Materials Chemistry and Physics 263 (2021) 124381

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Materials Chemistry and Physics

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

Novel topical and transdermal delivery of colchicine with chitosan based

biocomposite nanofiberous system; formulation, optimization,
characterization, ex vivo skin deposition/permeation, and
anti-melanoma evaluation
Hamed Morad a, c, Mohsen Jahanshahi b, *, Jafar Akbari a, Majid Saeedi a, Pooria Gill d,
Reza Enayatifard a, **
a
Department of Pharmaceutics, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
b
Nanotechnology Research Institute, Faculty of Chemical Engineering Babol Noushirvani University of Technology, Babol, Iran
c
Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran
d
Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences, Sari, Iran

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Colchicine-loaded chitosan-based opti­

mized nanofibers depict anti-melanoma
efficiency.
• Topical delivery of colchicine by nano­
fibers shows a significant skin
deposition.
• Significant transdermal permeation of
colchicine observed from composite
nanofibers.
• Transdermal release of colchicine from
nanofibers complies with the first-order
model.

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

Keywords: Melanoma bears the highest mortality rate in skin cancers. The unique properties of chitosan-based nanofibers
Melanoma provide a great potential to formulate an effective topical drug delivery system. With the emphasis on Colchicine
Nanofiber systemic toxicity as an obstacle against the administration in systemic chemotherapy and considering its supe­
Colchicine
riority over approved chemotherapeutic agents for melanoma management, it was chosen to be formulated in a
Topical
Transdermal
nanofiber based topical drug delivery system. The optimization assay was conducted by AFM and SEM based on
the morphology, topographic data, and mean diameter size of the nanofibers. Other characterization studies
include FTIR, XRD, STA, contact angle measurement, tensile test, ex vivo skin permeation, deposition analysis,
release kinetic and anti-melanoma efficiency against A-375 cell line. As a result, significant colchicine deposition
in the skin with remarkable cytotoxicity against melanoma cell line makes it a desirable formulation to be
administered as a topical or local reservoir system for neoadjuvant chemotherapy before other interventions and
adjuvant therapy of tumors after surgery and, also, for other skin diseases with dose adjustment. Besides, the

* Corresponding author.
** Corresponding author.
E-mail addresses: mjahan@nit.ac.ir (M. Jahanshahi), enayatifard_r@yahoo.com (R. Enayatifard).

https://doi.org/10.1016/j.matchemphys.2021.124381
Received 12 November 2020; Received in revised form 20 January 2021; Accepted 8 February 2021
Available online 10 February 2021
0254-0584/© 2021 Elsevier B.V. All rights reserved.
H. Morad et al.
observed first order release kinetic behavior through the skin could suggest it as a transdermal colchicine de­
livery system which improves its efficiency in systemic indications.
1. Introduction
Melanoma is the deadliest skin cancer. It bears a high recurrence
rate, rapid progression, and a very high metastatic potential compared
with other malignancies, whose fatality time is less than a year [1]. The
first-line and second-line FDA-approved drugs like Dacarbazine and
Paclitaxel-Carboplatin combination have not significantly altered fa­
tality period [1,2]. BRAF inhibitors like Vemurafenib or Dabrafenib and
MEK inhibitors like Trametinib showed very limited responses [3].
Evaluation of microtubule-mediated chemotherapeutic agents demon­
strates that the clinical efficiency of drugs interacting with vinca or
taxane binding cites of microtubules has been decreased. The observed
chemoresistance is related to over expression of P-glycoprotein (P-gp),
multidrug resistance-associated protein (MRP) and/or breast cancer
resistance protein (BCRP) [4]. Attractively, Colchicine (CL), with its
specific binding site on microtubules, reported to be less sensitive to the
mentioned resistance mechanism. Moreover, it has extra anti-vascular
activity, enhanced water solubility, and simple structure in compari­
son with the two other groups [5]. CL, as a well-known tricyclic pseu­
doalkaloid that could be extracted from Colchicum autumnale (autumn
crocus) and Gloriosa superba L, has numerous adopted indications in
systemic and topical diseases. In addition to gout, pseudogout, familial
Mediterranean fever, and amyloidosis, as old therapeutic indications,
recently a multitude of novel indications have been reported for
immunology, cardiology, oncology and dermatology. The dermatolog­
ical diseases include Actinic keratosis, Behcet’s disease, Chronic urti­
carial, Epidermolysis bullosa acquisita, Granuloma annulare,
Henoch–Schonlein purpura, Idiopathic plantar eccrine hidradenitis,
Linear IgA, Neutrophilic urticarial, Nodular vasculitis, Purpura annu­
laris telangiectoides, Pyoderma gangrenosum, Recurrent aphthous sto­
matitis, Relapsing polychondritis, Scleredema, Scleredema
diabeticorum, Sweet syndrome, and Urticarial vasculitis [6–8].
Applications of nanofiber-based fabrications are growing in a variety
of fields. However, one of the most notable areas is the use of nanofibers
(NFs) in drug delivery, including transdermal, topical, oral, ocular and
even local injection systems. There are numbers of advantages attrib­
uted to nanofibers over conventional transdermal drug delivery systems
(TDDS), like transdermal patches containing gel, metered liquid spray or
other topical formulations and also pressure sensitive adhesive (PSA)
matrix type TDDSs or even other nanostructures, like nanotubes,
nanocrystals, nanofilms, nanorods, nanospheres, liposomes, noisomes,
solid lipid nanoparticles, nanostructured lipid carriers, dendrimers, etc.
These features for NF-based scaffolds includes large surface area-to-
volume ratio, flexibility in achieved mat, great porosity, high entrap­
ment efficiency, the lowest polydispersity in fiber’s diameter size with
various compositions and bearing potential of being functionalized with
biological agents and simulating the condition of extracellular matrix of
skin and, on the other hand, the capability of controlling drug release
rate. These unique properties of nanofibrous carriers yield an effective
and special reservoir of drug over skin with optimum absorption and
great distribution of drug molecules from nanometric fibers over the
skin surface that would be dedicated to more parts of skin area per unit
of time, which increases the drug internalization and bioavailability as
topical or transdermal drug delivery systems, which improves the
therapeutic effects [9–16]. Moreover, NFs compared with other
mentioned TDDSs, not only could provide high occlusive effect, which
relates to its high surface area, but also brings great breathability to the
skin which refers to high porosity of the NFs [17]. The high porosity
could also help to absorb wounds exudates in topical formulations and
better cell attachment, which improves the healing period [17,18]. In
addition, drug crystallization is a major challenge of conventional
2
Materials Chemistry and Physics 263 (2021) 124381
transdermal patches. The encapsulation of drugs within the NFs in
amorphous manner would prevent this problem [17,19,20]. Besides,
NFs provide more stability for drugs and protects sensitive and potent
active pharmaceutical ingredients (API). Interestingly, Both hydrophilic
and lipophilic drugs could be loaded in various biocompatible and
biodegradable polymers with such inexpensive technology [18].
NF-based carriers for localized delivering of chemotherapeutic agents
have some additional advantages, like stability boosting of loaded
chemotherapeutic agents with maintenance of its cytotoxic property,
controlled and targeted tumor delivery, low systemic side effects and
toxicity, single-dose therapy, and reducing administered dose in the
formulation [21].
As found by latest studies, achieving a distinct and optimized
nanofibers and reducing their diameter size is critical for fabricating
applicable nanofibrous mats with perfect efficiency [22,23]. On the
other hand, in the recent study of Zhu.et al., the loaded Chitosan (CS) in
the structure of nanofibrous delivery system of 5-FU against melanoma
exhibited remedying effect on normal skin cells [24]. Therefore, the aim
of this study is to precisely optimize CL-loaded CS-based Composite-NFs
and fully characterize and evaluate it as an efficient topical and trans­
dermal nano drug delivery system. As a transdermal formulation, it
could be suggested for general diseases’ management with the dose
range of 0.015 mg/kg up to less than 0.1 mg/kg like gout, osteoarthritis,
pericarditis, Behcet’s disease, Familial Mediterranean fever, hepatic
diseases, atherosclerosis and cardiovascular diseases or even localized
targeting inflammations managements with minimum systemic side
effects. Although, as mentioned above, CL seems to be a great choice for
melanoma management, the main factor that has limited its being
applied in cancer therapies is that at high doses it could cause severe
toxicity to normal cells; thus, systemic administration of CL for cancer
therapy has been impossible so far. Consequently, there is the need to
jettison the obstacle against safe administration of the natural based
drug of CL [7,8,25]. Therefore, as one of the aims of this study, the
nanofiber based formulation of CL, using CS as polymer, may be sug­
gested for localized and targeted management of not only all mentioned
topical skin diseases, but also, especially, as a neoadjuvant and adjuvant
therapy in melanoma before and after other treatments, like tumor
excision, radiotherapy or systemic chemotherapies particularly for
effective inhibition of tumor recurrence with sustain releasing the lower
doses than the dose range of 0.1 mg/kg - 0.8 mg/kg in wide range of
cancers from melanoma to any carcinomas [8].
2. Materials
CL (HPLC grade), Chitosan (CS) with medium molecular weight,
Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose), 3-(4,5-
dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and
dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich Co. (St.
Louis, MO, U.S.A.). Poly vinyl alcohol (PVA) with 27000 molecular
weight and Acetic acid (glacial) were bought from Merck (Germany). A-
375 cell line was obtained from Pasteur institute (Medical research
center, Tehran, Iran). Ultra-pure water was utilized in all the proced­
ures. All the solvents and reagents applied had analytical grade in
accordance with United States’ pharmacopeia standards.
3. Methods
3.1. Optimization process of pristine and CL-loaded PVA/CS composite
NFs
The assembled electrospinning apparatus (PN 04, Pars Nano Ris Co,
H. Morad et al.
Iran) consisted of a 5 ml syringe with 140 mm inner diameter and
aluminum foil as the collector. The flow rate was kept constant at 0.2
ml/h and the process was performed at room temperature and 50%
relative humidity. The optimization was conducted in three steps based
on various voltages (10, 12, 15, 18 and 20 kv) and nozzle-collector
distances (10, 12, 15, 20 cm) of electrospinning. The qualified NFs
were chosen by considering the AFM analysis results, including mean
and distribution of diameters sizes, bead appearance, and surface
roughness.
Initially, different concentrations of CS (2.5%, 3%, 5% and 7% w/v)
and PVA (8%, 10% and 12% w/v) were electrospuned separately to find
the best concentration of each polymer.
In the second step, the selected concentrations of each polymer were
combined together in different PVA/CS ratios (60:40, 50:50, 60:40,
70:30 and 80:20 v/v) and electrospuned to reach the optimized ratio and
related apparatus variables for pristine Composite NFs.
Finally, the 20% w/w of CL was added to the prepared optimized
Composite solution and stirred for 8 h to ensure complete solvation of
drug. Then, each apparatus factor was respectively changed to achieve
optimized CL-loaded NFs.
The solvents for CS and PVA were 70% acetic acid and ultrapure
water, respectively. Each separate polymeric solution and the Composite
mixtures had an overnight stirring by magnetic stirrer.
3.2. Optimization assay via atomic force microscopy
Atomic force microscopy (AFM) images were obtained using a JPK
Instrument’s NanoWizard II (Berlin, Germany) mounted on an
OLYMPUS IX51 inverted microscope (USA). Images were performed in
contact mode (air) using ACTA silicon cantilevers (USA) with a tip
radius of 6 nm, a resonant frequency between 200 and 400 kHz, and
spring constant of 75 N m− 1. The scan rate was set to 1 Hz and the set
point was close to 1 V. The obtained images were analyzed with the JPK
image processing software (JPK Instruments), not only evaluating the
morphology of the NFs but also calculating the topographic character­
istics by measuring mean and distribution of diameters’ sizes and all
surface roughness values.
3.3. Scanning electron microscopy
Total and surface morphology of pristine and CL-loaded NF mats
were studied by field emission scanning microscopy (FE-SEM, MIRA3
TESCAN, 3.0 Kv, Czech Republic). The Mean diameter size of NFs was
measured by Image J software (1.51 J8, NIH, USA).
3.4. FTIR spectroscopy
The FTIR spectra of pure PVA, CS, CL, pristine PVA/CS NF mats and
CL-loaded PVA/CS Composite NF mats were evaluated by WQF 510A
spectrophotometer, China with a spectral range of 400–4400 cm− 1 and
resolution of 2 cm− 1.
3.5. XRD analysis
X-ray diffractometer (X’Pert-Pro MPD, PAN analytical, Netherlands)
equipped with a Cu X-ray Anode was used to investigate the crystallinity
of the NF Composites and CL loaded NFs. Continuous scanning was
carried out over a 2θ-angle range of 2◦ –70◦ at 40 kV and 40 mA with step
angle size of 0.02◦ and scanning rate of 43 s/step.
3.6. Thermal study
The simultaneous thermal analyses of pure CS, PVA, CL, PVA NF,
PVA/CS Composite NF and CL-loaded PVA/CS Composite NF were
carried out by BAHR thermoanalysis GmbH STA 503 (Hullhorst, Ger­
many) from 10 ◦ C to 700 ◦ C under argon atmosphere and heating rate of
3
Materials Chemistry and Physics 263 (2021) 124381
10 ◦ C/min.
3.7. Water angle measurement
The water contact angle of PVA mats alone, pristine PVA:CS Com­
posite mats and CL-loaded Composite mats were carried out in a contact
angle measurement apparatus (CA-500 M, Sharif solar Co, Iran) by
depositing a drop of water (5 μl) over the surface of the mats and
recording the drop shape by digital camera. The measurements were
repeated at least five times for each sample.
3.8. Tensile study (DTS)
The tensile parameters of PVA NFs, PVA/Cs Composite NFs and CL-
loaded PVA/CS Composite NFs were investigated by tensile testing
machine (STM- 5, SANTAM, Iran) with 500 kgl capacity at ambient
temperature based on ASTM international standard test method for
Tensile Properties of Polymer Matrix Composite Materials (ASTM, D
3039/D 3039 M). Briefly, the speed of moveable head was kept constant
at 5 mm/min with respect to stationary head, the NF mat pieces having
rectangular shapes (0.1 mm × 20 mm × 50 mm) were mounted in the
grids and after fixation, and the length of the specimen in the apparatus
was considered as initial length. The digital micrometer (DSWQ0,
China) was used to measure the thickness of the specimens. The stress-
strain response curves were studied for determination of tensile
strength as the maximum stress that was applied over the mats, elon­
gation at break as the optimum elongation at break point, and Young’s
modulus as the linear part slope of the curve. Five specimens were
evaluated for each test.
3.9. Skin permeation studies
The animal studies were approved by the Ethics Committee for An­
imal Experiments at Mazandaran University of medical sciences in
compliance with Ethical Guidelines for Investigations in Laboratory
Animals. The ex vivo skin permeation experiments were conducted to
evaluate the skin permeation parameters of CL-NFs compared with CL-
solution. The Franz glass diffusion cells were Composed of receptor
compartment filled by 30 ml phosphate buffered (PBS, 7.4) that was
continuously stirred at 150 rpm during the experiment, and donor part
contained a piece of CL-NFs mat with about 2*2 cm2 area (25 mg) and 5
ml of CL-solution (1 mg/ml) in each related cell over the stratum cor­
neum surface of the rat’s skins with 3.8 cm2 effective diffusion area and
sealed by sealing film. In priory, the excised clean shaved male Wistar
rat’s abdomen skin had an overnight immersion in a normal saline so­
lution and then mounted between the donor and receptor compart­
ments. The temperature was maintained at 32 ± 0.5 ◦ C utilizing a
constant thermostatic water circulation through the cell’s surrounded
jacket. Before initiation of the experiments, 30 min’ equilibration time
was considered. 2 ml aliquots were withdrawn from the receptor sam­
pling nozzle at 0.5, 1, 2, 4, 6, 12 and 24-h time intervals, filtered and
analyzed. An equal volume of fresh medium at the same temperature
was replaced into the receptor compartment. At the end of the experi­
ment, the part of the skin that was in exposure to the donor phase was
removed and the surface was washed three times with deionized water
to remove excess drug. Then the skin was cut into small pieces and
homogenized in 10 ml of PBS, 7.4. After being kept for 24 h in room
temperature and then 10 min centrifugation at 3000 rpm, the super­
natant was filtered by 0.22 μm syringe filter, and the CL content was
determined by HPLC method as mentioned above.
Please note that the animal skin model is a simulated model of
human skin and may over predict human skin CL delivery.
3.10. Transdermal release kinetic
Various kinetic models like zero order, first order, Higuchi,
H. Morad et al.
Korsmeyer-Peppas, Hixon-Crowell, etc. were evaluated to determine the
most similar kinetic model related to the CL transdermal release from
CL-NFs.
3.11. HPLC analysis
The CL concentration was quantified according to the described
assay method in colchicine monograph of USP pharmacopeia by HPLC
Knauer (Germany) using Knauer XDB-C18 column (5 μm, 4.6 × 250
mm). The mobile phase was a mixture of methanol and phosphate buffer
(pH 5.5 ± 0.05) with the ratio of 55:45 (v/v), and the flow rate was 1 ml/
min, and the drug monitoring was performed by UV detector at 254 nm.
The CL retention time was 7.7 min, which was in accordance with the
predicted range in the monograph (5.5–9.5 min) [26].
3.12. Encapsulation yield and drug content calculation
A specified piece of CL-NFs mat with about 2*2 cm2 area was
weighed and dissolved in 50 ml of 1% acetic acid, and 10 μl of the so­
lution was injected into HPLC with the same condition explained above
in HPLC analyses section (3.11). Encapsulation efficiency (EE) and drug
content (DC) percentages were calculated by following equations.
ACTUAL ​ DRUG ​ LOADING
EE⋅(%)⋅ = × ⋅100⋅%
PREDICTED ​ DRUG ​ LOADING
DC ⋅ (%)⋅ =
ACTUAL ​ DRUG ​ LOADING
× ⋅100 ​ %
MAT ​ WEIGHT
3.13. Cell viability assay
The A-375 cell line was selected due to its availability in our country.
The cells were seeded overnight into 96-well plates (1 × 104 cells/well).
Then the cells were treated with equal weighed pieces of pristine PVA/
CS composite NF-based carrier and CL-loaded PVA/CS composite NFs by
direct exposure for 72 h. Subsequently, the cell culture medium of each
well was drawn out and replaced with 100 μL MTT solution (0.5 mg/ml
in PBS), calculating to provide 0.05 mg per each well, and incubated for
4 h at 37 ◦ C in 5% CO2. Then, the culture media was removed carefully,
and 100 μL of DMSO were added to dissolve formazan crystals. The
optical density of wells was determined at 570 nm by microplate reader
(Epoch, Biotek, U.S.A.).
4. Results and discussion
4.1. Optimization of pristine and CL-loaded PVA/CS composite NFs
In the first step, none of the concentrations of CS solution led to
production of fiber in all attempted voltages and distances (Fig. 1 A, B, C
&D). This phenomenon was confirmed by Koosha et al. [27]. The elec­
trospun results of 8% w/v PVA in all voltages and distances from 10 cm
to 15 cm showed totally bead full NFs but in 20 cm at all applied volt­
ages. Approximately, half of the constructed NFs contained a lot of
spindles and few beads (Fig. 1 E & F). The study of Sargazi et al. also
confirmed that shorter distances could not provide sufficient time for
solvent evaporation. thus, the NFs did not form properly [28]. The
infeasibility of forming NFs in 20 cm distance with relatively higher
frequency could be attributed to other related parameters. The study of
Elkasaby et al. determined the polymer concentration as one of the most
significant parameters affecting NFs diameter. The 10% w/v and 12%
w/v of the PVA were electrospuned in a variety of distances and volt­
ages. The results showed that both concentrations in all voltages and
distances would form continuous NFs with great frequency, no beads or
spindles. The optimized parameters for both concentrations leading to
the thinnest PVA NFs were 20 cm distance and 20 kv voltage (Fig. 1 G
&I) [29.]. The mean diameter size with surface roughness (Sa) for
4
Materials Chemistry and Physics 263 (2021) 124381
optimized 10% w/v and 12% w/v PVA was 224 nm with 29 nm and 255
nm with 57 nm respectively. The diameter size distribution of 10% PVA
was a little better (Fig. 1. H& J).
In the second step, since both 10% and 12% PVA demonstrated
relatively similar characteristics, both concentrations were utilized for
preparing various ratios in compounding with CS. Besides, as illustrated
above, CS owns no electrospinnability alone. To increase the chance of
electrospinning, the lowest concentration of CS (2.5%) was selected to
be mixed with PVA. All compounding ratios in all distances and voltages
could form Taylor cone and acceptable jet, but the results were different.
All PVA 10%:CS ratios had formed totally bead full fibers. Additionally,
in higher voltages and distances, spindles appeared, too (Fig. 2. A, B&C).
It could be concluded that 10% PVA could not bring optimum
improvement in electrospinnability of CS solution even at 80:20 ratios
[27]. The PVA 12%:CS with 40:60 and 50:50 ratios in all apparatus
variable parameters showed beads and short pieces of fibers in back­
ground of constructed fibers (Fig. 2. D). As could be seen in Fig. 2. D, the
appearing fibers in the background have very small diameters about 50
nm and also small length in micrometer scale that could be named as
“micronanofibers” as a new term. The attained fibers from the ratio of
60:40 in all voltages and distances also contained some beads but the
mean diameter size of the best apparatus variables at 20 cm and 20kv
was fine and scarcely had some beads (204 ± 23 nm) (Sa = 91.72 nm)
(Fig. 2E &F). The 70:30 ratio led to achieving beadles fibers with
continuous structure in all apparatus variables and 20 cm distance with
20kv voltage showing desirable mean diameter size (243 ± 71 nm)
(Fig. 2. G &H). In the end, the electrospun resultant of 80:20 ratio gained
bead less fibers in all distances and voltages, but the mean diameter size
even at 20 cm and 20 kv was enormous (438 ± 157 nm) and caused
considerable surface roughness (Sa = 266 as a representative) in com­
parison with 70:30 ratio fibers (Sa = 44.08) (Fig. 2. I &J). Presence of
some beads in the AFM image of formulation with 60:40 ratio would
demonstrate that the auto-calculated mean diameter for this formula­
tion was not real and specific for nanofibers and seems to be an average
of both beads and nanofibers diameters. Comparison of the reported Sa
value of the formulation with 60:40 ratio, which was 91.72 with the
formulation of 70:30 ratio which was 44.08, would prove this hypoth­
esis. Investigation of the similar reported studies determines that
depending on the polymer’s nature (molecular weight and degree of
acetylation of CS and degree of polymerization and hydrolysis of PVA)
there would be an optimum ratio for CS to be mixed with PVA to reach
electrospinality that could be in the range of 11% up to 50%. Based on
our findings, the optimum ratio for the combination of utilized CS and
PVA was 70:30. It could be construed that at higher ratio of CS, more
than 30, the accumulated increase in cationic charge at the surface of the
Taylor cone and ejected jet which relates to CS nature would cause a
repulsive force between the polymer’s ionic groups and change the
surface tension that inhibits proper formation of continuous fibers
during electrospinning [30]. As another result, the diameter of the
Composite NFs would be influenced by changing in PVA:CS ratios.
Increasing the PVA:CS ratio would increase the diameter size of NFs.
This result was in agreement with previous works [27,28,30].
In the final step, CL was added to the solution of optimized PVA:CS
(70:30), and the achieved electrospun NFs in various distances and
voltages were evaluated. The thinnest fiber with the best diameter size
distribution was attained at 20 cm and 20kv (Fig. 2. K& L). The mean
diameter size was 247 ± 57 nm, and the surface roughness values were
Sa = 74.27, Sq = 98.56, Sy = 894.72, Sp = 646.07, Sv = − 248.6 and Sm
= 297.27 nm and also for pristine Nfs were Sa = 44.08, Sq = 64.26, Sy =
719.64, Sp = 598.22, Sv = − 121.43 and Sm = 297.21 nm. The loading of
drug into the fiber mats did not make any significant change in diameter
and surface roughness (p > 0.05). This would confirm the successful
incorporation of drug within the NFs [31].
The SEM micrographs were also corroborated by AFM and illustrated
beadles and continuous non-woven NFs with smooth surface and no
phase separation. The determined mean diameter size from SEM was
H. Morad et al. Materials Chemistry and Physics 263 (2021) 124381

Fig. 1. AFM micrographs of CS 2.5% (A), 3% (B), 5% (C) and 7% (D), PVA 8% at 10 cm, 20kv (E) and 20 cm, 18kv (F), PVA 10% at 20 cm, 20kv (G) with its size
distribution (H) and PVA 12% at 20 cm, 20kv (I) with its size distribution (J).

5
H. Morad et al.
Fig. 2. AFM micrographs of PVA 10%:CS 60:40 (A), 70:30 (B) and 80:20 (C) and PVA 12%:CS 50:50 (D), 60:40 (E) with its size distribution (F), 70:30 (G) with its
size distribution (H), 80:20 (I) with its size distribution (J), CL-loaded PVA 12%:CS (K) and its size distribution (L) all at 20 cm and 20kv.
195 ± 0.05 nm for pristine Composite NFs and 227 ± 0.03 nm for CL-
loaded Composite NFs. The thinner diameter size reported from SEM
in comparison with AFM was related to complete evaporation of residual
solvent conducted before SEM analysis by an overnight keeping in
vacuum oven at ambient temperature (Fig. 3 A, B, C & D).
4.2. FTIR molecular analysis
According to Fig. 4 A, the FTIR spectrum of pure CS showed a broad
band at about 3438 cm− 1 and significant peaks at 2922, 1653, and 1095
cm− 1 refers to overlapped N–H and O–H stretching, C–H bending and
stretching of alkyl groups, bending of amide type I, and C–O stretching
vibration frequencies, respectively [32–34].
Main characteristic peaks for pure PVA were observed at 3450 cm− 1
(O–H, stretching), 2921 (C–H of alkyls, stretching), 1652 (C– – O,
stretching) and 1101 (C–O, stretching) [34–37].
The PVA/CS Composite nanofibers illustrated a broad band around
3433 cm− 1 with higher intensity and a slight shift to higher wavelength
compared with the similar band in PVA spectrum, referring to increased
formation of hydrogen bonding by adding CS [33]. Furthermore, the
exhibited peak at 1652 cm− 1 compared with the same peak in pure CS
spectrum showed a very slight shift (from 1653 to 1652 cm− 1) with
decreased intensity, confirming the increase in consumption of CS’s
amide to interact with hydroxyl groups of PVA by hydrogen bonding
[34–36]. Other characteristic peaks related to PVA, and CS also existed
in the Composite spectra with very slight change.
6
Materials Chemistry and Physics 263 (2021) 124381
The spectra of CL exhibited two differentiated peaks at 3315 and
3429 cm− 1 which related to O–H and N–H stretching vibrations. In the
CL-loaded Composite NFs, these two peaks were combined and orga­
nized an individual band with more intensity in comparison with the
same peak in pristine Composite NFs. This phenomenon determines the
hydrogen bond formation between CL, which possesses poly oxygen
structure with an amide group, and the Composite’s O–H and N–H
groups. Also, the C–H stretching peak of CL at 2935 cm− 1 appeared in
the CL-loaded nanofibers spectra. Moreover, other distinctive peak of
CL, including 1733 (C– – O, stretching), 1653 (C– – O of amide, stretch­
ing), 1559 (N–H, bending), 1253 (C–O of ether, stretching), were
manifested in the CL-loaded nanofibers spectra with decreased intensity,
which confirms the participation of the drug’s N and O atoms in
hydrogen bonding.
No significant modification of drug’s characteristic peaks in the drug
loaded nanofibers spectrum might be due to homogeneous loading of
drug along the Composite [38].
4.3. XRD pattern
Based on Fig. 4 B, XRD diffractogram of PVA showed a high intensity
peak at 2θ = 19.6◦ with a shoulder at 22.7◦ and also two low intensity
peaks at 11◦ and 40.79◦ . These characteristic peaks are related to the
presence of crystalline lattice planes in the structure of PVA polymer.
The obvious crystalline peak of chitosan at 20.2◦ was also observed.
Evaluation of the spectrum of the pristine PVA/CS Composite
H. Morad et al.
Fig. 3. SEM micrographs of PVA:CS Composite NFs (A & B) and CL-loaded PVA:CS Composite NFs (C & D).
nanofiber in comparison with the raw polymers demonstrated that only
the main peak of pure PVA at 19.6◦ remained. Besides, this peak became
weaker and had a very short shift to a lower degree (19.4◦ ). The absence
of the polymer’s crystalline peaks in the Composite and intensity
reduction of the related main peak of PVA revealed that PVA/CS Com­
posite nanofibers have lower crystallinity compared to raw polymers.
The attained reduction in crystallinity could be related to hydrogen
bonding interaction occurring between the polymers molecules in the
Composite. This result was confirmed by above FT-IR analysis. More­
over, the absence of chitosan diffraction peak in the PVA/CS Composite,
considering the 70:30 compounding ratios and crystallinity reduction in
nanofibrous form, illustrates fine compatibility of chitosan and PVA [32,
34].
As shown in Fig. 4 B, loading of CL into the PVA/CS Composite
nanofibers led to enhancement of peak’s intensity. Since the CL mole­
cules are surrounded by oxygen atoms, which could be the receptor
atom of hydrogen bonding from the Composite donor groups like hy­
droxyl and amides, it seems that it has the potential to be entrapped by
developing all around hydrogen bonding interactions and also van der
waals interactions. Thus, it may stabilize the Composite molecular
structure and improve the crystallinity of the Composite. As could be
seen above, the FT-IR spectra evaluation also proved formation of these
hydrogen bindings. A spectroscopic analysis study on CL interaction
7
Materials Chemistry and Physics 263 (2021) 124381
with human serum albumin, which has many available donor groups for
hydrogen bonding, showed that CL has the potential to conduct such
spontaneous interactions [37,39].
Crucially, the sharp diffraction pattern of pure CL vanished in the
spectra of CL loaded NFs, which obviously demonstrates the dispersion
of drug in amorphous manner along the Composite nanofibers. The CL
amorphization was proved by STA analysis [40].
4.4. STA analysis
As Shown in Fig. 5, the DTA thermogram of pure CS exhibited two
characteristic peaks at 89 ◦ C (endothermic) and 309 ◦ C (exothermic)
attributed to the adsorbed moisture evaporation and polymer degrada­
tion, respectively. The TGA mass loss stages of CS were observed in the
area of 30 ◦ C–107 ◦ C and 240◦ C–370 ◦ C, confirming the thermal be­
haviors recorded by DTA related to water evaporation occurring in the
first stage and CS degradation, including dehydration of sugar rings,
decomposition and depolymerization of the polymer in the second stage
[41].
The DTA curve of pure PVA showed an endothermic peak at 81 ◦ C
that refers to glass transition temperature (Tg) followed by other two
endothermic peaks at 222 ◦ C and 297 ◦ C related to melting and
decomposition temperatures. The remaining four peaks represent the
H. Morad et al.
Fig. 4. FT-IR spectra (A) and XRD pattern (B) of pure powder of CS, PVA, CL and the attained mat of PVA/CS Composite NFs and CL-loaded PVA/CS Composite NFs.
Representative stress-strain curves (C) of PVA NFs, PVA/CS Composite NFs and CL-loaded PVA/CS Composite NFs. Ex vivo percutaneous permeation profile (D) of CL
per unit area of the rat skin from CL-loaded nanofibers and CL solution.
degradation process of PVA planes structure [34,36]. The TGA curve
also illustrated weight loss phases at these three ranges.
Comparison of the DTA curves of pure PVA and PVA NF alone
indicated that the first three peaks only remained and the four other
peaks vanished, which refers to crystallinity decrease during process of
electrospinning. However, the three phases of weight loss are still
obvious in the TGA thermogram, confirming all thermal behaviors
mentioned above [35].
The pristine PVA/CS Composite NFs compared with PVA NFs and
pure CS demonstrated the three main peaks of PVA in the DTA ther­
mogram. However, the melting peak became broad and shifted to lower
temperature due to reduction of PVA crystallinity with addition of CS.
Besides, the molecular hydrogen bonding between two polymers and
also plasticization of presenting water traces in nanofibers led to exhi­
bition of a broad glass transition temperature. Due to lower proportion
of CS compared with PVA in the Composite and also the crystallinity
reduction, the two peaks of CS were not obvious in the Composite. The
three stages of mass loss in TGA curve also confirm the presence of PVA,
and since the first two phases are common in both PVA and CS, the
observed decrease in thermal stability of Composites indicates crystal­
linity reduction of PVA via added CS [34,36].
The sharp endothermic peak of pure CL at 160 ◦ C, which relates to its
melting point, disappeared in CL-loaded PVA/CS NFs curve,
8
Materials Chemistry and Physics 263 (2021) 124381
determining the entrapment of drug in the nanofibers as an amorphous
manner. This result was also confirmed by XRD and FT-IR studies con­
ducted in this paper. The DTA curve of CL-loaded PVA/CS NFs, in
comparison with pristine Composite NFs, depicts the three main peaks of
PVA with a slight shift. The movement of degradation peak at 320 ◦ C
was a little more toward higher temperature that determines increase in
thermal stability. Two new peaks also appeared with a shift toward
higher temperatures that interpreted higher Composite crystallinity and
increased thermal stability via addition of CL. The mass loss temperature
also increased, which confirms the enhancement of thermal stability.
The FT-IR and XRD proved that the new hydrogen bond interactions
between CL and the Composite structure are responsible for this
phenomenon.
Overall, the three phase weight loss profile is similar in both pristine
Composite NFs and CL-loaded NFs. Furthermore, the represented main
peaks in DTA curves are similar, too, demonstrating homogeneous
dispersion of amorphous CL along the Composite nanofibers [35,42].
4.5. Water angle analysis
The mean water angle of PVA mats, alone, pristine PVA:CS Com­
posite mats and CL-loaded Composite mats were 43.69 ± 4.8◦ , 38.8 ±
5.5◦ and 31.35 ± 3.4 ◦ respectively (Fig. 6 A, B, C as representative).
H. Morad et al. Materials Chemistry and Physics 263 (2021) 124381

Fig. 5. TGA, blue curves and DTA, black curves of pure powders of CS (A), PVA (B), CL (C) and attained mat of PVA NFs (D), PVA/CS Composite NFs (E) and CL-
loaded PVA/CS Composite NFs (F). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6. Representative water angles images of PVA NFs (A), PVA/CS Composite NFs (B) and CL-loaded PVA/CS Composite NFs (C).

Obviously, addition of CS to PVA decreased the contact angle. As re­
ported above, the surface roughnesses of PVA (Sa = 57) and PVA:CS (Sa
= 44) are almost similar, so the enhancement of wettability after addi­
tion of CS does not relate to the surface roughness and could be attrib­
uted to increase in hydrophilicity of the Composite NFs in comparison
with the PVA NFs. In fact, the amino and hydroxyl groups of CS and also
newly formed hydrogen bonding with PVA are responsible for this
development. The incorporation of CL into the Composite NFs also
decreased the contact angle. Thus, loading of CL increased the wetta­
bility of the mat. Based on AFM findings, no significant change in surface
roughness was exhibited after drug loading (44 nm–66 nm). This affirms
that CL was not present over the surface of the NFs and the reported
slight change in roughness refers to the diameter size enhancement
developed by drug loading into the NFs. Thereby, increase in wettability
does not relate to surface roughness or presence of drug on the surface of
NFs. However, it relates to increase in NFs hydrophilicity due to ho­
mogenous dispersion of hydrophilic drug with surrounded poly oxygen
groups and hydrogen bonding with Composite all along NFs with high
surface area that provides easy penetration of water into the mat. These
modifications in angle values are in accordance with FT-IR, XRD and
DSC results and also previous similar studies [27,34].
4.6. Tensile evaluation
The mechanical features of the mats could be related to various
factors like composition, atomic interaction energies, morphology,
consistency and average diameter of fibers [43,44]. Fig. 4C illustrates
the representative stress-strain response curves. The tensile strength of
PVA NFs, PVA/CS NFs and CL-loaded PVA/CS NFs were 8.53 ± 1.45,
15.86 ± 3.71 and 16.91 ± 1.52 respectively. Also, their calculated
Young’s modulus were 0.7 ± 0.39, 4.27 ± 0.94 and 3.84 ± 0.45,
respectively. The difference of tensile strength and Young’s modulus
between PVA NFs group and PVA/CS NFs group were significant (p <
0.05). This shows that the addition of CS to PVA and making a fibrous
nano Composite could lead to produce stronger interactions, which
based on FT-IR, XRD and STA analyses relates to formation of new
hydrogen bonding between two polymers which provided fine unifor­
mity composition. The study conducted by Liu et al. also stated that
consistency without phase separation or agglomeration in the Com­
posite nanofibers could impact on mechanical properties of the mats
[45]. On the other hand, depending on previous studies, CS could
improve stiffness in the mats [46,47]. Moreover, the difference between
PVA/CS NF group and drug-loaded PVA/CS NF group was not signifi­
cant (p > 0.05), which demonstrates that the twenty-percent drug
loading did not significantly change the mechanical properties of

9
H. Morad et al.
PVA/CS Composite. As mentioned in FT-IR, XRD and STA studies, the CL
could form fine hydrogen bonding within the Composite, but it seems
that more than twenty percent drug loading may be needed to enhance
the mechanical stability of the nanofibrous Composite drug delivery
system. However, this non-significant alteration in mechanical proper­
ties after twenty-percent drug incorporation is desirable.
The elongation at break of PVA NFs was 97.93 ± 46.64, which was
significantly higher than PVA/CS NFs (29.92 ± 9.44) and CL-loaded
PVA/CS NFs (28.37 ± 7.92) (p < 0.05). This criterion was not signifi­
cantly different between PVA/CS NFs groups and CL loaded one (p >
0.05). This result also confirms that the PVA NFs possess less stiffness
than the fibrous nano Composites. This elasticity of PVA NFs refers to the
tendency of randomly aligned nanofibers to be aligned easier through
the axis of tensile pulling relative to PVA/CS Composite NFs. This
behavior that occurs in Composite NFs with more resistance refers to
stronger atomic interaction which needs more stress for slipping of
nanofibers along each other [48]. Therefore, the PVA/CS Composite NFs
and CL-loaded PVA/CS Composite NFs demonstrated more stable and
firmer mats compared with pristine PVA NFs.
4.7. Assuring encapsulation yield and drug content by HPLC
Despite the fact that electrospun nanofibers usually provide a com­
plete encapsulation of drug, considering the narrow therapeutic index of
CL, in order to ensure full loading of CL into nanofibers mat and precise
dose adjustment for further administrations of the formulations, the EE
and DC percentages were calculated based on the HPLC results. The EE
was 98.27 ± 1.85% and DC was 19.65 ± 0.37% after repeating the
procedure several times.
4.8. Ex vivo skin permeation and deposition studies
The cumulative permeated profiles of CL per unit area of the rat skin
from CL-loaded nanofibers and CL solution were illustrated in Fig. 4 D.
Total permeated drug during 24h from NF formulation was 45 ± 2.29
μg/cm2 and 15.29 ± 1.21 μg/cm2 from drug solution. This difference
was significant (p < 0.001). The drug flux from NF formulation was also
significantly (p < 0.001) higher than drug solution (more than three-
fold) (Fig D and Table 1).
This increase in drug permeation and flux could refer to the high
specific surface area to mass with very high aspect ratio, flexibility,
porosity structure and release sustainability of PVA/Chitosan nanofiber
scaffold that also improves dissolution rate and drug release profile by
providing a smaller amount of drug permeation through each unit of
skin surface per each unit of time and also possess occlusive property
with desirable breathability enhancing the drug permeation [17]. Be­
sides, the amorphous status of CL in NFs would enhance the drug flux
through the stratum corneum. In contrast, crystal status of CL in the
control solution would prevent drug penetrating into the rat skin [40].
Moreover, water angle measurements’ results proved the high tendency
of CL loaded-NFs to easily absorb even small quantity of skin moisture
after being applied over the skin surface and lead to desirable penetra­
tion of water molecules into the mat, thus providing gradual swelling
and leaching and consequently sustaining diffusion of drug in
Table 1
Skin permeation and deposition parameters of CL from CL-loaded composite NFs
and CL-solution.
Q24h (μg/cm2) Flux (μg/cm2/h) Skin deposited24h (μg/cm2)
CL-NF 45.00 ± 2.29 5.317 ± 0.159 51.72 ± 4.09
CL-solution 15.29 ± 1.21 1.638 ± 0.099 18.46 ± 1.00
Values determined as mean ± SD.
Q24, it is the cumulative amount of permeated CL per skin area into the receptor.
Flux, calculated from the curve slop of cumulative amount of permeated CL per
skin area versus time.
10
Materials Chemistry and Physics 263 (2021) 124381
amorphous manner to each unit of skin area [49].
As summarized in Table 1, significant differences in amount of CL
skin deposition from NF formulation and CL solution were observed (p
< 0.001). Total drug accumulated in the skin was found to be about
three-fold higher after applying CL-NFs compared to the CL solution.
The better accumulation of CL in skin from NF formulation could be
attributed to the higher amount of permeated CL and flux value. Pre­
vious study has proven that CL skin deposition is in direct relationship
with the amount and rate of CL permeation [50]. This greater affinity to
skin may also refers to the partition coefficient value of CL that is rela­
tively more desirable than some other drugs [14,51]. Skin accumulation
is beneficial for localized target delivery of CL in improved management
of all approved therapeutic properties of CL in dermatological diseases
like skin cancers, psoriasis, and so on or even could be suggested for
adjuvant or neoadjuvant local chemotherapy or localized post-surgery
tumor recurrence prevention by reducing the adverse effects caused
by the impact on unaffected areas or systemic absorption.
4.9. Transdermal release kinetic
The permeated amounts of CL per unit area of the rat skin from CL-
NFs were analyzed by applying the related equation of each kinetic
model. Depending on reported parameters in Table 2, the transdermal
release kinetic from CL-NFs was in accordance with first order model
with R2 value of 0.9858 ± 0.0021. In the previous study, the release
kinetic model of another drug (gentamicin) from a chitosan based
nanofibrous Composite membrane was also best fitted by first order
model [52].
4.10. Anti-melanoma activity
According to Fig. 7, the CL-loaded bio composite NFs exhibited a
significant anti-tumoral effect against A-375 human melanoma cell line
at the concentrations of 500 and 1000 μg/ml of the whole mat compared
with the control. Considering the 19.65 ± 0.37% DC concentration, that
was calculated in section 4.7, clearly shows better efficacy of the CL-
loaded bio composite NFs compared with pure CL. Additionally, the
pristine NFs and CL-loaded NFs in the lower concentrations shows a
relative cell proliferation compared with the control.
The significant reported cytotoxicity of CL-loaded composite NFs
could be ascribed to many reasons, including enhanced stability of CL
molecules that was provided in the nanofibers and large surface area
that was dedicated by the nanofiberous mat which would completely
attach to the cells. Moreover, the sustained release of CL from the
nanometer-scale fibers, with extensive cells contact level, ensures dis­
tribution and internalization into the melanoma cells that were
entrapped throughout the scaffold [21]. Additionally, the amorphous
manner of CL in the carrier and the desirable log-p of CL could be also
responsible for the reported high performance cellular entrance and
wiping off function subsequently [14,40].
The observed proliferation of the pristine nanofibrous carrier is
related to the presence of CS in the structure of nanofibers. The remained
CS based scaffold, after completion of CL sustain release, could provide a
desirable recovery for normal cells at the area after suffering from the
Table 2
Coefficients of determination (R2) and other parameters values of kinetics
models for transdermal release.
Kors- Zero Higuchi First order Hixson
peppas order
R2 0.907 ± 0.9756 ± 0.9355 ± 0.9858 ± 0.9828 ±
0.0066 0.0030 0.0074 0.0021 0.0025
Slope 21.0743 ± 2.5289 ± 9.0526 ± − 0.0129 ± 0.0436 ±
1.1124 0.1463 0.4747 0.0008 0.0027
Intercept 2.2911 ± 0.4827 ± − 4.5753 ± 1.9999 ± 0.0032 ±
0.1146 0.2568 0.4716 0.0014 0.0047
H. Morad et al.
Fig. 7. Cell viability of A-375 human melanoma cell line after 72h treated with various concentration of A) pristine chitosan based bio composite NFs,B) CL-loaded
chitosan based bio composite NFs and C) pure CL.
CL. The role of CS in post localized chemotherapy recovery was also
proved in the recent study [24].
Considering the significant skin depot of CL from the nanofibrous
carrier that was exhibited in section 4.8, and the observed residual wipe
out of melanoma cells, it could be suggested for safe localized target
chemotherapy as an adjuvant or neoadjuvant intervention or even for
post-surgery tumor recurrence prevention with the lowest systemic
cytotoxicity.
5. Conclusion
The chitosan based bio Composite nanofiberous drug delivery system
of CL was formulated, optimized, fully characterized and exhibited such
a desirable efficiency as topical and transdermal dosage forms. The
significant skin deposition and anti-melanoma performance, could
suggest it as a neoadjuvant chemotherapy before other interventions
and adjuvant therapy after tumor excision surgery for local delivery and
also as tumor recurrence inhibitor. Moreover, it may be suggested for
management of other types of external or internal tumors. It also could
be recommended for administration in other related skin diseases with
dose adjustment.
Additionally, based on the reported remarkable first order release
kinetic behavior through skin it could be suggested as an efficient
external nanofibrous CL delivery patch for systemic approved in­
dications by omission of first pass effect and providing more conve­
nience and compliance for patients.
Statement of human and animal rights
The animal studies were approved by the Ethics Committee for An­
imal Experiments at Mazandaran University of medical sciences in
compliance with Ethical Guidelines for Investigations in Laboratory
Animals.
Statement of biosafety considerations
All cell culture experiments were performed in accordance with the
approved biosafety level guidelines of Mazandaran University of Medi­
cal Sciences.
Data availability statement
Any raw/processed data required to reproduce these findings could
be shared on demands.
CRediT authorship contribution statement
Hamed Morad: Conceptualization, Methodology, Software, Valida­
tion, Formal analysis, Investigation, Resources, Data curation, Writing -
original draft, Writing - review & editing, Visualization, Supervision,
Project administration. Mohsen Jahanshahi: Supervision, Resources,
Project administration, Funding acquisition. Jafar Akbari: Supervision,
Resources, Project administration, Funding acquisition. Majid Saeedi:
11
Materials Chemistry and Physics 263 (2021) 124381
Supervision, Resources, Project administration, Funding acquisition.
Pooria Gill: Resources, Supervision. Reza Enayatifard: Supervision,
Resources, Project administration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.matchemphys.2021.124381.
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