Fabrication of new generation of co-delivery systems based on graphene-g-cyclodextrin/chitosan nanofiber

Article abstract of DOI:10.1016/j.ijbiomac.2019.11.144

Although many techniques have been devoted to promote therapeutic purposes of drug carrier systems, however, there are still many challenges in this area. Here, we designed co-loaded delivery systems, composed of curcumin loaded cyclodextrin-graphene oxid

Full text of DOI:10.1016/j.ijbiomac.2019.11.144

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Fabrication of new generation of co-delivery systems based on
graphene-g-cyclodextrin/chitosan nanofiber
Shabnam Sattari, Abbas Dadkhah Tehrani, Mohsen Adeli,
Khadijeh Soleimani, Marzieh Rashidipour
PII:
S0141-8130(19)34855-X
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.11.144
BIOMAC 13925
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
27 June 2019
6 November 2019
18 November 2019
Please cite this article as: S. Sattari, A.D. Tehrani, M. Adeli, et al., Fabrication of new
generation of co-delivery systems based on graphene-g-cyclodextrin/chitosan nanofiber,
International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/
j.ijbiomac.2019.11.144
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Fabrication of new generation of co-delivery systems based on graphene-g-
cyclodextrin/chitosan nanofiber
Shabnam Sattari 1a, Abbas Dadkhah Tehrani 2a*, Mohsen Adeli 3a, Khadijeh Soleimani 4a and
Marzieh Rashidipour1b
a Department of Chemistry, Faculty of Science, Lorestan University, Khorramabad, Iran
bRazi Herbal Medicines Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran
Abstract
Although many techniques have been devoted to promote therapeutic purposes of drug carrier systems,
however, there are still many challenges in this area. Here, we designed co-loaded delivery systems,
composed of curcumin loaded cyclodextrin-graphene oxide core (Cur@CD-GO) and gallic acid loaded
chitosan shell nanofibers (Cur-Ga NF), which can promote the therapeutic efficiency of drugs. The
synthesized nanofibres were fabricated by electrospinning technique with the coaxial system. Results
showed that co-loaded delivery systems (Cur-Ga NF) provide better performance over single drug-loaded
NFs (Cur@CD-GO). It was demonstrated that the nanofibers were successfully prepared, and the drugs in
the core and sell of nanofibers were released in a controlled and sustained manner. The produced Cur-Ga
NF, providing improved anti-cancer activity, antimicrobial activity, antioxidant activity and anti-
inflammatory outcome as compared to single drug-loaded NFs. Our investigations showed that such co-
delivery fiber systems could be employed as a promising nanocarrier for therapeutic applications.
Keyword: Graphene oxide, Curcumin, Gallic acid
* Corresponding author: Tel: (+98)913-2323706; fax: +98 6616200612.
E-mail address: A_dadkhahtehrani@yahoo.com; Dadkhah.a@lu.ac.ir
(A. DadkhahTehrani).
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1. Introduction
Nowadays, co-loading of therapeutic drugs into one drug delivery system has revolutionized in
the medical field to improve the therapeutic activity. Combination therapy provides an effective
strategy to achieve high therapeutic concentration compared with single drug therapy [1-3]. In
this regard, polymer nanocarriers with core–shell nanostructures are the most prevalent carrier in
these applications [4, 5]. Electrospinning technique is a versatile method to produce core–shell
nanocarrier with special structure and characterization by appropriate choice of the polymer
composition and using suitable reinforcing agents. Electrospun nanofibers with core–shell
structures provide an effective strategy for loading different types of active pharmaceutical
ingredient and have been proposed as one of the most desired systems for controllable and
sustainable delivery of drugs and bioactive molecules [6-11]. Especially, the preparation of
nanofibers with core–shell structures in a single step, using coaxial electrospinning, is one of the
most prominent strategies to effectively promote features of the drug delivery systems [12-16].
Previous studies have reported that numerous pharmaceutical systems can be fabricated by the
coaxial electrospinning. For example, song group [17], designed biofunctionalized scaffolds
based on ligned laminin core-polydioxanone/collagen shell (Lam-PDO/Col) fibers for promoting
neuritogenesis using a coaxial system. They suggested that a magnetic field-assisted
electrospinning technique with sustained release of laminin can improve neural tissue
regeneration. In another study, Yin and his colleagues [18], prepared Icariin (ICA)-Silk fibroin
(SF) /PLCL nanofibers by coaxial electrospinning. Icariin in the core of nanofibers was released
in two stages (burst stage and constant stage). The results demonstrated such systems could be an
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effective technique for bone defect regeneration. These studies inspired us to further to provide
nanofibers with improved therapeutic properties.
Although various studies of drug delivery systems based on nanofibers have been conducted[19,
20], the preparation of nanofibers co-delivery containing anti-cancer and antimicrobial drugs
along with two different release systems through a single step via co-axial electrospinning
process has rarely been reported. With this aim, in the present study, a new graphene oxid-
grafted polymer nanofibers with core-shell structure fabricated as synergistic drug delivery
system by the electrospinning process. The use of graphene-based drug delivery systems that are
compatible with physiological environments have been highly regarded in the wide range of
biological applications owing to their excellent physical and chemistry properties, high surface
area and having a surface amenable to modification [21-23]. According to the previous studies
generally, graphene-based nanomaterials may exert different degrees of toxicity in animals or
cell models [24-26] . However, a simple modification of graphene based nanomaterials with
biocompatible polymer especially bio-polymers such as cyclodextrin and chitosan, significantly
reduces their toxicity and enhanced their activity as a potent drug delivery nanosystems [27-31].
Herein, in this study for the first time, polyoxazoline is used for grafting cyclodextrine to
graphene nanosheets via nitrene chemistry. The conjugation of graphene nanosheets to polymer
chains through nitrene chemistry has many advantages due to the easy operation, simple
processing process and good yield. The hydrophobic drugs, Cur could be loaded onto the empty
spaces on graphene via π-π interactions as well as through inclusion interactions with β-
cyclodextrin (CD). Then, a core-sell drug delivery nanofibers fabricated simply by coating of the
graphene oxid-grafted-cyclodextrin@Cur drug delivery system (Cur@CD-GO) with gallic acid
loaded chitosan (Ga@Cs) via coaxial electrospinning. Further, the chemical structure,
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morphology, cytocompatibility, antibacterial and anti-inflammatory property of the nanofibers
were systematically investigated.
2. Experimental
2.1. Materials and Instruments
2-Ethyl-2-oxazoline (EOX) ≥99% used as the monomer, Polyvinyl alcohol (PVA, MW= 85,000-
124,000, 98-99% hydrolyzed), Polyvinylpyrrolidone (PVP, average MW= 40,000), Chitosan
with molecular weight 190–310 kDa were obtained from Sigma Aldrich chemical company. β-
cyclodextrin (CD), p-Toluenesulfonyl chloride, Graphite powder, Curcumin (Cur) and gallic acid
(Ga) as models drug also all other reagents and solvents used in the study were sourced from
Merck chemical company and purified by usual methods.
Fourier transforms infrared (FTIR) spectra of samples were recorded by Bruker-Tensor320
spectrometer with a KBr pellet. All UV-visible spectra were recorded using a Shimadzu UV–vis
1650 PC spectrophotometer. Thermogravimetric analysis was performed using Perkin Elmer
Pyris 1 TGA from 25 °C to 900 °C, at a scan rate of 10°C/min under nitrogen atmosphere. X-ray
diffraction (XRD) patterns of samples in the scan range of 2θ = 2° to 90°were recorded with a
Siemens diffractometer using Cu-KA radiation at 35 kV. The morphology of the nanofibers
studied by scanning electron microscopy LEO 440i scanning electron microscope under vacuum
at an operating voltage of 10 kV. Before analysis, all samples were sprayed with gold by
sputtering for 15 s. At least 100 fibers were measured for each sample, and their averages and
standard deviations were reported. The contact angle metering system (developed by the College
of Chemistry and Chemical Engineering, Lanzhou University, China) was used to evaluate the
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wettability of the nanofibers. To prepare nanofibers, an electrospinning device from Nano Azma
Company (Iran) was employed.
2.2. Synthesis of 6-O-Monotosyl-β-cyclodextrin (β-CD-OTs)
According to reported method in literature [32], NaOH solution (35.5 mL, 2.5 M) was added to a
suspension of β-cyclodextrin (8.8 mmol in 90 mL H2O). After 10 min, p-toluenesulfonyl
chloride (13.22 mmol) was added slowly over 8 min while it was stirred at 0–5 °C in an ice-
water bath. Then the mixture was continuously stirred for 5 h at 25 °C. The unreacted p-
toluenesulfonyl chloride was removed by filtration and the pH of the filtrated solution was
adjusted with pre-swollen cation exchange resin to be approximately pH = 8-9. After the
formation of a white precipitate, the exchange resin was collected and further purified with
distilled water three times. The reaction yield was 50%.
2.3. Synthesis of azido-terminated poly (2-ethyl-2-oxazoline)/β-cyclodextrin (CD-POX-N3)
The polymerization of EOX was performed using β–CD-OTs as initiator and NaN3 as a
terminator. Briefly, 2-ethyl-2-oxazoline (8.0 g, 80 mmol) and β–CD-OTs (300 mg, 1.6 mmol)
was added to acetonitrile (10 ml) with monomer/initiator ratio 20:1. After stirrer for 48 h under
an N2 atmosphere at 70 °C, polymerization of PEOX was terminated using NaN3 (430 mg, 6.6
mmol) and the reaction mixture was stirred for another 48h at 70 °C. The polymer was purified
by precipitation in diethyl ether and then was dialyzed against deionized water for 48 h.
Afterward, the obtained product dried under vacuum at 50°C. The reaction yield was 85%.
2.4. Preparation of β-Cyclodextrin-grafted graphene Oxide (CD-GO) via nitrene chemistry
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Graphene oxide (GO) was synthesized according to the reported procedure in our previous
literature [33]. In order to prepare of CD-GO, GO (0.03 g) was dispersed in 50 mL DMF in a
round-bottom flask and sonicated for 2 h. CD-POX-N3 (0.96 g) was dissolved in 50 ml DMF and
added into the GO mixture and reaction was continued at 110 °C for 48 h. Synthetic methods often
need to use specific solvents such as DMF as reaction media that despite their advantages in the
handling of reactions may be toxic and therefore should remove from the products even in very
small portions. In this context, the obtained product thoroughly washed with di ethyl ether
several times and then dialyzed against water for 72 h. Finally, the product was dried at 50 °C for
48 h. The reaction yield was 79%.
2.5. Preparation of core solutions of nanofiber (Cur@CD-GO NF)
In brief, Cur (0.1 mmol) was mixed with CD-g-GO (100 mg in an ethanol: water mixture with a
40:70 volume ratio) by stirring for 24 h. The resulting solution was centrifuged at 6,000 rpm for 10
min. Then, the supernatant was adding to PVP (8%) with a weight ratio of 70:30. The resulted
mixture was stirred for another 24h at 37 °C in darkness.
2.6. Preparation of shell solutions (Ga@Cs)
In brief, Cs (2%) acetic acid solution (100 ml) was added into PVA (8 wt %) aqueous solution
(with a weight ratio of 30:70) and continuously stirred at room temperature. Then, Ga (0.1 mmol in
1 mL ethanol) was added to the above solution (1000 mg) and stirring continued for another 5 h.
2.7. Fabrication of Cur-Ga nanofibers (Cur-Ga NF)
For fabrication of the core–shell nanofibers, the Cur loaded PVP/CD-GO solution used as the inner
fiber and subsequently coated through coaxial electrospinning using Ga@Cs solution. For coaxial
electrospinning, two individual syringes containing core solution (Cur@CD-GO) and shell solution
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(Ga@Cs) were connected to a coaxial spinneret and were injected at a flow rate of 0.5 ml/h and 1
ml/h with a high voltage of 20-30kV respectively. For the crosslinking, the prepared nanofiber,
were thermally treated at 70 °C for 5 h.
2.8. In vitro drug release
To determine the Ga and Cur content of the nanofibers, the nanofibers were dissolved in ethanol
and the UV-vis absorbance of the resulting solutions was measured at 285 and 450 nm for Ga and
Cur, respectively. The release profile of synthesized nanofibers was assayed at 37 °C and pH 5.4.
Briefly, nanofibers mats containing Cur and gallic acid were cut to 1 cm × 1 cm sheets, and
dispersed into 10 mL of phosphate buffered saline (PBS) solution (pH 5.4) under shaking rate of
120 rpm for 72 h. The amount of released curcumin and gallic acid were quantified by UV/Vis
spectrophotometry at 430 and 250 nm using curcumin and gallic acid standard calibration curves,
respectively. The release percentage of drugs were then calculated using Eq. (1):
퐏퐞퐫퐜퐞퐧퐭퐚퐠퐞 퐨퐟 퐝퐫퐮퐠 퐫퐞퐥퐞퐚퐬퐞퐝 = 퐂퐨퐧퐜퐞퐧퐭퐫퐚퐭퐢퐨퐧 퐨퐟 퐝퐫퐮퐠 퐫퐞퐥퐞퐚퐬퐞퐝 /퐂퐨퐧퐜퐞퐧퐭퐫퐚퐭퐢퐨퐧 퐨퐟 퐝퐫퐮퐠 퐥퐨퐚퐝퐞퐝 × ퟏퟎퟎ
Eq. (1)
2.9. Antimicrobial activity
Antimicrobial activity of synthesized nanofibers was assessed against two pathogens, the Gram-
positive bacterium Bacillus cereus (B. cereus) and the Gram-negative bacteria Escherichia coli (E.
coli) by disc diffusion method. In this method, bacteria were cultured on a nutrient agar plate, then
nanofibers mats were placed on the plate containing the bacteria and incubated at 37 °C for 24 h.
Finally, the inhibition zone for bacterial growth was measured accurately. Determination of
minimum inhibitory concentrations (MIC) was performed using a sterile 96-well plate by
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microdilution method. At first, 100 µl of Mueller-Hinton Broth medium (Merck, Germany) was
poured into 96 well microplates. Then, 50 µl of NFs extracted (1mg in 1 ml methanol:water
solvent mixture with a 1:1 volume ratio ) was added to the first well of row and from the second
house to the third and so on until the ninth house was diluted. In another raw, 50 µl of antibiotics
of gentamicin, vancomycin, and Amphotericin B with appropriate sensitivity was added. Finally,
50 µl of the diluted microbial suspension was added to all wells in equal of McFarland half-pipe.
After 24 h of incubation at 37°C plate was observed. The results related to the antibiotics were
compared with the standard table of the National Committee for Clinical Laboratory Standards
(NCCLS).
2.10. Antioxidant activity
Antioxidant tests for Cur@CD-GONF and Cur-Ga NF were performed via 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging assay[34]. Briefly, NFs extracted (1mg in 1 ml
methanol:water solvent mixture with a 1:1 volume ratio ) were mixed with 3 ml of 10−4 M DPPH
solution prepared in the methanol and the resulting mixtures was incubated for 30 min at room
temperature in darkness. Then the absorbance of the solutions were recorded by UV–Vis
spectrophotometer at the wavelength of 517 nm. The antioxidant activity (%AA) of the
nanofibers was expressed according to the following equation:
% AA=((AControl-Asample ))/AControl
Eq. (2)
Where Acontrol and Asample represent the absorbance values of control DPPH solution and DPPH
solution with nanofibers, respectively. The measurements were carried out in triplicate and the
results were given as average values ± standard deviation.
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2.11. Cytotoxicity assay
The Human alveolar epithelial cell line A549 and Human epidermoid mouth carcinoma cell line
KB cell line were obtained from the Pasteur Institute, Tehran-Iran. Cells were seeded in at a
density of 5×104 cells per well in RPMI medium supplemented with 10% (v/v) FBS and 1%
penicillin, then cultured in a humid atmosphere at 37°. After culture, Cur@CD-GO and Cur-Ga
NFs with different concentration were added to the medium-removed 96-well plates and
incubated for another 24 h. After that, 10 μl of MTT (5 mg/ml stock solution) was added to each
well and cells were incubated for an additional 4 hours. The cell viability curves were calculated
using an ELISA microplate reader (SLT, Austria) at 490 nm and with regard to the control cells.
All tests and investigates were run in triplicate.
2.12. Anti-inflammatory assay
Anti-inflammatory activities of the nanofibers were investigated by formalin-test-induced edema
test in the hind paws of mice according to the guidelines of the Institutional Animal Care and
Use Committee. (IACUC). [35-37]. Mice were allocated randomly into five groups, each one
contains five animals; (a) control (b) dosages of nanofiber and (c) diclofenac as the reference
drug. Approximately 10 µl of a 2/5 % suspension of formalin in normal saline was injected into
the plantar side of hind paws of the mice. Before induction of inflammation used of the formalin,
the nanofiber extracts at different concentrations and diclofenac in an appropriate volume
administered intraperitoneally into paws of the mice. The control group received only distilled
water-tween 80 (5%). The paw thickness was measured using a plethysmometer prior to formalin
injection and followed by measurement at 1-h intervals for 3h.
3. Results and discussion
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Polyphenolic compounds such as curcumin (Cur) and gallic acid (Ga) have been studied widely
for medicinal purposes owing to their potent physiological and pharmacological activities [38-
40]. However, their intrinsic hydrophobic, poor bioavailability, rapid systemic elimination and
their instability at high temperatures as well as in the presence of oxygen and light are major
obstacles to their use for various biomedical and clinical applications [41]. In an attempt to
overcome these limitations, the present study focuses on the synthesis of the Cur-Ga NF Co-
delivery with improved biological activity via coaxial electrospinning. The Cur-loaded
cyclodextrin-graft-graphene oxide fiber (Cur@CD-GO) was coated with chitosan-gallic acid
(Ga@CS) to increase the antibacterial, antioxidant and anticancer properties and to achieve a
sustained release of the Cur. More details on the construction of nanofibers can be found in
Figure 1 and Figure 2.
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Figure 1. Representation of the synthesis of CD-graft-GO nanocarrier.
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Figure 2. Electrospinning setup of the fabrication of Core-Shell nanofiber.
The synthesis of nanofibers was evaluated using, spectroscopy analyzes and microscopy image
as well as thermo gravimetric analysis. FT-IR spectroscopy of stepwise synthesis of CD-graft-
GO are shown in Figure 3A. It can be seen from Figure 3Aa that β-CD gave peaks at 3406, 1637,
2933 and 1030 cm-1 which were attributed to O–H stretching vibration, O-H in-plane bending
vibration, –CH2– stretching vibration and C–O–C anti-symmetric stretching vibrations,
respectively. The β-CD-OTs indicated all the characteristic peaks of β-cyclodextrin with a small
shift. Moreover, the β-CD-OTs spectrum represented new peaks at 1597,817,667 and 553 cm-1
which are attributable to benzene rings of tosyl groups and new peaks at 1365 and 1157 cm-1
which are assigned to anti-symmetric and symmetric stretching vibrations of the O=S=O bonds
(Figure 3Ab)[42]. In the IR spectrum of CD-PEox-N3 (Figure 3Ac), characteristic absorptions at
1639 cm-1 were related to the amidic carbonyl groups of POX chains. Also, the absorption band
observed at 2100 cm-1 is attributed to the azido group and indicating the introduction of azido
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group at the end of the POx chains. In the IR spectrum of graphene oxide (Figure 4Ad),
characteristic absorptions at 3360, 1728, 1626, 1421, 1278 and 1029 cm−1 were due to stretching
vibrations of O-H, C=O, C=C in the aromatic ring, O–H deformation, epoxy (C–O–C) and the
carboxyl (C–OH) groups, respectively [32, 36]. The disappearance of the absorption band of
azido group at 2250 cm-1 in the IR spectrum of CD-GO provides appropriate evidence that CD-
POX-N3 was grafted onto the surface of GO through nitrene chemistry by the formation of
aziridine rings.
XRD of synthesized samples to investigate the change in crystal structure of materials during the
reaction steps, are shown in Figure3B. Intensity peak of GO nanosheets at 2θ=11.87° after the
grafting of cyclodextrin through polyoxazoline was significantly reduced and showed an
amorphous pattern compared to those of GO (Figure 3Bb). On the other hand, the presence of the
main peaks of Cur (2θ = 12.3°, 14.5°, 17.2°, 18.2°), indicated a high degree of crystallinity of cur
[43]. Similarly, Ga powder showed highly crystalline structure and sharp diffraction peaks were
observed at diffraction angles of (2θ) 16.68°, 25.54° and 27.85°. The powder X-ray diffraction
patterns of the Cur@CD-GO NF and Cur@CD-GO coated Ga@Cs (Cur-Ga) NF are also
presented in Figure 3B. As can be seen, no such crystalline diffraction peaks (related to Cur and
Ga) were observed in the diffractograms of the nanofiber mats. This indicates that the crystalline
form of Cur and Ga has been disappeared and the drugs successfully have been loaded into the
nanofiber mats.
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Figure 3. A:IR spectra of a) CD, b) CD-OTs, c) CD-POX-N3, d) GO e) CD-GO. B: X-ray
diffraction pattern of a) GO, b) CD-GO, c) Cur, d) Cur@CD-GO NF, e) Ga and f) Cur-Ga NF.
Cur-Ga nanofibers were effectively prepared with different volume ratios of core solutions
(30:70, 50:50 and 70:30 of Cur@CD-GO:PVP nanofiber). The FE-SEM images were analyzed
to determine morphology and average diameters of produced fibers. Figure 4A represented the
FE-SEM images of Cur-Ga nanofibers with different volume ratios. It was found that formation
of defect-free, beadless and smooth morphology nanofibers with the diameters distributed
uniformly could be obtain when the volume ratio of Cur@CD-GO:PVP used was 30:70. On the
other hand, the formation of nanofibers with defects and beads were observed when the
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Cur@CD-GO volume ratio increased. Furthermore, the average diameter and diameter
distribution of nanofibers decreased when volume ratio of Cur@CD-GO increased. The observed
changes in the morphology may be due to increase of conductivity of the solutions containing
higher amounts of Cur@GO. The nanofiber containing 30:70 of Cur@CD-GO:PVA with
diameter distribution about 210 ± 5 nm was selected as the optimized fibers for further studies.
Figure 4B depicts the TGA curves of GO, CD-GO and Cur-Ga NF, respectively. The initial
°
weight loss at below 100 C is attributed to the moisture evaporation in all samples CD-GO
showed higher thermal stability than GO. Upon grafting of CD to the surface of GO through
nitrene chemistry, the main decomposition stage of GO at 200 °C [30] shifted to higher
temperatures around 330 °C (about 89%) which could be related to the pyrolysis of graphene
nanosheets and breakdown of the polymer chains. It can be observed that the Cur-Ga NFs do not
show a mass loss at temperatures below 300°, as shown in Figure 4B. Furthermore, the NF
showed three main weight-loss steps. The first weight-loss step, with the weight loss about 45%,
observed around 330°C, which can be related mainly to the breakdown of the polymer chains.
The second weight-loss step (about 10%) observed between 330 °C to 350 °C, which can be
related to the breakdown of the CD-GO. The third weight-loss step of about 38%, which can be
associated with the degradation of the carbon skeleton of polymer chains, GO and drugs.
Collectively, the incorporation of polymer chains (PVA, PVP and Cs) and drugs caused notable
changes in the thermal properties of the CD-GO and nanofiber.
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Figure 4. A) SEM images of a) different concentrations of core sulation a) 30:70, b) 50:50 and
c)70:30 of Cur@CD-GO:PVP NFs. B) TGA thermograms of GO, CD-GO and Cur-Ga NF. C)
The contact angle measurements of Cur@CD-GO, and Cur-Ga NFs.
The release characteristics of Cur and Ga from the Cur@CD-GO and Ga-Cur NF were evaluated
at pH 5.4 (Figure 5A). The release characteristics of drugs showed that nanofibers by preventing
of the growth of curcumin and Ga crystal in aqueous solutions could improve their solubility and
consequently their release behavior. Also, the results showed that the releases of Cur from the
Cur-Ga NF were slower than those of the Cur@CD-GO NF. Cur@CD-GO NF exhibited high
burst release of drug and almost 70% of Cur was released within 12 h while, Cur-Ga NF
exhibited the initial burst release at 12 h and the continuous release to 72h. From the release data
it was found that the total amount of the released Cur from Cur@CD-GO NF and Cur-Ga NF
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were approximately 91.15 and 60.71% after 24 h but reached 95.41% and 78.72 % after 72
hours, respectively. The burst release observed for Cur-Ga NF may arise due to the diffusion of
Cur near the fiber surface while the slower and sustained release may be attributed to greater
compression of the coating which increases the release time of the Cur through the coated layer.
Furthermore, a rapid release of GA drug was observed of Cur-Ga NF at the first 12 h, almost 80
%, and the continuous release to 72h. Overall, the core/shell nanofibers displayed sustained-
release profiles for Cur drug and a high release for Ga drug for more than 48 h.
The results obtained from the contact angle test also indicated that the Cur@CD-GO NF surface
properties has been changed after coating with Ga@Cs. The surface coating of Cur@CD-GO NF
with Ga@Cs increases the contact angle of water with the surface of the NF that could
effectively retard the penetration of medium into the core layer, thereby delaying the Cur release
(Figure 4C).
The bacterial inhibitory efficacy of Cur-Ga and Cur@CD-GO NF against the Gram-positive
bacterium Bacillus cereus (B. cereus) and the Gram-negative bacteria Escherichia coli (E. coli) were
evaluated based on disc diffusion assay. The antibacterial activity of various nanoformulations of
nanofibers was depended on their composition as well as the type of bacteria. The average
diameter of the zones of inhibition against B.cereus were 18mm and 21.6 mm for Cur@CD-GO
NF and Cur-Ga NF, and against E.coli were 14.6 mm, and 16.4 mm, respectively (Figure 5b).
Based on the observations, a bigger inhibitory zone is observed for Cur-Ga NFs for both of
bacteria investigated in comparison with Cur@CD-GO NFs. As expected, Cur-Ga NF showed
enhanced antibacterial activities compared to CD-GO@Cur. This phenomenon was accordant with
the fact that the Cur-Ga NF could provide a synergistic effect of Cur and shell solution (Ga and Cs)
towards bacterial with the negatively charged surfaces, resulting in loss of membrane permeability,
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cell leakage, and finally cell death which further enhanced the antibacterial activity of NFs. Also,
the minimum inhibitory concentrations (MIC) for NFs and CD-GO have been presented in Table
1. Most significantly, Cur-Ga NF showed the lower minimum inhibitory concentration towards
E.coli and B. cereus bacteria than that of Cur@CD-GO NFs. As a result, the ability of the
synthesized NFs for incapacitate bacteria qualify them for the future therapies.
Therefore, the antioxidant capacity of Cur@CD-GO and Cur-Ga NFs contains phenolic
compounds was also investigated using DPPH radical scavenging assay (Figure 5C, D). As
expected, Cur-Ga NF represented higher antioxidant activities than Cur@CD-GO probably due to
the presence of polyphenol and polycation compounds. UV-visible spectrum of DPPH solution in
the methanol exhibited an absorption peak at around 517 nm but there was obvious decrease in the
intensity of DPPH absorption peak after adding the nanofibers, which means that these nanofibers
had good antioxidant ability. The photographs of the DPPH solution before and after the reaction
with Cur@CD-GO and Cur-Ga NFs are shown in Figure 5C. The visual investigation shows that
with the addition of NFs into the solution, violet color of DPPH solution turns into yellow which
might be explained by transforming a DPPH radical into its reduced form DPPH˙-H in the
presence of NFs. Furthermore, the antioxidant activity of 50 µg/mL of Cur@CD-GO and Cur-Ga
NFs extracted using methanol: water (1:1) was calculated according to Eq. (3) as 58.8±3% and
97.4±3% respectively. Efficient concentrations (EC50) were also determined between 40±5 and
15±3 µg/mL for Cur@CD-GO and Cur-Ga NFs, respectively. Lower EC50 shows high antioxidant
capacity of Cur-Ga NFs. In fact, the results showed that the antioxidant ability of curcumin and
gallic acid was retained during electrospinning process.
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Figure 5. A) In vitro release profile of curcumin and gallic acid from Cur-Ga and Cur@CD-Go
NFs at 37 °C in pH 5.4. B) The inhibition test of B. cereus and E.coli growth after 24 h of
incubation, in presence of Cur@CD-GO and Cur-Ga NFs. C) UV–vis spectra of a) DPPH (before
the reaction); DPPH solutions in which b) Cur@CD-GO and, c) Cur-Ga NFs ere immersed (after
the reaction). D) Percentage antioxidant activity of NFs.
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Table 1: Investigation of MIC values of nanofibers
Nanofibers (NFs)
Cur@CD-GO NF
Cur-Ga NF
E.coli
B. cereus
54.5 µg/ml
42.6 µg/ml
63.25 µg/ml
51.3 µg/ml
The cytotoxicity of the NFs against the Human alveolar epithelial cancer cell was evaluated by
MTT assay after treated with nanofibers. For this purpose, the extraction medium of nanofibers
were cultured with cancer cell for 72 h. The cell viability for the Cur@CD-GO and Cur-Ga NFs
are shown in Figure 6a. Cell viability was decreased in a dose dependent manner. MTT assay
indicates decreases in cell viability at increasing concentrations of NFs. Furthermore, significant
growth inhibition was observed in cells treated with Cur-Ga NF at all concentrations compared
with Cur@CD-GO NFs after 72h of incubation. The viability of cancer cell was decreased to
78.6% by Cur-Ga NF (80µg/ml), while it was decreased to 49.58% by Cur@CD-GO NFs at
same concentration. Significantly, an IC50 of 30 µg/ml of Cur-Ga NF was observed against
Human alveolar epithelial cancer cell representing almost 5-fold reduction in the required potent
dose as compared to Cur@CD-GO NFs. The results showed synergistic inhibition of growth by
Cur-Ga NF against the tested cell with higher potency as compared to Cur@CD-GO NFs. The
anticancer efficacy of NFs has largely been attributed to their potent antioxidant and antibacterial
as well as their anti-inflammatory activities. Importantly, cytotoxicity test of NFs was also
conducted against Human epidermoid mouth carcinoma cell line KB (healthy cell). Remarkably,
cell viability studies showed that prepared NFs has negligible toxicity to Healthy cells (Figure
6b). The results showed that even after 72 h of treatment with 100 µg/mL of Cur-Ga NFs and
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Cur@CD-GO NFs, ~70–75% and 55-60% of cells are alive, respectively. Also, the results
showed the dose-dependent cell viability with NFs and indicates high cell viability at low
concentrations of NFs. Compared with Cur-Ga NFs, which showed high cellular viability,
Cur@CD-GO NF showed relatively higher cytotoxicity. This result may be due to the toxicity of
graphene [44]. Most significantly, Cur@CD-GO NF coated with Cs@Ga has reduced the
toxicity of Cur@CD-GO NF to cells. These data suggest that the prepared nanofibers has
excellent biocompatibility and have the potential to be developed in medical applications.
Due to high biocompatibility, Cur-Ga NF selected as the optimized fibers for anti-inflammatory
studies. The formalin-induced paw edema model was used to investigate the anti-inflammatory
effects of Cur-Ga NF in experimental mice [35]. This experiment is a simple model of sub
chronic inflammation and induced a significant inflammation in the hind paw of the mice. The
anti-inflammatory activity of nanofibers extracts was studied in comparison with diclofenac
sodium (as a reference drug) and normal saline (Control). At dose of 50 µg/ml of the Cur-Ga NF
exhibited significantly reductions in edema development (75.5%) compared with reference drug
at same concentration (53.8%) after 4 hours of formalin injection. As shown in Figure 6C,
development of formalin-induced paw edema significantly was prevented by the NF extract,
while inflammation was still observed in the control and treated groups with diclofenac sodium
drug even after 4 hours of formalin injection. Specifically, nanofibers showed a dose-dependent
anti-inflammatory effect that increase with increasing concentrations of nanofibers at dose of 10-
50 µg/ml. Previous investigations confirm that polyphenolic compounds such as curcumin and
gallic acid with inhibit the expression and the release of a number of pro-inflammatory mediators
and proteolytic enzymes, and the production of reactive oxygen species, have potent anti-
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inflammatory properties[45-47]. Consequently, this results suggested that the prepared NFs
could be used as a potential safe and effective anti-inflammatory agents.
Figure 6. The cytotoxicity of the NFs against the a) Human alveolar epithelial cell line A549
(cancer cell). b) Human epidermis mouth carcinoma cell line KB (normal cell) incubated to 72 h
(Mortality% of cancer cell without treated with NF was 85±3 % and normal cell viability %
without treated with NF was 82±2% after 72 h as control groups). c) Anti-inflammatory of NFs
in experimental mice.
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4. Conclusion
In summary, we have reported a novel approach to design co-delivery systems of curcumin and
gallic acid by the electrospinning process. This co-loaded delivery system successfully fabricated
by electrospinning technique with the coaxial system, and can provide synergistic treatment by
promoting therapeutic efficiency of drugs. The designed systems have appropriate structural and
physicochemical properties for use as drug delivery scaffolds. The proposed system, Cur-Ga NF,
showed high potency against Human alveolar epithelial cancer cell, compared to single drugs
nanofibers. Upon further elucidation, after the modification of CD-GO@Cur NF with Cs@Ga ,
the higher biological activities were observed in the Cur-Ga NF system, owing to the presence of
gallic acid, curcumine, chitosan and reducing the toxicity of graphene oxid. Our study further
confirmed that the new formulation of curcumin and gallic acid can serve as one of the most
promising approaches for fabrication of anti-inflammatory, antimicrobial, tumor inhibition and
tissue regeneration agents.
Acknowledgments
The authors wish to gratefully acknowledge the support of Lorestan University.
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