MSC-07020; No of Pages 9

Materials Science and Engineering C xxx (2016) xxxxxx

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Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Ooxacin loaded gellan/PVA nanobers - Synthesis, characterization and

evaluation of their gastroretentive/mucoadhesive drug delivery potential
Priya Vashisth, Navdeep Raghuwanshi, Amit Kumar Srivastava, Harmeet Singh, Hemant Nagar, Vikas Pruthi
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India

a r t i c l e i n f o a b s t r a c t

Article history: The purpose of this investigation is to formulate a gastroretentive sustained drug release system for ooxacin to
Received 3 August 2016 improve its retention time, pharmacological activity, bioavailability and therapeutic efcacy in the stomach.
Received in revised form 23 September 2016 Ooxacin loaded gellan/poly vinyl alcohol (PVA) nanobers were fabricated using a simple and versatile
Accepted 23 October 2016
electrospinning technique. The fabricated nanobers were evaluated for percent drug encapsulation efciency
Available online xxxx
and in vitro drug release in simulated gastric medium (pH 1.2). The in vitro release prole and kinetic studies
Keywords:
for drug indicated the sustained release of ooxacin from the nanobers through Fickian diffusion kinetics. The
Gellan antimicrobial activity of the ooxacin loaded nanobers was assessed in comparison to the pure ooxacin by
Nanobers means of minimal inhibitory concentrations (MIC) against microbial strains of Enterococcus faecalis, Escherichia
Electrospinning coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The optimized ooxacin loaded gellan/PVA nanobers
Gastric retention displayed biphasic drug release prole with considerable mucoadhesion and gastric retention in the rat's gastric
Drug delivery mucosal membrane. Data obtained, suggested that the developed gastroretentive drug delivery can potentially
enhance the pharmacological activity of ooxacin and can also serve as a viable alternative for improving drug
bioavailability via oral route.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Electrospun nanobers possess high surface area to volume ratio,
which is an advantageous feature for drug delivery as it enhances the
total drug release in sustained manner which in turn improves drug ef-
cacy [1]. The importance of sustained drug delivery systems has long
been recognized in the pharmaceutical eld [2,3]. However, the applica-
tions of such sustained release technology for oral drug delivery has
been limited as the actual time for effective drug release is restricted
by gastrointestinal transit time [4,5]. In such case, prolonging the gastric
retention time of a drug delivery system is often desirable for achieving
maximum therapeutic benet of drugs.
Gastroretentive/mucoadhesive drug delivery systems are valuable
as they prolong the gastric residence time of oral dosage and increase
contact time between drugs and gastric mucosa which eventually im-
proves the bioavailability and absorption of drugs in the upper part of
the gastrointestinal tract (GIT). Various delivery systems based on
swellable, oating, mucoadhesive and high-density formulations have
been developed to achieve maximum gastric retention. Though due to
limited success in this area, researchers worldwide exploring new
methods to synthesize an effective gastro-retentive drug delivery sys-
tem [6,7].
Corresponding author.
E-mail addresses: vikasfbs@gmail.com, vikasfbs@iitr.ernet.in (V. Pruthi).
In the same direction, in the present work, we have developed gellan
based nanobrous system as a novel gastroretentive drug delivery sys-
tem. Gellan is a mucoadhesive polymer of microbial origin which can
prolong the drug residence time by offering a simple and practical ap-
proach to achieve gastric retention [8]. Our previous analysis showed
superior biocompatibility of gellan based nanobers as compared to
its other formulations (hydrogels or lms) [9]. Due to the mucoadhesive
nature of gellan, gellan based nanobers could stay in gastric medium
for a long time which in turn can improve the efcacy of the encapsulat-
ed drug in it.
This work highlighted the use of gellan/PVA nanobers for sustained
release of ooxacin drug in contrast to the pure drug. Ooxacin is con-
sidered as one of the potent antibiotics of uoroquinolone family
which is commonly used as an antibiotic against a wide range of
Gram-positive and Gram-negative microorganisms [10,11]. It is also
renders as safe if inserted in the human body either by oral, intramuscu-
lar or intravenous administration. It has a good tissue distribution as
well as is approved by the Food and Drug Administration (FDA) for
use in drug delivery systems. Besides these useful characteristics, this
drug has some shortcoming such as short half life and premature degra-
dation in gastric medium due to which it requires frequent administra-
tions [12]. The frequent use of antibiotics can cause unwanted side
effects and also increase the risk of antibiotic resistance.
Hence, the primary rationale of this study is to overcome these
drawbacks associated with ooxacin by encapsulating it in a protective

http://dx.doi.org/10.1016/j.msec.2016.10.051
0928-4931/ 2016 Elsevier B.V. All rights reserved.

Please cite this article as: P. Vashisth, et al., Ooxacin loaded gellan/PVA nanobers - Synthesis, characterization and evaluation of their
gastroretentive/mucoadhesive drug..., Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.10.051
2 P. Vashisth et al. / Materials Science and Engineering C xxx (2016) xxxxxx
polymeric nanobrous matrix. Since ooxacin has site-specic absorp-
tion, incorporation of ooxacin in the polymeric nanobers can improve
its gastrointestinal solubilization, absorption, retention time, pharmaco-
logical activity, bioavailability and therapeutic efcacy in GIT.
2. Experimental section
2.1. Materials
Gellan (GELRITE; average Mw 1000 kg/mol) and ooxacin drug were
procured from Sigma Aldrich (St. Louis, MO, US). PVA (Mw = 140,000),
phosphate buffer saline (PBS), Mueller-Hinton (MH) broth, potassium
bromide (KBr) and other chemical were purchased from HiMedia
(India).
2.2. Fabrication of ooxacin loaded gellan/PVA nanobers
Electrospun gellan/PVA nanobers were fabricated as reported ear-
lier by our group [9,13]. Briey, for fabrication of ooxacin loaded
gellan/PVA nanobers, an optimized concentration of ooxacin
(25 mg/ml) was dissolved in pre-prepared gellan/PVA (1:1) solution.
Water was used as a solvent to prepare electrospun nanobers as both
the polymers show complete solubility in water. Addition of ooxacin
in aqueous gellan/PVA solution created an amorphous dispersive solu-
tion which was electrospun in a high voltage environment (18 kV)
through a needle of 21G. The solution ow rate was maintained at
0.1 ml/h using a syringe pump (Harvard apparatus 11 plus syringe
pumps). The grounded aluminum collector was utilized to collect the
gellan/PVA and ooxacin loaded gellan/PVA nanobers. The distance
between the needle tip and grounded collector was xed (1618 cm).
Longer tip-to-collector distance was desirable in this case as the aque-
ous solution take much time to evaporate as compare to other volatile
electrospinning solvents. The whole electrospinning process was car-
ried out under ambient temperature (25 2 C) and relative humidity
(30% 1%) conditions. The electrospun nanobers were kept in desic-
cators lled with silica for the complete removal of residual solvent.
2.3. Crosslinking of gellan/PVA nanobers
Gellan is a biocompatible thermo-stable polymer which has limited
applications due to its hydrophilicity. Similarly PVA (supporting poly-
mer used in this study) is also a thermo-stable hydrophilic polymer
which shows instability in aqueous media. Our previous study shown
that the gellan/PVA nanobers treated with heat possessed good me-
chanical strength, swelling properties as well as biocompatibility in
comparison with non-crosslinked nanobers [14]. Hence, in this study,
heat crosslinking at 150 C for 15 min was performed to stabilize the
gellan/PVA and ooxacin loaded gellan/PVA nanobers in aqueous
media and body uids.
2.4. Physiochemical characterization of ooxacin loaded gellan/PVA
nanobers
The morphology of crosslinked gellan/PVA and ooxacin loaded
gellan/PVA nanobers was examined using FESEM (Quanta 200F
Model, FEI, Netherland) at an accelerated voltage of 5 kV. The samples
(1 1 cm) were sputter coated with gold using Baltech SC005 sputter
coater (Balzers, Switzerland) for 60 s and the average diameters of the
nanobers were measured over 50 different points of FESEM images
using Image J analyzer (Image J 1.49 software, NIH, USA).
Specic surface area and pore width of gellan/PVA and ooxacin
loaded gellan/PVA nanobers were measured using Brunauer
EmmettTeller (BET) and Barrett-Joyner-Halenda (BJH) methods, re-
spectively. For analysis, plot of nitrogen adsorption/desorption isotherm
were used to investigate adsorption points in the relative pressure
range P/P0 of 0.050.30 (Micromeritics ASAP 2020 software).
Please cite this article as: P. Vashisth, et al., Ooxacin loaded gellan/PVA nanobers - Synthesis, characterization and evaluation of their
gastroretentive/mucoadhesive drug..., Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.10.051
The surface static contact angles of the gellan/PVA and ooxacin
loaded gellan/PVA nanobers before and after crosslinking were inves-
tigated through sessile drop method using a drop shape analysis device
- DSA30 (Krss, Hamburg, Germany). For analysis, deionized water
(30 l) was rst dropped on to the surface of completely dried nano-
bers through a microsyringe needle tted in the system. The contact
angle was measured after 60 s of incubation time in order to avoid dis-
crepancies in contact angle measurement due to position and time.
FTIR (Fourier transformed infrared) analysis was performed using
dried nanobrous samples (gellan/PVA and ooxacin loaded gellan/
PVA nanobers) that were nely crushed, mixed with potassium bro-
mide (KBr) and pressed to form a disc. Subsequently, the infrared ab-
sorptions of ooxacin, gellan/PVA nanobers and ooxacin loaded
gellan/PVA nanobers were recorded in the mid-infrared scanning
range from 4000 to 400 cm1 with the resolution of 4 cm1 to validate
the chemical changes or interactions occurred between the drug and
polymeric matrix (gellan/PVA) during the electrospinning process.
The physical state of ooxacin in gellan/PVA nanobers was ob-
served using powder XRD technique (X-Ray Diffrectometer, Bruker D8
Advance). The XRD patterns were recorded with Cu K at a range
from 5 to 100 with the scanning rate of 2/min.
Thermal analysis (TGA, DTG and DTA) of ooxacin, gellan/PVA nano-
bers and ooxacin loaded gellan/PVA nanobers was performed for
determining the physical state and thermal stability of ooxacin drug
in nanobers. The analysis was performed using a TGA instrument
(EXSTAR, TG/DTA 6300, Hitachi, Tokyo, Japan). The samples (pure
ooxacin, gellan/PVA nanobers and ooxacin loaded gellan/PVA nano-
bers) were kept under vacuum for 24 h prior to testing. Subsequently,
the precisely weighed samples (810 mg) were heated from 23 C to
500 C at a scanning rate of 2 C/min under nitrogen gas atmosphere.
2.5. Determination of drug loading efciency
The percent encapsulation efciency (%EE) of ooxacin in gellan/
PVA nanobers was quantied by thoroughly dissolving the ooxacin
loaded gellan/PVA nanobers in aqueous solution [15]. After the com-
plete dissolution of the polymeric nanobers in the aqueous medium,
the ltrate was used to determine the amount of ooxacin using UV
vis spectrophotometer (Lasany double beam LI-2800) at 291 nm. The
amount of ooxacin in nanobers was calculated from the obtained
data against a predetermined calibration curve for the drug. The encap-
sulation efciency of ooxacin was determined as follows:
EE % Actual ofloxacin content in nanofibers mg=
Theoretical ofloxacin content in nanofibers mg  100
2.6. In vitro drug release study
The in vitro drug release study for ooxacin from gellan/PVA nano-
bers was carried out by incubating them in dissolution medium contain-
ing 0.1 N hydrochloric acid of pH 1.2, for a period of 24 h. For
assessment, the nanobrous samples were incubated in 30 ml of disso-
lution medium and maintained in a thermostat (37 C) at 50 rpm. At the
predetermined incubation time, 3 ml of samples was withdrawn from
the dissolution medium and replaced with the fresh medium. The with-
drawn medium was used to measure the OD of solution at the 291 nm
using UVvis spectrophotometry for determining the cumulative drug
release [16]. To further explore the drug release kinetics of ooxacin
drug from gellan/PVA nanobers, various kinetic models were imple-
mented and observed using DD solver Add-In software [17,18].
2.7. In vitro antimicrobial activity
The antimicrobial activity of ooxacin loaded gellan/PVA nanobers
and pure ooxacin was determined by measuring MIC with micro-
 P. Vashisth et al. / Materials Science and Engineering C xxx (2016) xxxxxx
dilution assay [19]. First, the ooxacin loaded nanobers were added in
the wells of 96-well microtiter plate in such a way that the nal concen-
trations of ooxacin remain 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9
and 1 g/ml followed by addition of MH broth. Subsequently, 100 l of
1 106 cells from each set of standard culture (E. coli, E. faecalis, S. aureus
and P. aeruginosa) was added to the respective wells of 96-well plate.
The MIC of pure ooxacin was also achieved in the same manner for
the direct comparison of the possible outcomes and to evaluate the ef-
fectiveness of the ooxacin loaded gellan/PVA nanobers compared to
pure ooxacin. The plates were incubated at 37 C and the MIC was de-
termined as the lowest inhibiting concentration at different time inter-
vals (12, 24 and 48 h). Each assay was repeated three times (n = 3) with
each antimicrobial agent formulation to ensure reproducibility of
results.
2.8. In vitro mucoadhesion studies
The in vitro mucoadhesion study of ooxacin loaded gellan/PVA
nanobers was assessed by mounting a strip of rat's gastric mucosal
membrane on a glass slide followed by sprinkling of accurately weighed
(50 mg) powdered nanobrous formulation on it. The glass slide was
then incubated at 37 C for 20 min so that the gellan/PVA nanobers
can interact with the gastric mucosa. After incubation, the slide was
tilted at an angle of 45 with the help of a stand and washed carefully
using PBS of pH 6.4 [20]. The detached nanobers were collected,
dried and weighed. The percentage mucoadhesion was determined as
follows:
%Mucoadhesion Dry weight of sprinkled nanofibersDry
weight of detached nanofibers=Dry weight of sprinkled nanofibers  100
2.9. In vivo gastric retention studies
The gastric retention of fabricated nanobers was evaluated by
preparing a suspension of ooxacin (5 mg/ml) in 0.5% carboxy meth-
yl cellulose solution with distilled water and fed orally using a can-
nula. Gellan/PVA and ooxacin loaded gellan/PVA nanobrous mats
were rst crushed and then suspended in distilled water for oral ad-
ministration. The experiment was carried out on healthy wistar albi-
no rats, weighing between 180 and 200 g. Animals were provided
from the authorized animal house of Sapience Bio-analytical Re-
search Lab, Bhopal, Madhya Pradesh, India. Animals were acclima-
tized to the standard laboratory conditions in cross ventilated
animal house at temperature 25 2 C, relative humidity 4456%,
and light and dark cycles of 12:12 h, fed with standard pellet diet
and water ad libitum during the study. The experimental protocol
for animal studies was approved by the Institutional Animal Ethics
Committee (IAEC) in accordance with the Committee for the Purpose
of Control and Supervision of Experiments on Animals (CPSCEA),
India, (CPCSEA; 1413/PO/a/11/CPCSEA) guidelines. Twenty four
overnight fasted healthy wistar albino rats were divided into four
groups consisting of six animals in each group for the present inves-
tigation. Group-I served as control and received normal saline,
Group-II served as standard group and were treated with standard
drug ooxacin (5 mg/ml). Group-III and Group-IV (test groups)
were treated with gellan/PVA and ooxacin loaded gellan/PVA nano-
bers, respectively.
2.10. Histopathological study
All treated rats were anesthetized under mild anesthesia and
sacriced after 6 h. The abdomen of rats was then cut open and the
stomach was excised out from each animal. Excised stomachs were
washed with 0.9% (w/v) NaCl solution, and the antrum regions were
xed in 10% neutral buffered formalin. Histological sections (about
Please cite this article as: P. Vashisth, et al., Ooxacin loaded gellan/PVA nanobers - Synthesis, characterization and evaluation of their
gastroretentive/mucoadhesive drug..., Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.10.051
3
5 m thickness) were prepared by microtomy and stained with hema-
toxylin-eosin (H&E) dye. The xed, stained tissue sections were exam-
ined under a digital microscope (Motic DMWB series) at 40
magnication to analyze the gastro-retention of ooxacin loaded
gellan/PVA nanobers.
3. Results and discussion
3.1. Physiochemical characterization of drug loaded nanobers
The comparative FESEM (Field emission scanning electron microsco-
py) micrographs of gellan/PVA and ooxacin loaded gellan/PVA nano-
bers with their average diameter depicting histograms have been
presented graphically in Fig. 1. Data obtained from FESEM characteriza-
tion demonstrated smooth nonwoven and uniform morphology of
ooxacin loaded nanobers. In addition, the surface morphology of
nanobers loaded with ooxacin was found to be cylindrical (similar
to the gellan/PVA nanobers) and slightly entangled at some places.
No drug crystals were noticed on the nanobers surface which suggests
the homogenous dispersion of ooxacin within the gellan/PVA nano-
bers. Data also depicted a slight decrease in average ber diameter
from 40 nm to 25 nm in ooxacin loaded gellan/PVA nanobers as com-
pare to gellan/PVA nanobers (Fig. 1c, f). Reduction in average ber di-
ameter with the incorporation of ooxacin could be attributed to the
increase in conductivity of polymer solution after incorporation of
ooxacin drug [12].
Observations based on the BrunauerEmmettTeller (BET) analysis
indicated that the fabricated nanobers exhibited high surface area
and high porosity (Table 1). The gellan/PVA nanobers without drug
were found to have surface area of 19.02 m2/g and average pore width
of approximately 3.8 nm. In contrast, the ooxacin loaded gellan/PVA
nanobers exhibited high surface area of 21.55 m2/g and average pore
width of about 4.2 nm. An increase in average surface area and average
pore width was recorded upon loading of ooxacin drug in the gellan/
PVA nanobers which found to be in agreement with the outcomes of
FESEM analysis.
3.2. Analysis of hydrophilicity of nanobers by contact angle measurement
Assessment of hydrophilicity of a biomaterial is a signicantly
important parameter in drug delivery sector as the drug delivery for-
mulations often come in contact with various biological uids during
drug delivery. Therefore, to determine the hydrophilicity of nano-
bers, water contact angles on gellan/PVA and ooxacin loaded
gellan/PVA nanobers, before and after crosslinking were measured
using a commercial drop shape analysis system (Fig. 2). Non-
crosslinked nanobers exhibited hydrophilic nature for which the
contact angle value of about 29 was recorded. Whereas contacts an-
gles values ranging from 57 to 62.2 were observed for crosslinked
nanobers. Crosslinked nanobers showed an increase in the contact
angle value as compared to non-crosslinked gellan/PVA nanobers
which reects the improved stability of crosslinked nanobers in
aqueous medium. The contact angle of gellan/PVA nanobers was re-
corded to be decreased after loading of ooxacin. Data showed an in-
crease in hydrophilicity of the drug after incorporation in the gellan/
PVA nanobrous matrix. This could be attributed to the change in to-
pographic features of nanobers due to enhancement in surface
roughness and increased surface area after incorporation of drug
[21].
3.3. Analysis of secondary interactions among ooxacin and gellan/PVA
nanobers
FTIR analysis was carried out to validate the interactions between
drug and polymeric matrix (gellan/PVA nanobers). FTIR spectrums of
pure ooxacin, gellan/PVA nanobers and ooxacin loaded gellan/PVA
4 P. Vashisth et al. / Materials Science and Engineering C xxx (2016) xxxxxx

Fig. 1. FESEM micrographs and corresponding diameter distribution histograms of (a, b, c) gellan/PVA (d, e, f) and ooxacin loaded gellan/PVA electrospun nanobers fabricated at
optimized electrospinning conditions (Voltage = 18 kV; Feeding rate = 0.1 ml/h; tip-to-collector distance = 18 cm). Scale bar = 1 m; AD = average diameter; SD = standard deviation.

nanobers are shown in Fig. 3. The IR spectra for ooxacin exhibited
characteristic bands at 14001350 cm1 which represent the bending
vibration of hydroxyl group. The sharp peaks found at 1450
1400 cm1 are attributed to the presence of methylene group in ben-
zoxazine ring. Stretching vibrations from 1550 cm11500 cm1 are
assigned to CH2 groups of aromatic ring, whereas the bands present in
the range from 1660 cm 11575 cm 1 are due to the presence of
amide groups of quinolones. Absorption peaks for carbonyl group and
carboxyl group were observed at 1776 cm1, 1700 cm1, respectively.
The data obtained is in line with earlier reports on characterization of
polymeric formulation containing ooxacin [22]. Furthermore, the
peaks found around 2700 cm1 are assigned to methyl (CH3) groups
and the stretching peaks noticed at 3055 cm1, 3000 cm1 are due to
vibration of hydroxyl group as well as due to the presence of

Table 1
In vitro characterization of ooxacin loaded gellan/PVA nanobers.

SNo. Formulation Polymer Drug concentration Fiber diameter BET surface area Average pore width Encapsulation efciency
ratio (mg/ml) (nm) (m2/g) (nm) (%)

1 Gellan/PVA Nfs 1:1 40 12.9 19.02 3.8

2 Ooxacin loaded 1:1 25 25 15.8 21.55 4.2 78.47
Nfs

Please cite this article as: P. Vashisth, et al., Ooxacin loaded gellan/PVA nanobers - Synthesis, characterization and evaluation of their
gastroretentive/mucoadhesive drug..., Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.10.051
 P. Vashisth et al. / Materials Science and Engineering C xxx (2016) xxxxxx 5

Fig. 2. The shape of water drops and contact angle measurement for (a) non-crosslinked gellan/PVA (b) non-crosslinked ooxacin loaded gellan/PVA (c) crosslinked gellan/PVA and (d)
crosslinked ooxacin loaded gellan/PVA nanobers. Abbreviation: ~CA - approximate contact angle.

intermolecular hydrogen bonding in the ooxacin structure. The spec-
trum of gellan/PVA nanobers exhibited characteristic absorption
bands at 3500, 3273 cm1 and from 1611 cm1 to 1263 cm1 which at-
tributed to the hydroxyl groups of glucopyranose ring of gellan and car-
boxyl groups of the gellan/PVA matrix, respectively [9,23,24]. A broad
peak for hydroxyl group was observed at 3273 cm1 which represents
the hydrogen bonding between hydroxyl groups of gellan and PVA. In
case ooxacin loaded gellan/PVA nanobers, a slight shifting in the
peak positions ranging from 1638 cm1 to 1095 cm1 was observed
which could be attributed to entrapment of ooxacin between polymer-
ic chains. In addition, on comparing the spectrum of gellan/PVA nano-
bers with the spectrum of ooxacin loaded gellan/PVA nanobers, a
new peak at around 2743 cm1 and a signicant change in intensities
of peaks present at 1686 cm1, 851 cm1 was noticed which also indi-
cated the entrapment of ooxacin drug in the gellan/PVA nanobers.
3.4. X-ray diffraction (XRD) analysis
Pure ooxacin and ooxacin loaded gellan/PVA nanobers were
comparatively examined through XRD technique in order to reveal the
physical state and distribution of the ooxacin drug in the electrospun
nanobers. XRD patterns of pure ooxacin, gellan/PVA and gellan/
PVA-ooxacin nanobers are shown in Fig. 4. Diffraction peaks for
ooxacin were observed at 2 = 6.0, 10.9, 13.9, 15.8, 20.7, 21.9
and 26.6 which indicated the crystalline nature of pure ooxacin. It
was noticed that ooxacin encapsulation did not signicantly inuence
the crystalline nature of the gellan/PVA nanobers [25]. However, a
major peak lying between 2 = 6070 in case of diffractograms

Fig. 3. FTIR spectra of pure ooxacin drug, gellan/PVA nanobers and ooxacin loaded Fig. 4. XRD diffraction patterns of pure ooxacin drug, gellan/PVA and ooxacin loaded
gellan/PVA nanobers with in the scanning range from 4000 to 400 cm1. gellan/PVA nanobers.

Please cite this article as: P. Vashisth, et al., Ooxacin loaded gellan/PVA nanobers - Synthesis, characterization and evaluation of their
gastroretentive/mucoadhesive drug..., Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.10.051
6 P. Vashisth et al. / Materials Science and Engineering C xxx (2016) xxxxxx

Please cite this article as: P. Vashisth, et al., Ooxacin loaded gellan/PVA nanobers - Synthesis, characterization and evaluation of their
gastroretentive/mucoadhesive drug..., Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.10.051
 P. Vashisth et al. / Materials Science and Engineering C xxx (2016) xxxxxx 7

Fig. 6. (a) In vitro drug release prole of ooxacin drug from gellan/PVA nanobers in gastric medium and (b) tting curve of Korsmeyer-Peppas model for drugs loaded gellan/PVA
nanobers. Data presented is the mean () standard deviation of at least three measurements (n = 3).

representing gellan/PVA nanobers was found to be disappeared along
with an emergence of new characteristic peak at around 2 = 21 in the
XRD patterns of ooxacin loaded gellan/PVA nanobers. These observa-
tions suggested the semi crystalline state of loaded ooxacin drug in the
gellan/PVA nanobers. The data obtained was also found to in line with
the FTIR analysis which further conrmed the successful encapsulation
of ooxacin in the gellan/PVA nanobers.
3.5. Thermal stability of fabricated nanobers
Thermal stability of the pure ooxacin, gellan/PVA and ooxacin
loaded gellan/PVA nanobers was evaluated by DTA/TGA/DTG (differ-
ential thermal analysis/thermogravimetric analysis/differential thermo-
gravimetric analysis) characterization techniques (Fig. 5). From the
thermal curves, the degradation of ooxacin was noticed in four major
stages with the rst decomposition phase ranging from 100 C
300 C, second from 300 C400 C, third from 400 C500 C and fourth
from 500 C578 C. However, the decomposition of gellan/PVA and
ooxacin loaded gellan/PVA nanobers were recorded in six successive
stages. The initial weight loss was noticed in the range of 100 C200 C
which could be ascribed to the evaporation of residual absorbed water
molecules (moisture vaporization). Another stages of decomposition
were in the range from 200 C300 C, 300 C350 C, 350 C400 C,
400 C500 C, 500 C571 C. The degradation zone in the range of
300 C400 C was found to be the highest thermal degradation zone
which corresponds to a complex process including depolymerization
as well as decomposition of bonds and units of both the polymers. The
result obtained, suggested that there was no signicant difference in
the thermal stability of gellan/PVA and ooxacin loaded gellan/PVA
nanobers.
The peak observed in DTG thermograms give an idea about the Tmax,
which is corresponds to the maximum degradation rate. Pure ooxacin
drug exhibits a peak of maximum degradation at 275 C while gellan/
PVA nanobers exhibited a degradation peak at 306 C. The peak for
maximum decomposition for ooxacin loaded gellan/PVA nanobers
was observed at 324 C. The slight shifting of temperature peak toward
the high temperature was perceived in case of ooxacin loaded gellan/
PVA nanobers as compared to pure drug. This shifting indicated the
presence of weak interactions between polymeric matrix (gellan/PVA)
and ooxacin as well as the thermal stability of ooxacin drug in the
nanobrous matrix.
3.6. In vitro drug dissolution and release kinetics studies
The encapsulation efciency (EE) and in vitro drug release assess-
ment is necessary in order to determine the bioavailability and extent
of drug assimilation due to its direct inuence on drug therapeutic ef-
ciency [26]. The %EE of ooxacin in gellan/PVA nanobers was recorded
as 78.47%. The in vitro drug release study for ooxacin over a period of
24 h was performed in 0.1 N hydrochloric acid (pH 1.2) as shown in
Fig. 6a and the release kinetics of the drug is depicted in Fig. 6b. As
both the polymers used to fabricate nanobrous matrix were hydrophil-
ic, the drug release mechanism was mainly attributed to the swelling of
the surrounded polymeric (gellan/PVA) nanobrous matrix followed by
its slow diffusion or dissolution. As soon as the dissolution medium pen-
etrates the polymeric matrix it leads to diffusion of drug molecule into
the external environment. A biphasic release prole of ooxacin from
the nanobers was observed. An initial rapid drug release phase
(burst effect) with N 50% of drug released within 4 h was followed by
a slow and sustained release phase. The sustained release phase gradu-
ally extended up to 24 h. The initial burst release of ooxacin from the
nanobrous matrix could be due to the diffusion of drug molecules
adsorbed or accumulated on the surface of nanobrous matrix [27,28].
Initial burst release is usually desirable to achieve an optimum thera-
peutic drug levels in a timely manner in vivo. The successive sustained
release phase occurs primarily due to the diffusion of drug molecules
entrapped in the core region of nanobrous matrix. This phase leads
to the maintenance of drug level at the desired site for a prolonged pe-
riod, which results in the enhanced efcacy of the drug at the site of
action.
The kinetics of ooxacin release was studied using various
established mathematical models to ensure the mechanism of drug re-
lease [17]. The ooxacin release prole from gellan/PVA nanobers
was found to be closely tted with Korsmeyer-Peppas model with expo-
nent (n) value of 0.30 (Fig. 6b). The obtained exponent value (b0.5)
clearly indicates Fickian diffusion release process. Fickian diffusion of
drug usually takes place from cylindrical formulation or from the swol-
len polymer matrix [18]. The drug release prole and tted kinetic
model suggested that, in the initial phase of incubation, the hydrophilic

Fig. 5. DTA, TG and DTG curves of thermal decomposition of pure ooxacin drug, gellan/PVA and ooxacin loaded gellan/PVA nanobers at a scanning rate of 2 C/min under nitrogen gas
atmosphere.

Please cite this article as: P. Vashisth, et al., Ooxacin loaded gellan/PVA nanobers - Synthesis, characterization and evaluation of their
gastroretentive/mucoadhesive drug..., Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.10.051
8 P. Vashisth et al. / Materials Science and Engineering C xxx (2016) xxxxxx

Table 2 after that the MIC was found to be decreased for the ooxacin loaded
In vitro antimicrobial activity of ooxacin loaded gellan/PVA nanobers against reference nanobers as compared to pure ooxacin. Gellan/PVA nanobers with-
strains at different incubation periods.
out ooxacin drug did not show antimicrobial activity against any of the
MIC (g/ml) bacterial strains tested throughout the experiments. Data obtained, sug-
E. coli E. faecalis S. aureus P. gested that gellan/PVA nanobrous matrix acted as a preservative agent
aeruginosa for ooxacin and provide it the physiochemical and thermal stability
Time (h) 12 24 48 12 24 48 12 24 48 12 24 48 which resulted in the improved efcacy of the drug against the treated
Native ooxacin 0.1 0.2 0.4 1 1.4 2 0.2 0.5 2 1 4 8 microorganisms.
Ooxacin loaded 0.1 0.1 0.2 0.1 0.2 0.5 0.2 0.3 0.8 0.5 2 3
nanobers 3.8. In vitro mucoadhesion studies

A mucoadhesive formulation provides prolonged residence time to

gellan/PVA nanobers get swell due to permeation of surrounding me-
dium and drug release taken place majorly due to the diffusion of drug
and dissolution of the polymeric matrix. The obtained regression (R2 =
0.9791) value for the drug release model indicates the signicance of
the adopted model for ooxacin drug release from the gellan/PVA
nanobrous matrix.
3.7. Antimicrobial activity
The in vitro antimicrobial activity of pure ooxacin and ooxacin
loaded gellan/PVA nanobers against test organisms (E. coli, E. faecalis,
S. aureus and P. aeruginosa) is briefed in Table 2. The data obtained sug-
gested that the MIC for pure ooxacin increased with the elongation of
incubation time. This may be attributed to the fact that the pure drug (in
powdered form) steadily loses its activity and the microorganisms that
escaped from the drug action can grow/multiply rapidly [29]. Ooxacin
loaded gellan/PVA nanobers showed lower or equal MICs at all the in-
cubation time points compare to those obtained for the pure ooxacin.
In particular, ooxacin loaded gellan/PVA nanobers results in decrease
of twofold MIC with respect to pure ooxacin in case of P. aeruginosa,
while the highest MIC decrease was achieved in the case of E. faecalis
(three fold decrease). In case of E. coli and S. aureus at 12 h, ooxacin
loaded nanobers showed the same MIC as pure ooxacin, however
the drug on desired site and therefore enhances the bioavailability as
well as the therapeutic response of the drug. In the prepared
nanobrous formulation both the polymers exhibits signicant
mucoadhesive properties [8,30]. Moreover, the increased surface area
of the nanobers is reported to provide additional sites for contact
with mucin [31,32]. Exhibiting these properties, the fabricated drug
loaded nanobers are expected to have better mucoadhesion. In this
study, the ooxacin loaded gellan/PVA nanobers had shown signi-
cant mucoadhesive capacity toward gastric mucosa with a percent
mucoadhesion of 76.48 1.27% (n = 3). Along with the high surface
area, the fabricated gellan/PVA nanobers bear negative charge on
their surface due to the presence of gellan and as reported earlier, the
anionic polymeric formulations with high charge density possess
more efcient bioadhesive properties than cationic or nonionic formu-
lation. Hence, the data obtained from mucoadhesion studies and physi-
ochemical characterization of these fabricated nanobers is strongly in
agreement with the earlier ndings [33,34].
3.9. In vivo gastro-retention studies
Gellan is widely reported to have high potential for developing
gastroretentive drug delivery systems since it has a noteworthy capabil-
ity of mucoadhesion [35]. In this investigation, the in vivo gastro-

Fig. 7. In vivo gastro-retention and localization studies of ooxacin loaded gellan/PVA nanobers in rat stomach tissue (a) control (b) standard (c) gellan/PVA nanobers and (d) ooxacin
loaded gellan/PVA nanobers.

Please cite this article as: P. Vashisth, et al., Ooxacin loaded gellan/PVA nanobers - Synthesis, characterization and evaluation of their
gastroretentive/mucoadhesive drug..., Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.10.051
 P. Vashisth et al. / Materials Science and Engineering C xxx (2016) xxxxxx
retention studies proved the ability of ooxacin loaded gellan/PVA
nanobers to reside in the rat gastric mucosa. Microscopic examination
of rat stomach tissue (antrum region) for control and standard group
(group I & II) animals showed normal localization of the gastric mucosal
tissue after 6 h (Fig. 7a, b) whereas a noteworthy initial mucoadhesion
followed by a good amount of localized nanobers near gastric epitheli-
al cells of antrum region was noticed in the case of gellan/PVA and
ooxacin loaded gellan/PVA nanobers (Fig. 7c, d). Visible gastro-reten-
tion and localization of ooxacin loaded gellan/PVA nanobers indicates
that these nanobrous formulations can efciently serve as a reservoir
for drug and can increase its residence time at the site of absorption
as well as allow the constant gradual release of the drug. The
gastroretentive property of the fabricated ooxacin loaded gellan/PVA
nanobers and sustained release of the drug from these nanobers
can potentially improve the pharmacokinetics of dosage form in the
gastrointestinal tract as compared to free drug.
4. Conclusions
Ooxacin was successfully incorporated in the gellan/PVA nano-
bers using electrospinning technique. The physiochemical characteriza-
tion of ooxacin loaded gellan/PVA nanobers suggested the presence
of weak interaction in between the polymeric matrix and ooxacin
drug as well as semi-crystalline state of the drug in the nanobers.
The gellan/PVA nanobers showed an initial burst release followed by
sustained release of ooxacin drug up to 24 h in contrast to pure drug.
In addition, the fabricated nanobers demonstrated an enhanced in
vitro antimicrobial activity against test microorganisms with signicant
mucoadhesion and gastro-retention properties. The obtained data sig-
nies that fabricated nanobers could be employed as a potential vehi-
cle for oral delivery of ooxacin to improve its bioavailability and in vivo
efcacy.
Conict of interests
The authors declare that they have no competing interests.
Acknowledgements
This study is nancially supported by Council of Scientic and Indus-
trial Research (09/143(0779)/2010-EMR-1), Government of India. We
are thankful to Sapience Bioanalytical Research Lab for providing us fa-
cilities for in vivo studies. Authors are also thankful to Dr. Narayan C.
Mishra (Associate Professor), Department of polymer & process engi-
neering and Institute Instrumentation Centre (IIC), IITR for providing re-
search facilities for this work.
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