MUCOADHESIVE PROPERTY OF CHITOSAN COVALENTED FUNCTIONALIZE
WITH GRAPHENE OXIDE (GO-CS)
Pantipa Koraviyotin1,*, Nattaya Ngamrojanavanich2,#, Noungnuj Muangsin2,#
1
Program of Petrochemistry and Polymer Science, Faculty of Science, Chulalongkorn University,
Thailand
2
Biomaterial and Bioorganic Chemistry Research Group, Chulalongkorn University, Thailand
*e-mail: pa.jung@hotmail.com, #e-mail: nongnuj.j@chula.ac.th
Abstract
The mucus membrane is moist membranes found in alimentary canal therefore the
mucodahesive polymer influenced for oral drug delivery carrier. The chitosan modified with
graphene oxide (GO-CS) that have rich hydrogen functional group on GO surface and flexible
chain of CS is a capability structure for mucoadhesive property. The GO-CS synthesis by Hammer
method via amidation process and characterized by FTIR, TGA and NMR. The mucoadhesion of
polymer are measured by The Periodic Acid Schiff (PAS). The covalent attachment of chitosan
with graphene oxide are showed adsorbed mucin of GO higher than pure chitosan in PBS solution
at pH 1.2 and 6.4. The GO-CS displayed a 12.64 and 2.53-fold stronger mucoadhesion than that
without GO both at pH 1.2 and 6.4. Therefore, GO-CS is an interesting mucoadhesive polymer for
biomedical application.
Keywords: mucoadhesion, chitosan, graphene oxide, mucus membrane, mucoadhesive polymer
Introduction
The mucoadhesive polymers are an attractive material in drug delivery system for
mucosal membranes that improve prolong period of attachment, high drug concentration and
reduce the dosage. Beside the potential of structure for affixed with mucus including chain
flexibility, strong hydrogen bonding group, ionic charge and thiol group [1-3]. The mucosa
membrane be consist of the mucin glycoproteins which have rich of sialic acid, sulfate, and
carboxylate content leading to a negative surface charge [4]. Chitosan (CS) is naturally linear
cationic polysaccharide consist of N-acetyl-D-glucosamine and D-glucosamine units found good
mucoadhesive property, biodegradability, non-toxicity and high biocompatibility. Owing to
cationic and chain flexibility would be able to interact more strongly with the mucus glycoproteins
via hydrogen bonds of primary amino (NH2) and hydroxyl (OH) groups of chitosan backbone and
electrostatic effect however its poor solubility in neutral and basic pH [5-6]. Graphene oxide
(GO), the honeycomb structure of graphene consists of oxygen functional groups, has prepared by
oxidation which dispersion and exfoliation in aqueous due to its strongly hydrophilic. The
advantages of GO is oxygen functional groups on surface to approach chemical functionalization
via covalent or non-covalent. Moreover, the biocompatibility, sp2 structure, large surface area,
strong hydrogen bonding group and particularly low cost precursor therefore GO is able to use in
biomedical application and have functional group for mucoadhesive property [7-8]. The graphene
oxide modified with chitosan displayed high loading camptothecin and showed cytotoxicity in
HepG2 and HeLa cell lines [9]. The graphene oxide loaded doxorubicin encapsulation by folic
acid conjugated chitosan (GO-DOX-CHI-FA) as drug delivery carrier [10].
The aim of this work is to synthesis the modified covalent attachment of chitosan with
graphene oxide via amide linkage as drug delivery carrier that enhance mucoadhesion of hybrid
polymer due to chain flexibility, covalent and non-covalent (such as electrostatic interaction and
hydrogen bond) able to attach with mucin.
Methodology
Synthesis of GO and GO -CS
The graphite powder were oxidized to form GO by a modified Hummer method [11] The
GO-CS was prepared by the amidation process used of EDC as a coupling agent. In a typical
process, mixed 1 g CS and 0.1g GO into 100 ml 1% (v/v) acetic acid and sonicated for 2 h then
loaded EDC as coupling agent and stirring for another 16 h at room temperature finally sonicated
for 3 h. Afterward, precipitation suspension with de-ionized water and acetone. The final product
was dialyzed in the ice bath for 3 days and lyophilized [9].
Characterization
The CS, GO and GO-CS are characterized by A Fourier transform infrared spectra
(FTIR) was recorded on Nicolet 6700 using KBr pellets.
1
H Nuclear Magnetic Resonance
spectroscopy (NMR) was obtained from Bruker NMR operated at 400 MHz. Thermgravimetric
analysis (TGA) was done under nitrogen using PerKin-Elmer Pyris Diamond TG/DTA instrum
ent at heating rate 10°C/min from 50 to 600°C.
Adsoption of mucin on GO and GO-CS
The Periodic Acid Schiff (PAS) is evaluated the amount of free mucin. A 0.5% (w/v)
mucin solution in PBS buffer solution at pH 1.2 and pH 6.4 were prepared. The CS, GO and
GO-CS were dispersed (at 10 mg/1mL final) in the above mucin solutions and shaken at 37 ◦C for
2 h then centrifuged at 12,000 rpm for 2 min. The mucin concentration was calculated by
reference to the calibration curve of mucin solution, and the amount of mucin adsorbed was
calculated as the difference between the total amount of mucin added and the free mucin content
in the supernatant [12].
Results
The GO-CS was obtained via amide bond of amine group of CS chain and carboxyl group on
GO surface, using EDAC as a coupling agent as shown in Fig. 1.
CH OH
CH OH
CH OH
O OH2
O OH2
O OH2
O
O
NH
NH2
HN
O
O
H3C
O
O
HO
O
OH
OH
O
C H 2O H
O
OH
OH
HO
+
O
C H 2O H
O
OH
NH
O
O
NH2
C H 2O H
O
OH
O
1% acetic acid,
EDAC , 24h
O
OH
OH
NH2
O
CH3
OH
HO
HO
O
HO
O
Graphene oxide (GO)
Chitosan (CS)
O
O
Graphene oxide-Chitosan (GO-CS)
Fig. 1. Synthesis scheme of Graphene oxide-chitosan (GO-CS)
Fourier transformed infrared spectroscopy (FTIR)
The FTIR spectrum of CS, GO and GO-CS was presented in Fig. 2. the spectra of CS peak at
1635 and 1078 cm-1 presented of C=O amide, C-O stretching, as a characteristic band of CS
backbone, respectively. The absorption band of GO at 1723, 1624, 1225 and 1058 cm-1 attributed
to C=O (COOH), C=C, C-OH and C-O-C stretching of the oxygen functional groups on GO
surface after grafted CS on GO the intensity of C=O (amide) and C-O stretching were increased
and no band showed at 1723 cm-1 (C=O) so that confirm CS are grafted with GO via amide bond.
1H Nuclear Magnetic Resonance spectroscopy (NMR)
The 1H NMR show in Fig. 3 of chitosan presented the characteristic peaks at 4.53, 3.57 –
3.38, 2.84 and 1.73 ppm that attributed to H1, H2, H3-H6 and acetyl proton in CS skeleton. The
GO is not present spectra of 1H NMR however GO-CS show the appearance of H3-H6 proton of
CS backbone implied that CS are successfully grafted on GO.
Thermogravimetric analysis (TGA)
The results of TGA data show in Fig. 4 represented the thermal decomposition of CS started
to lose around 259 ₒC and 60% weight loss that attributed to chitosan backbone degradation. The
GO show TGA data had 62% weight loss and mass loss at 208 ₒC due to the pyrolysis of oxygen
functional group on GO. However, the TGA data of GO-CS present two stages, the first stage was
indicated to the degradation of oxygen functional group on GO while the second stage attributed
to decomposition of CS skeleton. The weight loss of GO-CS are 46% lower than GO suggested
that the thermal stability of GO increase after grafted CS part.
(a) CS
1078
Transmittance (%)
1635
(b) GO
1723
1225
1620
1058
(c) GO-CS
1635
1021
4000
3500
3000
2000
2500
1500
1000
500
-1
Wavenumbers (cm )
Fig. 2. FTIR spectra of (a) CS, (b) GO, (c) GO-CS (1:10 (w/w)
GO:CS
A
6
C H 2O H
O
(a) CS
OH
H3-H6
O
4
O
D2O
OH
2
3
NH
H2
C H 2O H
O
5
1
O
C H 2O H
O
OH
c
NH2
NH2
CH3
Ac
H1
6
(b) GO-CS
O
O
O
5 CH OH
CH OH
2
O
1
O OH2
O 4 2 OH O
3
NH
HN
2
HO
H2
4.0
OH
OH
O
4.5
OH
3.5
3.0
O
O
2.5
2.0
ppm
Fig. 3. 1H NMR of (a) CS, (b) GO, (c) GO-CS (1:10 (w/w) GO:CS)
(a) CS
60%
(b) GO
62%
(c) GO-CS
46%
100
200
300
400
500
600
ₒ
Temperature ( C)
Fig. 4. TGA data of (a) CS, (b) GO, (c) GO-CS (1:10 (w/w)
GO:CS)
NH
H3C
HO
H3-H6
5.0
CH OH
O OH2
1.5
O
Table 1. The comparison adsorption mucin of CS, GO and GO-CS (1:10 (w/w) GO:CS)
Adsorbed of
Adsorbed of
mucin at pH 1.2
mucin at pH 6.4
(mg/ml) ± SD
(mg/ml) ± SD
CS
0.03 ± 0.08
0.17 ± 0.06
GO
0.37 ± 0.04a
0.43 ± 0.01a
GO-CS
0.35 ± 0.04 a,b
0.43 ± 0.01 a,b
Batch
a
The mean difference is not significant (P > 0.05) compared to chitosan using
t-test function.
b
The mean difference is not significant (P > 0.05) compared to graphene
oxide using t-test function.
Adsorption of Mucin on CS, GO and CS-GO at pH 1.2 and 6.4 in PBS buffer solution
The comparison of adsorption mucin show in Table 2 and the Fig. 5 observed the adsorbed
mucin of GO and GO-CS higher than CS. At pH 1.2, the acidic condition in stomach, display the
adsorption GO and GO-CS are significant increase 13.2- and 12.6-fold comparing with pure
chitosan because of the rich hydrogen bonding between hydroxyl and carboxylic group of GO and
on mucin. Furthermore, at pH 6.4, the condition in small intestine, showed the adsorbed of mucin
on GO and GO-CS are higher than CS presented about 2.5-fold attributed to hydrogen bonding
between hydroxyl group on sialic acid, which is subdomain of mucus, and amine group on CS
chain and hydroxyl group on GO surface. Moreover, GO-CS has covalent bond attachment from
carboxylate of mucin glycoprotein with amine group on chitosan side chain. Therefore, at pH 6.4
expresses strong mucoadhesive property comparing with unmodified chitosan.
The advantage of this research in pharmaceutical application show the model in Fig. 6 that
able to use GO-CS loading aromatic insoluble drug due to the sp2 structure of GO that able to
occur π-π stacking with aromatic drug and hydrogen bonding from hydroxyl and carboxylic group
of GO surface interact with hydrogen boning group in drug structure. It is improvement of the
encapsulation efficiency. Beside, GO-CS has mucoadhesion with mucosa membrane and pH
dependent that make it suitable as drug delivery carrier.
pH 1.2
0.5
0.45
pH 6.4
Adsorption of mucin (mg/ml)
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
CS
GO
GO-CS
Fig. 5. Comparison adsorbed mucin of CS, GO and GO-CS (1:10 (w/w)
GO:CS)
NH
O
NH2
NHCOCH3
= GO-CS
HO
O
GO
OH
COOH
= Aromatic drug
Mu c u s
Fig. 6. Pharmaceutical model of Graphene oxide- chitosan (GO-CS)
(GO-CS)
Discussion and Conclusion
The modified chitosan with graphene oxide grafted (GO-CS) are synthesized by amidation
process which using EDC as a coupling agent. The GO and GO-CS have significant increased
mucoadhesive property comparing with CS in PBS buffer solution at pH 1.2 and 6.4. Because
of rich hydrogen boning from the part of GO, in addition it has amide bond between amine
group on CS chain and carboxylate group on mucin at pH 6.4.
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Acknowledgement
This research was financially supported by The 90th Anniversary of Chulalongkorn University
Fund (Ratchadaphiseksomphot
Endowment Fund) to Miss.
Pantipa
Koraviyotin,
the
Chulalongkorn University Centenary Academic Development Project (Under the Center of
Innovative Nanotechnology, Chulalongkorn University) to N.M., and Integrated Innovation
Academic Center: IIAC Chulalongkorn University Centenary Academic Development Project to
N.M. (PPS560530064) and the Biomaterial and Bioorganic chemistry Research group of
Chulalongkorn University.