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Drug functionalized microbial polysaccharide

based nanofibers as transdermal substitute

Article in Nanomedicine Nanotechnology Biology and Medicine March 2016

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 Nanomedicine: Nanotechnology, Biology, and Medicine
12 (2016) 1375 1385

nanomedjournal.com

Drug functionalized microbial polysaccharide based nanofibers as

transdermal substitute
Priya Vashisth, PhD a , Amit Kumar Srivastava, MPharm b , Hemant Nagar, MPharm b ,
Navdeep Raghuwanshi, MTech a, b , Shruti Sharan, MSc a , Kumar Nikhil, PhD a ,
Parul A. Pruthi, PhD a , Rajesh P. Singh, PhD a , Partha Roy, PhD a , Vikas Pruthi, PhD a,
a
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
b
Sapience Bio-Analytical Research Lab Bhopal, Madhya Pradesh, India
Received 22 April 2015; accepted 31 January 2016

Abstract

In order to promote the natural healing process, drug-functionalized nanofibrous transdermal substitute was fabricated using gellan as chief
polymer and polyvinyl alcohol (PVA) as supporting polymer via electrospinning technique. These fabricated nanofibers physiochemically mimic
the extracellular matrix (ECM) which supports the cell growth. For neo-tissue regeneration in a sterilized environment, amoxicillin (Amx) was
entrapped within these nanofibers. Entrapment of Amx in the nanofibers was confirmed by FESEM, FTIR, XRD and TG analysis. In vitro cell
culture studies revealed that the fabricated non-cytotoxic nanofibers promoted enhance cell adherence and proliferation of human keratinocytes. A
preliminary in vivo study performed on rat model for full thickness skin excision wound demonstrated the prompt re-epithelialization in early phase
and quicker collagen deposition in later phases of wound healing in case of Amx-functionalized gellan/PVA nanofibers. Data collectively
confirmed the potential usage of gellan based electrospun nanofibers as transdermal substitute for faster skin restoration.
2016 Elsevier Inc. All rights reserved.

Key words: Gellan; Transdermal substitute; Nanofibers; Electrospinning; Wound healing

Wound healing is a complex multi-step process which
involves the reestablishment of dermal and epidermal tissues
by the means of various cellular and biochemical process. This
process includes successive cascade of events involving
inflammation, migration, proliferation, and maturation phases. 1
Though skin itself has the natural ability of wound healing, open
wounds are soft target for microorganisms which cause infection
at wound site as well as affect the nearby healthy tissues which
consequently delays the wound healing process. Therefore, an
antibiotic treatment along with an appropriate wound dressing is
often helpful to eliminate infection. 2,3
We wish to confirm that there are no known conflicts of interest
associated with this publication.
This study is supported by Council of Scientific and Industrial Research
(CSIR), Government of India.
Corresponding author. Tel.: +91 1332 285530; fax: +91 1332 286151x273560.
E-mail addresses: vikasfbs@iitr.ernet.in, vikasfbs@gmail.com
(V. Pruthi).
http://dx.doi.org/10.1016/j.nano.2016.01.019
1549-9634/ 2016 Elsevier Inc. All rights reserved.
Apart from protecting microbial invasion, the ideal wound
dressings should have the swelling capability for absorbing excess
wound exudates and high porosity for gas permeability. 4,5
Recently, electrospun biomaterials have gained increasing atten-
tion as wound dressing material as they fulfill the above described
criteria of an ideal wound dressing and also possess nonwoven
structural resemblance to skin. 6,7 The high porosity and variable
pore-size distribution of electrospun nanofibers effectively provide
the required air for cell respiration whereas its high surface area to
volume ratio could quickly activate the cell signaling pathway. 8
Most significantly the morphological resemblance of nanofibers to
the natural extracellular matrix (ECM) could support fibroblasts
growth in order to repair the damaged tissue. 9
At present, nanofibers synthesized from natural biodegradable
polymers have gained a special attention for wound healing as they
are histocompatible, non-antigenic and can easily be washed off
from the wound surface. 10 Gellan is a natural anionic exocellular
polymer secreted from Pseudomonas elodea which mainly
consists of repeated tetrasaccharide units of glucose, glucuronic
acid and rhamnose residues in 2:1:1 ratio joined in a linear
1376 P. Vashisth et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 13751385
11,12
fashion. It is an economically abundant biopolymer with
remarkable properties such as biodegradability, biocompatibility
and swelling capabilities which make it an appropriate candidate
for effective wound healing. 1315
This investigation is the first attempt to develop gellan based
nanofibrous transdermal substitutes using a versatile technique
electrospinning. The electrospinning of gellan solution alone
was challenging as it shows a complex gelling behavior even
at low concentration (1 wt% in deionized water). Therefore, to
overcome this limitation, PVA was used as a copolymer of
gellan. PVA was found to lower the repulsive forces of highly
negatively charged gellan solution and subsequently allowed its
electrospinning. PVA is a biodegradable, nontoxic, non-carcinogenic,
biocompatible polymer with appropriate mechanical and swelling
properties which make it a suitable candidate for wound
dressing. 16,17 This study reports on the fabrication of cytocompa-
tible drug functionalized gellan/PVA nanofibrous transdermal
substitutes and their preclinical trial on rat excision model to prove
its efficacy for wound healing.
Methods
Materials
Gellan (Gelrite; average Mw 1000 kg/mol) was purchased
from Sigma Aldrich. PVA (Mw = 140,000), 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell culture-
grade dimethyl sulfoxide (DMSO), phosphate buffer saline
(PBS), Dulbeccos Modified Eagle Medium (DMEM), and all
analytical grade chemicals were obtained from Himedia (India).
Human keratinocytes (HaCaT) cell line was procured from
National Center for Cell Science (NCCS), Pune, India.
Fabrication of nanofibrous transdermal substitute
via electrospinning
Aqueous gellan solution (1 wt%) was prepared in deionized
water at 90 C. Aqueous PVA (10 wt%) solution was prepared
separately and mixed with gellan solution in equal ratio. For the
preparation of Amx-functionalized gellan/PVA nanofibers, Amx
drug (5 mg/ml) was mixed with the prepared gellan/PVA
solution. Electrospinning process was carried out in a high
electric voltage (21 kV) environment and at a fixed solution flow
rate of 0.1 ml/h under ambient temperature (25 2 C) and
humidity (30% 1%) conditions as reported earlier by our
group. 18 The random uniform gellan/PVA nanofibers were
collected on aluminum collector placed at a horizontal distance
of 18 cm from the needle tip. The electrospun nanofibers were
kept overnight in desiccator for complete removal of moisture.
Physiochemical characterization of nanofibrous
transdermal substitute
Field emission scanning electron microscopy (FESEM)
The morphological features of the fabricated nanofibers were
examined using FESEM (Quanta 200F, FEI). For analysis, the
nanofibrous samples (10 10 mm) were sputter coated with gold
using a sputter coater (Bal-tech SC005, Balzers, Switzerland) for
60 s and observed at an accelerating voltage of 10 kV. The average
diameter of the nanofibers was calculated over 100 different points
from the FESEM images using image analysis software (ImageJ,
NIH, USA).
Fourier transformed infrared spectroscopy (FTIR)
The presence of secondary interactions between drug and
polymeric nanofibrous matrix was determined by FTIR analysis
(Thermo Nicolet Nexus 6700, US). For the same, the dried test
samples (Amx, PVA nanofibers, PVA-Amx nanofibers, gellan/
PVA nanofibers and gellan/PVA-Amx nanofibers) were first
finely crushed, mixed with potassium bromide (KBr) and
pressed to form discs. Subsequently, the infrared absorptions
of test samples were recorded in the mid-infrared scanning range
of 4000-400 cm 1 with the resolution of 4 cm 1.
X-ray diffraction (XRD) analysis
Physical state of test samples was further examined by using
powdered X-Ray diffractometer (Bruker, AXS D8 Advance).
For analysis, the test samples were first mounted on the metal
sample holder. Subsequently, the XRD patterns were recorded
by using a slit detector with Cu K radiation over the 2 range
from 5 to 100 with the scanning rate of 2 min 1.
Thermogravimetric analysis (TGA)
Thermogravimetric analysis was carried out using a TGA
instrument (EXSTAR, TG/DTA 6300, Hitachi, Tokyo, Japan)
under nitrogen atmosphere. A sealed empty pan was used as the
reference. Firstly, the samples of about 8-10 mg were kept under
vacuum for 24 h prior to testing and then the precisely weighed
samples were sealed in aluminum pans. The samples were heated
from 25 C to 500 C at a scanning rate of 10 C/min.
In vitro cell culture of human keratinocytes on nanofibrous
transdermal substitute
The cytocompatibility of the nanofibrous transdermal substi-
tute was evaluated by keeping the UV sterilized test formulations
(PVA nanofibers, Amx-functionalized PVA nanofibers, gellan/
PVA nanofibers and Amx-functionalized gellan/PVA nanofibers)
in 24-well microtiter plates individually. Subsequently, the HaCaT
cells (1 10 5 cells/ml) were added to each well along with
DMEM medium supplemented with 10% fetal bovine serum
(FBS), 50 IU/ml penicillin and 50 g/ml streptomycin. 19 The
culture plates were incubated at 37 C in a CO2 incubator
maintaining 5% CO2. Cellular harvests were collected after fixed
incubation period and washed thrice with PBS (pH 7.4) to remove
the unattached cells.
In vitro cell proliferation assay
Cell proliferation on different nanofibrous samples was
quantified using colorimetric MTT assay. In this assay, the
yellow tetrazolium salt (MTT) is reduced to form intracellular
purple formazan crystals by the activity of dehydrogenase
enzymes secreted by mitochondria of metabolically active cells.
The purple formazan crystals can be solubilized to mark the
absorbance at 490 nm. The amount of formazan crystals formed
is directly proportional to the number of viable cells. Briefly,
after culturing the keratinocytes on different nanofibrous
samples for a period of 1, 3, 5 and 7 days, they were incubated
 P. Vashisth et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 13751385
Figure 1. FESEM micrographs and corresponding diameter distribution histograms of (A) gellan/PVA and (B) Amx-functionalized electrospun nanofibers.
Scale bar = 200 nm; AD = average diameter.
with MTT solution (60 l of 5 mg/ml stock) at 37 C for 4 h.
Consequently, the solution was replaced with 400 l of DMSO
to dissolve the formed formazan crystals. 20 Cell proliferation
was quantified by spectrophotometric means at 570 nm (A570 nm)
using microplate reader (Fluostar optima, BMG labtech,
Germany).
In vivo wound healing studies
The in vivo experiments were approved by Institutional
Animal Ethical Committee (IAEC; SBRL/IAEC/June/2014/15)
and performed as per guidelines of the Council for the Purpose of
Control and Supervision of Experiments on Animals (CPCSEA;
1413/PO/a/11/CPCSEA), Government of India. In this study,
thirty Wistar albino male rats (220-250 g) were used for full
thickness in vivo skin wound healing studies. The animals were
randomized into five groups consisting of six animals in each
group. Animals were shaved and topically anesthetized with
ketamine. A 6 mm full-thickness circular wound was created
on the back of each rat using a sterilized surgical blade.
Subsequently, the excised wounds were randomly covered with
PVA nanofibers, Amx-functionalized PVA nanofibers, gellan/
PVA nanofibers and Amx-functionalized gellan/PVA nanofibers
(n = 6). The change in wound area was observed as the
percentage of the original wound area on 1, 3, 6 and 9th day
1377
after initial wounding using a caliper. After two weeks, the
animals were euthanized and biopsies including wound area
were removed and fixed in 10% neutral formalin solution.
Histological examination
The formalin fixed tissue samples were processed in
ascending concentration of alcohol (25%, 50%, 75%, 90% and
100%) for dehydration and embedded in paraffin wax. Paraffin
embedded 5 m thick skin tissue sections were dried in oven
overnight for hematoxylineosin (H & E) and Masson's
Trichrome staining. The stained sections were observed under
the microscope (Evosfl, AMG groups, USA).
Biochemical estimation of wound healing
The biochemical estimation of wound healing involves the
estimation of collagen content synthesized at the wound site.
Collagen is the protein comprising approximately 10%-15%
hydroxyproline which is used as an index to measure collagen
content at the wounded site. The hydroxyproline content of the
re-epithelized wounded site was quantified as described
previously. 21 Briefly, tissue samples were hydrolyzed in Parex
tubes with 6 N HCl for 3 h at 130 C. The hydrolysate was
neutralized to pH 7.0 and was subjected to Chloramine-T
1378 P. Vashisth et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 13751385

Figure 2. (i) FTIR spectra, (ii) XRD spectra, (iii) TGA and (iv) DTG spectra of (a) amoxicillin drug, (b) PVA, (c) PVA-Amx, (d) gellan/PVA and (e) gellan/
PVA-Amx nanofibers.

oxidation for 20 min. After oxidation, 2.5 ml of Ehrlich reagent
was poured into test tubes which were immersed in a water bath
at 60 C. The test tubes were transferred to an ice bath after
25 min and then 6.6 ml of isopropyl alcohol was added to each
tube. After thorough stirring, the samples were analyzed using
spectrophotometer at a wavelength of 557 nm. The hydroxy-
proline content in each test sample was analyzed using
hydroxyproline calibration curve. 21
mean standard deviation (SD). Statistical analysis was per-
formed using one-way ANOVA test. Statistical significance
values were defined and considered at P b 0.05.
Results
Morphology and physiochemical characterization of
nanofibrous transdermal substitute

Statistical analysis FESEM images of electrospun Amx-functionalized gellan/PVA

nanofibers exhibited smooth, uniform and bead free nonwoven
Each experiment was carried out at least three times (n = 3) structure with an average fiber diameter of 60 37.4 nm while
and the quantitative data of the experiment are presented as gellan/PVA nanofibers without drug have an average nanofibers
 P. Vashisth et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 13751385
Figure 3. Cell proliferation assessment of human keratinocytes cells (HaCaT)
cultivated on blank coverslips (control), PVA, PVA-Amx, gellan/PVA and
gellan/PVA-Amx nanofibers after predetermined time period using MTT
(A570nm) assay.Error bars represent mean standard deviation for three
independent experiments (n = 3). P b 0.05.
diameter of 76 18.7 nm (Figure 1). The average fiber diameter
was found to be decreased with incorporation of Amx in gellan/PVA
nanofibers. This may be attributed to the increase in conductivity of
polymeric solution after incorporation of Amx drug. 22
Analysis of secondary interactions between drug and
nanofibrous transdermal substitute
FTIR spectra of (A) Amx, (B) PVA nanofibers, (C) Amx-functionalized
PVA nanofibers, (D) gellan/PVA composite nanofibers, and (E)
Amx-functionalized gellan/PVA nanofibers are shown in Figure 2, i.
The spectra for Amx exhibited stretching bands at 1687 cm 1 and
1516 cm 1 for amide I and amide II group, and absorption peaks for
carbonyl group and carboxyl group at 1776 cm 1, 1396 cm1,
respectively, which are in line with the earlier report.23 The stretching
peaks noticed at 3529 cm 1, 3462 cm 1 are due to the vibration of
amino group and hydroxyl group in the Amx structure. Spectra of
gellan/PVA nanofibers exhibit characteristic absorption bands at
3500 cm 1, 3273 cm 1 and from 1611 cm 1 to 1263 cm 1 which
are attributed to the hydroxyl groups of glucopyranose ring of gellan
and carboxyl groups of the gellan/PVA matrix, respectively.24,25 A
broad peak of hydroxyl group at 3273 cm 1 represents the hydrogen
bonding between hydroxyl groups of gellan and PVA. However, in
case Amx-functionalized PVA and Amx-functionalized gellan/PVA
nanofibers, a change in peak positions ranging from 1638 cm 1 to
1095 cm 1 was noticed due to the entrapment of Amx between
polymeric chains. On comparing the spectrum of gellan/PVA and
PVA nanofibers with the Amx-functionalized nanofibers, new peaks
were observed at 1686 cm 1,851 cm 1 in the spectrum of
Amx-functionalized gellan/PVA and PVA nanofibers suggesting
the successful encapsulation of Amx in the nanofibrous matrix.
Analysis of physical state of drug in the designed nanofibrous
transdermal substitute
The crystalline patterns for native Amx and PVA, PVA-Amx,
gellan/PVA, gellan/PVA-Amx nanofibers, obtained from XRD
1379
technique were compared and depicted in Figure 2, ii. Native
Amx showed characteristic sharp peaks at 2 = 12.06, 15.02,
16.12, 17.08 and 17.94. 26 The diffraction peaks recorded for
PVA-Amx nanofibers (Figure 2, ii, c) and gellan/PVA-Amx
nanofibers (Figure 2, ii, e) were similar to those of PVA
nanofibers (Figure 2, ii, b) and gellan/PVA nanofibers (Figure 2,
ii, d), respectively. However, the intensity of peak present
at 2 = 19.8 was found to decrease in Amx-functionalized
gellan/PVA nanofibers which could be due to the adsorption
of the Amx drug molecules on to the surface of gellan/PVA
nanofibers. The data indicated that Amx entrapment in PVA or
gellan/PVA nanofibers does not change the crystalline structure
of nanofibers. However, all the peaks that correspond to Amx are
not visualized in the XRD spectra of Amx-functionalized
nanofibers which may be attributed to the fact that the entrapped
concentration of Amx drug was too low to be measurable via
XRD technique.
Thermograms (TG) and derivative thermograms (DTG) of the
test samples (native Amx and PVA, PVA-Amx, gellan/PVA,
gellan/PVA-Amx nanofibers) are depicted in Figure 2, iii and iv,
respectively. The thermal curves of native Amx exhibited a sharp
peak for maximum degradation (Tmax) at 208 C indicating its
melting temperature. The degradation of Amx was observed in
four major phases with the first decomposition range of around
25 C to106 C, second from 106 C to 200 C, third from
200 C to 300 C and the fourth from 300 C to 500 C. Our
observations are in line with the previous studies which reported
the thermal decompositions of Amx. 23,26 PVA nanofibers
possessed two weight loss endothermic peaks at 331 C and
438 C which represent its Tmax and polymer chain degradation,
respectively as well as four stages of decomposition
in the temperature range 22 C-200 C, 200 C-300 C,
300 C-400 C, 400 C-500 C were recorded. Amx function-
alized PVA nanofibers exhibited endothermic peaks with slight
shifting on 342 C and 496 C and similar degradation steps as
PVA nanofibers.
The thermograms of gellan/PVA electrospun nanofibers
showed decomposition peak at 306 C and 436 C whereas
Amx functionalized gellan/PVA nanofibers showed an addition-
al endothermic peak at around 278 C which suggested the
encapsulation of Amx drug and presence of weak molecular
interactions between the drug and nanofibrous matrix. The initial
weight loss for gellan/PVA and gellan/PVA-Amx nanofibers
was recorded in the temperature range of 25 C-200 C which
could be attributed to breakage of strong H2O bonds. Other
stages of degradation were documented in the decomposition
stages ranges of 25 C-200 C, 200 C-300 C, 300 C-400 C,
400 C-473 C and 473 C-571 C. The data obtained from
thermal analysis suggested the thermal stability of the Amx drug
in the nanofibrous matrix as compared to native Amx.
In vitro cell proliferation assay
The cell proliferation potential of fabricated electrospun
nanofibrous scaffolds was determined using MTT assay on 1st,
3rd, 5th and 7th day. Data presented in Figure 3, revealed the
cytocompatibility of the fabricated nanofibers and an increased
cell growth in case of electrospun nanofibers as compared to the
1380 P. Vashisth et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 13751385
Figure 4. Wound healing evaluation of full thickness excision wound in rats. Macroscopic appearance of wound closure at 0, 3, 5, 7 and 9th day after treatment
with different fabricated formulations.
control. This may be attributed to the hierarchical features of
nanofibers which are responsible for the enhanced cell
adherence, motility and cell proliferation. No statistically
significant difference was found in respect of cell growth
among gellan/PVA and Amx-functionalized gellan/PVA nano-
fibers after 7 days of culture, indicating that both type of
nanofibers display similar adhesion and proliferation properties.
In vivo wound healing evaluation
The tissue regeneration potential of gellan based formulations
such as films, microcarriers and hydrogels has been evaluated
earlier by various researchers. 2730 However, in this study, for
the first time, the potential of gellan based nanofibers as
transdermal substitute is evaluated via an in vivo wound healing/
closure assay on the dorsal excised wounds of rats. The wound
appearance after treatment with different type of fabricated
nanofibrous sample was observed at predetermined time
intervals (Figure 4). Gellan based nanofibers (gellan/PVA and
gellan/PVA-Amx) were found to attach on wound bed easily as
compared to PVA nanofibers because of the bio-adhesive
properties of gellan. Since gellan and PVA are water soluble
polymers, all electrospun nanofibers facilitated the hydration of
wound which is an important requirement of wound healing
process. The percent wound healing/closure as a function of time
is depicted in Figure 5. Wounds treated with gellan/PVA and
Amx-functionalized gellan/PVA nanofibrous substitute
exhibited a significant augmented wound healing rate as
compared to control. The wound closure rate observed at day
5, in case of gellan based nanofibers was found to be much
higher (gellan/PVA-AmxNgellan/PVA: 86% N 77.5%) than
control (untreated wound) where only approximately 42.2%
wound closure was observed.
Histological analysis of the wound site
The efficacy of gellan based nanofibrous transdermal substitute
for wound healing purpose in terms of re-epithelialization and skin
regeneration was evaluated using H&E and Massons Trichrome
staining assay. 31 The histological sections (H&E) of untreated and
treated wounds are represented in Figure 6. After two weeks of
wound formation, the sectioned dermis tissue from the wound site,
treated with gellan/PVA and Amx-functionalized gellan/PVA
nanofibers (Figure 6, D and E) were found to be composed of
reorganized epithelial layer with evident stratification, whereas in
the case of control and wound treated with PVA nanofibers,
disorganized granulation as well as inappropriately formed
epidermal layer was noticed (Figure 6, A-C). The characteristic
 P. Vashisth et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 13751385
Figure 5. Percent wound closure with respect to time for different fabricated
formulations; control, PVA nanofibers, PVA-Amx nanofibers, gellan/PVA
nanofibers and gellan/PVA-Amx nanofibers.
properties of wound healing such as proper re-epithelialization
with longer epithelial tongue, fully developed connective tissue in
dermis, presence of dermal papillae and well-developed stratum
corneum were observed in the histological sections of wound
treated with gellan based nanofibers. Moreover, the presence of
small blood vessel in the dermis (Figure 6, D and E) at the wound
bed treated with gellan based nanofibers renders these nanofibers
as a potential substitute for wound healing.
Formation of collagen is another hallmark sign of tissue
regeneration which is necessary for a healthy skin and their
normal functioning. 32 The histological sections of Massons
trichrome stained untreated wound and the wound treated with
PVA, PVA-Amx, gellan/PVA and gellan/PVA-Amx nanofibers
are depicted in Figure 7. A significant dense amount of collagen
fibers was observed at the wound site of rats treated with gellan/
PVA (Figure 7, D) and gellan/PVA-Amx (Figure 7, E)
nanofibers as compared to untreated and PVA nanofibers treated
wound (Figure 7, A-C) where collagen fibers are largely missing
even after two weeks of treatment. A number of inflammatory
cells were also noticed near the granulation surface in case of
PVA-Amx nanofibers (Figure 7, C). In contrast, the wound
treated with gellan/PVA-Amx nanofibers showed significant
levels of collagen deposition as well as higher amount of
myofibroblast formation. In addition, the wounds treated with
gellan/PVA nanofibers showed well organized and matured
collagen deposition which helps in reconstruction of ECM, the
presence of dermal appendages such as hair follicles (Figure 7,
D and E) as compare to untreated wounds where no such
appendages were noticed throughout the study.
Biochemical estimation of wound healing
The biochemical estimation of wound healing mainly
depends on the quantification of hydroxyproline content at the
wound site which is directly proportional to the synthesis of
collagen fibers. 33,34 The presence of higher hydroxyproline
1381
content at the wound site confers the higher rate of wound
healing. The data obtained from the biochemical hydroxyproline
assay evidently suggested that the rats treated with Amx-func-
tionalized gellan/PVA nanofibers exhibited the highest concen-
tration of hydroxyproline/collagen content in contrast with the
untreated wound (hydroxyproline content: Control b PVA-Amx
nanofibers b PVA nanofibers b gellan/PVA nanofibers b
gellan/PVA-Amx nanofibers = 19.91 b 29.02 b 29.9 b 33.91 b
34.2 g/100 mg) after two weeks (Figure 8). The hydroxyproline
quantification data support the results obtained by Massons
trichrome staining.
Discussion
The tissue engineered nanofibrous transdermal grafts facili-
tate the architectural similarity of natural ECM to the cells which
maintain the proliferation of replicating cells in site-specific
cellular manner. 35 In this investigation, gellan, an exocellular
microbial polysaccharide is explored to produce nanofibrous
transdermal substitute using the facile electrospinning process.
The gellan based nanofibers physiochemically support the cell
attachment, growth, and proliferation due to their inimitable
rheological properties. 14,29,36 However, despite their well-
known beneficial properties, the fabrication of uniform porous
nanofibers of gellan has been a major issue because of its
polyanionic nature, limited solubility, and highly coiled chain
conformations. 11 To counter this challenge, gellan was blended
with another water soluble, non-toxic and biocompatible
polymer i.e. PVA. 16 The addition of PVA at an optimized
weight ratio (1:1) to the gellan solution reduced the viscosity as
well as the repulsive forces of resulted solution and led to
successful fabrication of highly porous uniform nanoscale fibers.
The fabricated gellan/PVA nanofibers were also functional-
ized with Amx to eliminate any infection at wound site. Gellan
possesses unique hygroscopic property due to which the
fabricated gellan/PVA nanofibrous scaffold can absorb high
water content. The swollen gellan/PVA nanofibrous scaffolds
could play an important role in wound healing by providing a
rapid drug release profile at the wound site as compared to PVA
nanofibers alone. 16,37
Subsequently, to examine the cell proliferation efficacy of the
fabricated nanofibers, human keratinocytes were seeded on them
for different time periods. Data obtained proved that the gellan/
PVA nanofibrous scaffolds provided an ideal environment for
cultivation of HaCaT cells. The proliferation rate of keratino-
cytes cultured on gellan/PVA and gellan/PVA-Amx nanofibers
was found to be significantly higher than that on the PVA
nanofibers, PVA-Amx nanofibers and control (P b 0.05) which
may be attributed to the underlying porous fibrous structure as
well as the available active binding sites on gellan/PVA
nanofibers which naturally resemble the cellular cytoskeleton. 38
As cellular cytoskeleton is responsible for the adhesion of cells, it
is realistic to conclude that the underlying biomimetic gellan/
PVA nanofibers enhance the migration of attached cells along
with the fiber direction which in turn supports wound healing
process. 39 These observations suggest that the gellan/PVA
nanofibers have more potential as growth scaffold compared to
1382 P. Vashisth et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 13751385

Figure 6. Micrographs of H&E stained wounded tissues for (A) control (untreated wound), (B) PVA nanofibers, (C) PVA-Amx nanofibers, (D) gellan/PVA
nanofibers and (E) gellan/PVA-Amx nanofibers after 2 weeks of treatment. Re-synthesized blood capillaries (BC) and hair follicles (HF) are indicated
by arrows.

PVA nanofibers, and the incorporation of Amx drug does not
compromise their cytocompatibility. It can also be concluded
that the released Amx from the nanofibers does not interfere with
the cell proliferation and viability; conversely, it provided a
sterilized environment for cell growth. These outcomes are in
agreement with the work done by Kataria et al who evaluated the
wound healing performance of antibiotic loaded sodium alginate
nanofibers. 2
It is worth noting that when the fabricated nanofibers grafted
on full thickness wounds in Wistar albino rats, gellan based
nanofibrous transdermal substitute with or without drug (Amx)
considerably stimulated efficient rates of skin engraftment which
could be due to the cytocompatibility, biochemical and
topographical features of gellan that help in re-epithelialization.
In addition, the fabricated gellan/PVA-Amx nanofibers main-
tained a moist environment at the wound interface by absorption
of exudates and also provided microbial evasion. 40 Another
important feature of this faster healing may be attributed to the
degradation of gellan which led to increase in glucuronic acid
levels at wound site. The high level of glucuronic acid at wound
site can facilitate the cells to synthesize ECM proteins which
eventually promote early remodeling and faster healing.
The histological data obtained from the H & E and Massons
trichrome staining procedures noticeably suggested the presence
of well-organized collagen fibers, blood vessels and dermal
appendages which further confirmed the candidature of gellan
based nanofibers for wound healing application. ECM regener-
ated at the wound site can be monitored by biochemical
 P. Vashisth et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 13751385 1383

Figure 7. Micrographs of Massons trichrome stained wounded tissue after two weeks for (A) control, (B) PVA nanofibers, (C) PVA-Amx nanofibers,
(D) gellan/PVA nanofibers and (E) gellan/PVA-Amx nanofibers. Magnification: 100 . Abbreviations: Re-synthesized collagen fibers (CF) hair follicles (HF),
blood capillaries (BC) are denoted by arrows.

estimation of the synthesized amount of hydroxyproline/collagen
content therein. 41,42 The highest amount collagen was recorded at
wound site in case of Amx-functionalized gellan/PVA nanofibers
which successively supported the fact that gellan/PVA nanofibers
exhibit a natural mode of tissue regeneration, higher rate of
re-epithelialization and accelerated wound healing efficiency.
Conclusively, gellan nanofibrous transdermal substitute was
fabricated and a broad spectrum antimicrobial drug (Amx) was
loaded in these fabricated nanofibers to combat microbial infection at
wound site. The in vitro cell proliferation assay confirmed that gellan
based electrospun nanofibers can promote augmented cell adhesion
as well as proliferation of human keratinocytes as compared to
control and PVA nanofibers. In vivo studies revealed that gellan
based nanofibers provided appropriate physiological environment
for re-epithelialization and revascularization at wound site. The
fabricated Amx functionalized gellan/PVA nanofibers provide not
only a synergistic carriage of therapeutic agents, but also a familiar as
well as anti-infection environment to support the cell proliferation
and healing process. Thus, the fabricated novel transdermal
substitute could serve as an all in one candidate for ideal wound
healing and skin regeneration applications.
Acknowledgment
Authors are also thankful to Dr. Narayan C. Mishra, and
Institute Instrumentation Center, Indian Institute of Technology,
Roorkee for providing research facilities for this work.
1384 P. Vashisth et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 13751385
Figure 8. Hydroxyproline (collagen) content quantification after two weeks at
the wounded sites treated with control, PVA nanofibers, PVA-Amx nanofibers,
gellan/PVA nanofibers and gellan/PVA-Amx nanofibers.
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