Carbohydrate Polymers 186 (2018) 282–289

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Biocompatible nanocomposite of carboxymethyl cellulose and T

functionalized carbon–norfloxacin intercalated layered double hydroxides
⁎
N’guadi Blaise Alloua,b, Archana Yadavc, Mintu Palb,c, Rajib Lochan Goswameea,b,
a
Advanced Materials Group, Materials Science and Technology Division, CSIR–NEIST, Jorhat 785006, Assam, India
b
Academy of Scientific and Innovative Research, Jorhat Campus, India
c
Biotechnology Group, Biological Science and Technology Division, CSIR–NEIST, Jorhat 785006, Assam, India

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

Keywords: In recent years, the development of systems with progressive drug release properties, which is an effective
Layered double hydroxides technique for the use of drugs, has aroused great interest in the field of controlled release formulations. In this
Sodium carboxymethyl cellulose work, hybrid materials containing citric acid cross–linked carboxymethyl cellulose (CMC) and norfloxacin (NOR)
Functionalized carbon intercalated layered double hydroxide (LDH) deposited over the surface of functionalized carbon (AC) were
Citric acid cross–linking
prepared. The synthesized CMC@AC–LDHeNOR nanohybrids were characterized using different techniques and
Norfloxacin
in vitro NOR release behaviors were investigated in phosphate buffer saline, pH 7.4 at 37 °C. On the basis of the
Controlled release
release profiles, it was found that NOR release was delayed when it was intercalated in AC–LDH which in
presence of modified CMC decreases further. The nanohybrids indicated enhancement of antibacterial activity
against gram–negative and gram–positive bacteria. The MTT assay showed their non–toxic behavior against
ovarian normal epithelial and cancer cells, suggesting their potential use as drug carriers.

1. Introduction anionic exchange property and easy synthesis in laboratory. However,

During the past few decades, one of the most important and inter-
esting area of research in materials science field remains the develop-
ment of controlled drug delivery systems. Indeed, in order to overcome
many adverse effects caused by the conventional therapeutic systems,
the new drug formulations should ideally be biocompatible, biode-
gradable, stable in physiological conditions, comfortable for the pa-
tient, capable of achieving a high drug loading capacity, capable of
releasing drug at the target sites in a controlled manner and easy to
fabricate (Barkhordari & Yadollahi, 2016; Liu, Yang, Xiong, & Gu,
2016). It is in this context that, over the last decades, a great number of
nanoparticles have been developed to facilitate drug delivery and
among them layered double hydroxides (LDHs).
LDHs or anionic clays represent an important class of layered in-
organic materials formed by a network of divalent and trivalent metal
cations containing hydroxide anions and exchangeable anion between
the layers. Considerable attention has been given to the use of LDHs in
recent studies as catalyst (Saikia, Saikia, Sarmah, Allou, & Goswamee,
2017), adsorbent (Zubair, Daud, McKay, Shehzad, & Al-Harthi, 2017),
anticorrosion agent (Liu et al., 2015), flame retardant (Guo et al.,
2017), sensors and electrodes (Lu & Zhao, 2010; Zhang, Chen, Zhang, &
Lu, 2014) because of their good biocompatibility, low cytotoxicity,
due to the fact that the interlayer space of the LDHs can serve as a
reservoir where drugs are stored and released in a controlled manner,
these materials have great potential as drug delivery agents in the
pharmaceutical field (Allou, Saikia, Borah, & Goswamee, 2017; Bi,
Zhang, & Dou, 2014; Senapati et al., 2016; Wang et al., 2013).
Carboxymethyl cellulose (CMC), a natural polymer and water so-
luble polysaccharide, remains one of the most popular biomaterials
used in the formulation of extended release drug carrier (Butun, Ince,
Erdugan, & Sahiner, 2011; Elumalai et al., 2015; Park et al., 2016). This
is due to the fact that the hydrophilic polymer matrix is relatively
flexible and usually provides a reproducible release profile (Kamel, Ali,
Jahangir, Shah, & El-Gendy, 2008). Besides this, it must also be added
that CMC possesses other properties as interesting as the others, such as
nontoxicity, renewable nature, good biocompatibility and biodegrad-
ability and low cost compared to other natural polymers (Juncu, Stoica-
Guzun, Stroescu, Isopencu, & Jinga, 2016; Kondaveeti, Damato,
Carmona-Ribeiro, Sierakowski, & Petri, 2017).
Although the intercalation of Norflaxacin (NOR) into the interlayers
of LDH have shown good results (Li et al., 2016), the rapid aggregation
of LDH in culture media such as phosphate buffer saline remains one
the problems of its use (Allou, Saikia et al., 2017). Therefore, in this
contribution, inexpensive activated carbon from Ipomoea carnea stems

⁎
Corresponding author at: Advanced Materials Group, Materials Science and Technology Division, CSIR–NEIST, Jorhat 785006, Assam, India.
E-mail address: goswamirl@neist.res.in (R.L. Goswamee).

https://doi.org/10.1016/j.carbpol.2018.01.066
Received 30 September 2017; Received in revised form 11 January 2018; Accepted 20 January 2018
0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
N.B. Allou et al. Carbohydrate Polymers 186 (2018) 282–289

was combined with LDH to produce improved colloidal dispersion of Table 1

hybrid composite which was dispersed in CMC matrix. CMC was chosen Composites composition and code.
as a polymer to improve the protection of LDH–NOR nanohybrids ob-
Sample CMC (g) AC–LDH (mg) NOR (mg) AC–LDH–NOR (mg) Citric
tained by intercalation of NOR between the interlayers of LDH de- Code Acid
posited over the activated carbon surface through a co–precipitation (mg)
method. The linear CMC chains prepared in further need to be inter-
C0 1 – – – 50
connected by a cross–linking agent promoting the network formation in
C1 0.25 – – 50 50
order to produce a system mechanically stable and to release the drug C2 0.5 – – 50 50
in a controlled manner (James, John, Alex, & Anoop, 2014). The CMC C3 1 – – 50 50
network, thus formed, will allow increasing of the slow release prop- C4 1 32.90 17.10 – 50
erties of the final drug formulation obtained. Citric acid was chosen as C5 1 – 17.10 – 50
C6 1 50 – – 50
cross–linking agent due to its nontoxicity and large availability. NOR
was used as a test drug in this study not only because of its frequent
prescription, but also because of its high–dose taken for the long term
may cause a decrease in spermatogenesis, testicular atrophy, and Len-
ticular opacities (Roberts, 2008; Fernández et al., 2006). Since the
complete dissolution and absorption of NOR happen only in the middle
and lower small intestines (pH = 7.4), in vitro release tests of NOR were
investigated using phosphate buffer solution pH = 7.4 as a release
medium to prove the effectiveness of the synthesized hybrid composite
as slow release agent for antibiotic molecules.

2. Experimental

2.1. Materials

Raw material, Ipomoea carnea stems, was collected from Aleng Mora
region of Jorhat district, Assam, India. Sodium carboxymethyl cellulose
(CMC) sodium with high viscosity, magnesium chloride hexahydrate
(MgCl2·6H2O), sodium hydroxide (NaOH), nitric acid (> 69%) and
potassium dihydrogen orthophosphate monobasic (KH2PO4) all A.R
grade were obtained from Himedia Laboratories, Mumbai, India.
Aluminium chloride hexahydrate (AlCl3·6H2O) A.R grade, was provided
by M/s Otto Chemie, India. Citric acid was purchased from Fig. 1. XRD patterns of (a) AC–LDH, (b) AC–LDH–NOR, (c) C0, (d) C3.
Sigma–Aldrich. Cell culture Medium 199, MCDB M105, Minimum
Essential Medium Eagle (MEM) (alpha Modification), penicillin–-
streptomycin, fetal bovine serum (FBS), 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) were purchased from Himedia
(India). Norfloxacin (NOR) powder was kindly provided by M/s Viva
Pharma Jorhat, India. The molecular structure of the antibiotic is
shown in Fig. S1. Distilled water was used to prepare all the solution.

2.2. Synthesis of antibiotic anion intercalated LDHs over Ipomoea carnea

stem carbon surface

LDH functionalized NOR deposition over Carbon surface was pre-

pared via a conventional co–precipitation method (Li, He, Evans, &
Duan, 2004). A solution containing 0.02 mol MgCl2·6H2O and 0.01 mol
AlCl3·6H2O was added dropwise into a vigorously stirred solution
(140 ml) containing 0.075 mol NaOH, 1 g NOR and 0.5 g AC at pH 10,
followed by stirring during 30 min under a nitrogen atmosphere to
minimize the contamination by atmospheric CO2. The reactor tem-
perature was maintained at 50 °C. Then, the suspension was transferred
into a Teflon–line autoclave and kept in the oven at 80 °C for 24 h. The
resulting solid was filtered, washed several times with distilled water,
dried at 60 °C and denoted AC–LDH–NOR. The total concentration of Fig. 2. FT–IR spectra of (a) AC–LDH, (b) C0, (c) C6, (d) NOR, (e) AC–LDH–NOR, and (f)
NOR into AC–LDH galleries is measured to be 17.10%. C3.

2.3. Preparation of CMC@AC–LDH–NOR nanocomposites
CMC@AC–LDH–NOR nanocomposites were synthesized according
to the method described by Demitri et al. (2008) with different contents
of CMC. In a typical process, the desired amount of CMC and 50 mg
AC–LDH–NOR were dispersed in 50 ml distilled water. The mixture was
stirred at room temperature during 24 h. Then, citric acid (powder) was
added to the polymeric solution so that its final concentration
amounted to 1 g/l. The solution was homogenized by vigorous stirring
for 30 min and submitted to ultrasonication for 10 min to disperse the
AC–LDH–NOR particles and reduce aggregation. The homogeneous
solution obtained was transferred to petri dish and dried in oven at
40 °C to evaporate water for 24 h. Finally, the curing treatment was
achieved by heating the dried polymeric composite at 80 °C for 24 h and
kept in a desiccator for analytical tests. For comparison purpose,

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N.B. Allou et al.
Fig. 3. FE–SEM micrographs of (a) AC–LDH, (b) AC–LDH–NOR, (c) C0 and (d) C3.
composites containing AC–LDH, NOR and both AC–LDH and NOR not
intercalated were prepared following the same process. The composi-
tions of the composites as well as their codes are reported in Table 1.
2.4. Characterization
The powder X–ray diffraction patterns (XRD) of the samples were
collected using a Rigaku Ultima IV model Advance diffractometer using
Cukα radiation (λ = 1.7902 Å) at 40 kV, 40 mA and a scan rate of 0.2°
2θ/min with a Bragg angle ranging from 2° to 80° 2θ. The morphologies
of the nanocomposites were observed by field emission scanning elec-
tron micrographs (FE–SEM) were taken by using a FE–SEM apparatus
from Carl Zeiss (Sigma VP). Fourier transform Infrared (FT–IR) spectra
were recorded using a Shimadzu MS–11–Iraffinity–1 spectro-
photometer at a spectral resolution of 4 cm−1, using KBr pellets tech-
nique with a sample: KBr mass ratio of 1:100, scanned from 4000 to
400 cm−1. The thermal behavior of the samples was determined by
thermogravimetric analysis (TGA) on an SDT Q600 thermal analysis
system under argon atmosphere with a flow rate of about 100 ml/min
and a heating rate of 10 °C/min from room temperature to 600 °C.
2.5. Drug release behavior study
The in vitro release of NOR from CMC@AC–LDH–NOR as a function
of time was carried out by immersing 100 mg of the composite in
100 ml of phosphate buffer solution (PBS) pH 7.4 at a constant tem-
perature of 37 °C under a slow speed stirring of 100 rpm. At appropriate
time intervals, an aliquot of 5 ml of the solution was withdrawn and
immediately replaced with an equal volume to keep the volume con-
stant. The accumulated amount of NOR released into the solution was
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Carbohydrate Polymers 186 (2018) 282–289
measured at regular intervals during 12 h by UV–vis spectro-
photometeric scans at 277 nm. The release behavior studies were per-
formed three times and the results presented as mean values with the
corresponding standard deviation (SD) error bars.
2.6. Antibacterial activity assessment
The antibacterial activity of the CMC@AC–LDH–NOR nanocompo-
sites were tested against gram negatives Escherichia coli (E. coli,
ATCC–11229), Pseudomonas aeruginosa (P. aeruginosa, MTCC–2583)
and gram positives Bacillus subtilis (B. subtilis, MTCC–441),
Staphylococcus aureus (S. aureus, ATCC–11632) using an agar plate
diffusion method. First of all, the bacterial strains were grown in
Mueller Hinton broth for 24 h at 37 °C (Sezonov, Joseleau-Petit, &
D’Aril, 2007). Then, Mueller Hinton agar plates were prepared, ster-
ilized and solidified. 80 μl of the microbial culture suspension in
Mueller Hinton broth containing ∼3.22 × 109 colony forming units
(CFU) per ml was streaked over the dried surface of the agar plates and
spread uniformly, using a sterilized glass rod. Then, 100 μl of each
sample (0.5 mg/ml PBS, pH 7.4) was carefully transferred into the
wells, prepared with sterile barrier on a solidified nutrient agar plate in
petri dishes inoculated with test gram negative E. coli, P. aeruginosa and
test gram positive B. subtilis, S. aureus, respectively. After inoculation,
petri dishes were kept in an incubator at 37 °C for 18 h. The zone of
inhibition (ZOI) for release of antibiotic into the films was measured
with the help of Hi Antibiotic zone scale in mm. All the antibacterial
tests were done in triplicate and the error were given as standard de-
viation.
N.B. Allou et al.
Fig. 4. Thermal analysis of (a) AC–LDH, (b) C0, (c) C6 and (d) C3.
2.7. In vitro cytotoxicity assay of the composites
The in vitro cytotoxicity test of C3 and C6 against human ovarian
cancer cell line (PA–1) and normal ovarian epithelial cell line
(IOSE–364) was also investigated. IOSE–364 was grown in 1:1 Medium
199 and MCDB M105 media; and PA–1 was grown in Minimum
Essential Medium Eagle (MEM) (alpha Modification). Mediums were
supplemented with 10% fetal bovine serum, and 1% penicillin strep-
tomycin antibiotic solution at 37 °C in 5% CO2 incubator. Cells at a
concentration of 5000 cells/well were seeded in 96 well microtiter
plates and exposed to various concentrations (0, 5, 10, 20, 30 and
40 μg/ml in PBS) of those compounds. Relative cell survival percentage
was determined using MTT assay. After treatment for 24 h and 48 h,
MTT (5 mg/ml) was added into each well and incubated for 4 h at 37 °C.
The formazan crystals formed were dissolved in DMSO. The intensity of
colored formazan formed was determined by measuring the absorbance
at 570 nm using ELISA reader (BioTek, USA). The percentage cell via-
bility was calculated by following formula: % Cell Viability = (Mean
OD of Test/Mean OD of control) × 100. The experiments were re-
plicated three times and the cell viabilities were presented as the
mean ± SD.
3. Results and discussions
3.1. X–ray diffraction analysis
The synthesized composites were characterized by powder XRD
(Fig. 1). The XRD patterns of AC–LDH exhibited the typical reflection of
hydrotalcite–like materials with the planes (003), (006), (009), (110)
and (113), indicating a well–crystallized structure of the prepared
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Carbohydrate Polymers 186 (2018) 282–289
composites. However, the characteristic peak (002) corresponding to
AC can also be observed in the XRD pattern of AC–LDH. The most
important reflection peak corresponding to the basal spacing (d003) was
observed at 2θ value of 11.40° which represents a d–spacing of 0.77 nm.
The same value was also reported in the literature (Zheng & Chen,
2017).
A decrease of the main reflection peak from 11.40° to 6.62° 2θ value
was observed when LDH grows on the surface of the AC in the presence
of NOR, keeping the (110) and (113) diffraction peaks at the same
position. This shift of the basal reflection peak indicates the successful
intercalation of NOR into the interlayer space of LDH deposited over
the surface of the AC. Due to the antibiotic loading, the d–spacing in-
creases from 0.77 to 1.33 nm. The brucite–like LDH main layer has a
thickness of 0.48 nm (Liang et al., 2015). Accordingly, the gallery space
for AC–LDH–NOR material is found to be 0.85 nm, which is smaller
than the length of NOR, 1.16 nm (Ni et al., 2007). Therefore, it can be
assumed that the NOR molecules are arranged as a monolayer in a tilted
configuration within the brucite layers of LDH with a tilted angle of
42.6° as depicted in Fig. S2.
The XRD pattern of neat CMC composite (Fig. 1c) shows a broad
reflection centered at 2θ value of about 20°, indicating the amorphous
structure of CMC (Baniasad & Ghorbani, 2016). In comparison with the
neat CMC, the X–ray pattern of composite C3 (Fig. 1d) shows the typical
amorphous pattern of CMC suggesting that AC–LDH–NOR is well dis-
persed in the polymer matrix. The absence of new peak means that
there was no intercalation of the polymer chains between the layers of
LDH, hence the total exfoliation of AC–LDH–NOR particles.
N.B. Allou et al.
3.2. Infrared spectroscopy
Fig. 2 displays the IR spectra of AC–LDH, neat CMC,
CMC@AC–LDH, NOR, AC–LDH–NOR, and CMC@ AC–LDH–NOR. In all
the spectra, a strong and broad absorption band in the region of around
3350–3500 cm−1 can be observed, which is due to the OeH stretching
vibration of water molecules on the layer surfaces as well as in-
tramolecular and intermolecular hydrogen bonds (Huang et al., 2015;
Singh & Khatri, 2012). In the FT–IR spectrum of AC–LDH (Fig. 2a), the
bands corresponding to the stretching and bending vibration of carbo-
nate anions in the layers, MeO and MeOeH stretching vibrations were
observed at around 1364, 778, 682 and 553 cm−1 (Parida &
Mohapatra, 2012). The bands at around 2321, 1521 and 1111 cm−1
were attributed to stretching of carbon–oxygen and carboxylate groups.
The FT–IR spectrum of CMC (Fig. 2b) shows the peak related to
CeH stretching vibration associated with the methane hydrogen atoms
at 2913 cm−1. The peaks at around 1626 and 1412 cm−1 can be as-
signed to the symmetrical and asymmetrical stretching vibrations of the
carboxylate groups (Yadollahi, Namazi, & Aghazadeh, 2015). The
bands at 1075 and 1033 cm−1 may be attributed to CeOeC stretching
on polysaccharide skeleton. The FT–IR spectra of the neat CMC and
CMC@AC–LDH are almost similar outside of the peak at 524 cm−1
related to the metal–oxygen bond in the brucite–like lattice. This in-
dicates the presence of LDH in the prepared composite.
As shown in the FT–IR spectrum of NOR (Fig. 2d), the peaks at
3399, 2845, 1729 and 1619 cm−1 correspond to NeH stretching vi-
brations, CeH (from eCOOH group) stretching vibration, the eCOOH
group and C]O vibration, respectively. The two peaks observed at
1581 and 1395 cm−1 were attributed to antisymmetric and symmetric
stretching vibrations of eCOOe group. The peaks at 1487 cm−1
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Carbohydrate Polymers 186 (2018) 282–289
Fig. 5. Antimicrobial studies against (a) S. aureus,
(b) P. aeruginosa, (c) B. subtilis and (d) E. coli of: (1)
C1, (2) C2, (3) C3 and (4) control antibiotic (NOR).
represented υOeCeO of acids and at 1249 cm−1 suggested bending vi-
bration of the OeH group, indicate the presence of carboxylic acid.
In the FT–IR spectrum of AC–LDH–NOR, the characteristic peaks
related to AC, LDH and intercalated NOR are shown at 3447, 2852,
1629, 1588, 1492, 1383, 1256 and 1040 cm−1. As indicated in
Fig. 2(e), the band at 1364 cm−1 in the AC–LDH spectrum shifted to
1383 cm−1 and the peak related to C]O vibration in the spectrum of
NOR shifted to 1629 cm−1 due to the interaction between NOR and
LDH layers. The characteristic peaks related to intercalated NOR, AC,
LDH and CMC are shown at 2913, 1618, 1421, 1074, 1028 and
556 cm−1 in the spectrum of CMC@AC–LDH–NOR (Fig. 2f).
3.3. Electron microscopic analysis
FE–SEM analysis was performed to investigate the surface mor-
phology of AC–LDH, AC–LDH–NOR, neat CMC and
CMC@AC–LDH–NOR nanocomposites and the resulted images are
shown in Fig. 3. The deposited LDHs over the surface of functionalized
carbon (Fig. 3a) were in irregular hexagonal form, sheet–like shape,
having a particle dimension between 40 and 80 nm, which is the
characteristic of LDHs (Wang, Zhang, Bao, Wu, & Wan, 2012). As seen
in Fig. 3b, the incorporation of NOR produces the aggregation of LDH
particles due to the strong interactions between them and NOR mole-
cules. Fig. 3c indicates a smooth surface of neat CMC composite. In
contrast, the addition of AC–LDH–NOR makes disappear the smooth
surface of CMC (Fig. 3d) with well dispersed of AC–LDH–NOR particles
in the CMC matrix. The length of particles less than 300 nm can be
observed in the CMC matrix.
N.B. Allou et al.
Fig. 6. (A) Release profiles of Norfloxacin from C1, C2, C3, C4, and C5 and (B) Schematic
representation of NOR release behavior from CMC@AC–LDH–NOR composite.
3.4. Thermal analysis
Fig. 4 displays both TG and derivative thermogravimetric (DTG)
curves obtained under nitrogen flow for AC–LDH, C0, C6 and C3. In the
thermogravimetric curve of AC–LDH, three events of decomposition
were observed (Fig. 4a). The first step in the range of 20–110 °C cor-
responds to the loss of absorbed water on the layer surfaces (Allou,
Bordoloi, & Goswamee, 2017). The second step observed in the range of
110–240 °C is attributable to the elimination of the interlayer water and
the dehydroxylation of the octahedral layers in LDH. The last step of
decomposition corresponding to the major mass loss (about 38%),
which is due to the elimination of the interlayer anions occurred in the
range of 240–725 °C.
Two step degradation behavior was observed in thermal analysis of
neat CMC. The first step of weight loss with a maximum degradation
peak at 196 °C is due to the evaporation of adsorbed moisture
(Yadollahi & Namazi, 2013). The decarboxylation and decomposition of
CMC were observed in the temperature range of 260–450 °C associated
with a weight loss of 26% (Baniasad & Ghorbani, 2016).
The decomposition of C3 and C6 was similar and took place in three
steps of degradation. The first step observed is attributed to the loss of
absorbed water molecules. The following weight loss in the range of
250–450 °C is due to the decomposition of CMC and the elimination of
the interlayer water and the dehydroxylation of LDH. The last weight
loss (∼3%) centered at 476 °C for C6 and 490 °C for C3 corresponds to
the decomposition of the interlayer anions of LDH, precisely the de-
gradation of the intercalated NOR in the case of C3.
3.5. Antibacterial properties
The antibacterial activity of all prepared composites was evaluated
against Gram–negative bacteria (E. coli and P. aeruginosa) and
287
Carbohydrate Polymers 186 (2018) 282–289
Gram–positive bacteria (B. subtilis and S. aureus). The digital photo-
graphs of the diffusion bacterial test were shown in Fig. 5 and the in-
hibition zone values were listed in Table S1. As expected, no inhibition
zone was observed in the cases of neat CMC and AC–LDH, indicating
that citric acid does not contribute to the synthesized composites an-
tibacterial activity. The composites incorporating NOR inhibit the
growth of both gram positive and gram negative bacteria. However,
their effect against S. aureus is very low. For composite C3, in which
NOR was intercalated in AC–LDH, compared to C4 and C5, the zone of
inhibition (ZOI) was less (Table S1). This may be assigned to the slower
release of NOR when intercalated into AC–LDH. Moreover,
ZOIC3 < ZOIC4 < ZOIC5 suggesting that the filler delays release of
NOR and this delay is more when intercalated into AC–LDH (Campos-
Requena, Rivas, Pérez, Garrido-Miranda, & Pereira, 2015; Scaffaro,
Botta, Maio, Mistretta, & La Mantia, 2016). In addition, it is worthy to
note that increasing the amount of CMC decrease the inhibition zone.
3.6. Release profiles
The in vitro cumulative release of Norfloxacin from C1, C2, C3, C4,
and C5 was determined in PBS, pH 7.4 at 37 °C, as depicted in Fig. 6(A).
A comparison between the release profile of composite containing in-
tercalated (C3) and those not intercalated (C4 and C5) was made. The
NOR release from C5 shows an initial burst with 67% of NOR released
after 90 min, followed by a slower release where 93% of the drug were
released after 720 min. However, addition of AC–LDH in C5 gives a
different release behavior. The burst release was reduced (46%) after
incorporation of AC–LDH and it is observed within the first 60 min. This
suggest that addition of AC–LDH slow downs the release of NOR. This
behavior may be due to the interaction between the NOR anions with
the positively charged layers of AC–LDH (Ambrogi et al., 2017). In
contrast to the previous two composites, it was observed that the
slowdown of NOR release is much higher in C3. The release started by a
moderate burst effect with a 21% of NOR released within 30 min and
continued slowly but did not reach even the 50% of release after
720 min of study. Fig. 6(B) indicates the drug release mechanism from
C3. Thus, the slower release of NOR from C3, could be attributed to the
fact that before the NOR anions diffuse towards the release medium, the
anions of the release medium (phosphate anions) should penetrate into
the CMC network first and allow the release of the intercalated NOR
anions from AC–LDH into CMC matrix by an ion–exchange process.
After the ionic exchange, NOR anions can diffuse through the CMC
matrix towards the release medium (Barkhordari & Yadollahi, 2016).
Moreover, a comparison study was carried out on the effect of the
amount of CMC added during the composite preparation. As shown in
Fig. 6(A) (composites C1, C2 and C3), increase of the amount of CMC
causes decrease in the drug release rate. Therefore, on the basis of this
observation, the amount of CMC matrix in the composite could be ad-
justed in order to control the rate of the drug release.
3.7. Cytotoxicity assay
Although the biocompatibility of LDH and CMC were already re-
ported (Wang, Wang, & Wang, 2017; Donnadio et al., 2016), an in vitro
MTT assay was investigated to check the cytotoxicity of the composites
C3 and C6 against IOSE–364 cells and PA–1 cells at 24 and 48 h, re-
spectively. Our results indicated that carrier (C6) as well as
CMC@AC–LDH–NOR (C3) has around 85% cell viabilities, suggesting
that both of those composites are non–toxic to the ovarian cancer and
normal ovarian epithelial cells at the indicated doses used for this study
for 24 and 48 h treatment as shown in Fig. 7. In addition, we also ob-
served there was no significant difference on the cell viabilities at 24
and 48 h in the case of C3 treatment to the cells, suggesting that within
this time scale at indicated doses, the NOR is not released out to show
the toxic effect.
N.B. Allou et al.
Fig. 7. The cytotoxicity assay of C3 and C6 at different indicated concentrations using (a–c) IOSE–364 cells and (b–d) PA–1 cells at 24 and 48 h, respectively.
4. Conclusions
In summary, composites based on carboxymethyl cellulose cross-
linked with nontoxic citric acid and layered double hydroxide modified
norfloxacin deposited over the surface of functionalized carbon from
Ipomoea carnea stems were successfully prepared. The prepared com-
posites were characterized by different experimental techniques such as
X–ray diffraction, FT–IR, TGA and FE–SEM to investigate their structure
and morphology. The incorporation of AC–LDH in the CMC matrix af-
fected the release of NOR without hindering the antibacterial activity of
the obtained composites. The antibacterial studies have shown effec-
tiveness of the composites against both gram–negative and gram–po-
sitive bacteria. The addition of AC–LDH slow downs the release of NOR
from the polymer matrix and this slowdown was much higher when
NOR is stored between the interlayer of LDHs. In addition, the increase
of the amount of CMC causes decrease in the drug release rate.
Therefore, the amount of CMC in the composite could be adjusted in
order to control the rate of the drug release. The MTT assay has shown
that our synthesized composites are non–toxic to the ovarian normal
epithelial and cancer cells at the indicated doses used for this study at
24 and 48 h treatment. Consequently, the prepared
CMC@AC–LDH–NOR may be a promising candidate for its application
as controlled drug release system.
Conflict of interest
The authors declare no competing financial interest.
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Carbohydrate Polymers 186 (2018) 282–289
Acknowledgements
The authors gratefully acknowledge Dr D. Ramaiah, Director, CSIR-
NEIST, Jorhat for the funding and facilities for the work and for his kind
permission to publish this work. We express our heartfelt thanks to Dr.
P. Sengupta, Head of Materials Science and Technology Division for
support. The authors are also grateful to Dr. H.P. Deka Boruah, Head of
Biological Science and Technology Division for his kind help and to Dr.
Sibsankar Roy, CSIR–IICB, Kolkata, India for providing cell lines. N.B.A.
acknowledges CSIR-India and TWAS-Italy for award of the CSIR–TWAS
fellowship (FR number: 3240280453) for postgraduate studies at
CSIR–NEIST, Jorhat and AcSIR India for giving the opportunity of re-
gistration and CSIR India for funding the work under CSC–104 Net
Work project. This work is also financially partly supported by
SERB–Department of Science & Technology (SERB–DST), Government
of India for providing the Ramanujan Fellowship (SB/S2/RJN–087/
2014) to Dr. Mintu Pal.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.carbpol.2018.01.066.
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