scheme for
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hydrogel. Inset right panel picture shows hydrogel formation and left panel shows the injectable
property.
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