International Journal of Biological Macromolecules 181 (2021) 82–98

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Vitamin D3-loaded electrospun cellulose acetate/polycaprolactone

nanofibers: Characterization, in-vitro drug release and
cytotoxicity studies
Mohammed Ahmad Wsoo a,b, Saiful Izwan Abd Razak c,d,⁎, Siti Pauliena Mohd Bohari a, Shafinaz Shahir a,
Rabiu Salihu a,d,e, Mohammed Rafiq Abdul Kadir c, Nadirul Hasraf Mat Nayan f
a
Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, 81300 Skudai, Johor, Malaysia
b
Department of Chemistry, College of Science, University of Raparin, 46012 Rania, Kurdistan Region, Iraq
c
BioInspired Device and Tissue Engineering Research Group, School of Biomedical Engineering and Health Sciences, Faculty of Engineering, Universiti Teknologi Malaysia, 81300 Skudai, Johor,
Malaysia
d
Centre for Advanced Composite Materials, Universiti Teknologi Malaysia, 81300 Skudai, Johor, Malaysia
e
Department of Microbiology and Biotechnology, Federal University Dutse, PMB 7156 Ibrahim Aliyu Bypass, Dutse, Jigawa State, Nigeria
f
Faculty of Engineering Technology, Universiti Tun Hussein Onn Malaysia, 86400 Batu Pahat, Malaysia

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

Article history: Vitamin D deficiency is now a global health problem; despite several drug delivery systems for carrying vitamin D
Received 19 February 2021 due to low bioavailability and loss bioactivity. Developing a new drug delivery system to deliver vitamin D3 is a
Received in revised form 16 March 2021 strong incentive in the current study. Hence, an implantable drug delivery system (IDDS) was developed from
Accepted 19 March 2021
the electrospun cellulose acetate (CA) and ε-polycaprolactone (PCL) nanofibrous membrane, in which the core
Available online 23 March 2021
of implants consists of vitamin D3-loaded CA nanofiber (CAVD) and enclosed in a thin layer of the PCL membrane
Keywords:
(CAVD/PCL). CA nanofibrous mat loaded with vitamin D3 at the concentrations of 6, 12, and 20% (w/w) of vitamin
Cellulose acetate D3 were produced using electrospinning. The smooth and bead-free fibers with diameters ranged from 324 to
Polycaprolactone 428 nm were obtained. The fiber diameters increased with an increase in vitamin D3 content. The controlled
Implantable drug delivery system drug release profile was observed over 30-days, which fit with the zero-order model (R2 > 0.96) in the first
Kinetic drug release stage. The mechanical properties of IDDS were improved. Young's modulus and tensile strength of CAVD/PCL
Electrospun nanofiber (dry) were161 ± 14 and 13.07 ± 2.5 MPa, respectively. CA and PCL nanofibers are non-cytotoxic based on the
Vitamin D3 results of the in-vitro cytotoxicity studies. This study can further broaden in-vivo study and provide a reference
for developing a new IDDS to carry vitamin D3 in the future.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction
Vitamin D is a fat-soluble Seco-hormone that plays a crucial role in
regulating physiological functions in the human body [1] and has a
meaningful role in reducing the risk and severity of COVID-19 and
other acute respiratory tract infections [2–4]. Vitamin D testing and sup-
plements of vitamin D2 or D3 uses have increased substantially in recent
years [5,6]. Amrein, Karin, et al. recommended avoiding vitamin D defi-
ciency because vitamin D3 concentrations below 30 nmol/L (or
12 ng/mL) increase the risk of over-mortality infections and other dis-
eases dramatically [5]. Vitamin D deficiency is now a global public
health problem in all age groups, even in those living in countries
⁎ Corresponding author at: BioInspired Device and Tissue Engineering Research Group,
School of Biomedical Engineering and Health Sciences, Faculty of Engineering, Universiti
Teknologi Malaysia, 81300 Skudai, Johor, Malaysia.
E-mail address: saifulizwan@utm.my (S.I.A. Razak).
with inadequate exposure to sunlight [7]. The global public health strat-
egy to prevent severe vitamin D deficiency significantly involves vita-
min D supplements and systemic food fortification with vitamin D for
some groups.
Despite many approaches currently available for delivering vitamin
D [8], the low bioavailability and loss of bioactivity of the vitamin D dur-
ing formulation and distribution are significant challenges. Introducing
a new drug delivery system to improve the bioavailability and bioactiv-
ity of vitamin D3 is a great motivation in the current study. A study re-
ported IDDS has many advantages over other drug delivery systems
[9]; the main advantages are targeted drug delivery, reducing the level
of medicines needed to treat, enhancing bioavailability and efficacy
due to avoid the first-pass metabolism, and improving patient compli-
ance. Previously many implantable delivery systems have been devel-
oped and used for a range of clinical applications, including Jadelle®
[10], Nexplanon® [11], Vantas® [12], Psuprefact® Depot, and Zoladex
[13].

https://doi.org/10.1016/j.ijbiomac.2021.03.108
0141-8130/© 2021 Elsevier B.V. All rights reserved.
M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al.
Given the facts mentioned above and the global prevalence of vita-
min D deficiency, a rod implantable drug delivery system inserted in
the subcutaneous tissue to deliver vitamin D3 over a long time to treat
vitamin D deficiency can resolve low bioavailability and bioactivity of
the vitamin D3 due to direct delivery into the body. In the formulation
of new IDDS, tailoring long-termed drug release, mechanical properties,
and biocompatibility are the key challenges. Electrospun polymer nano-
fiber has been produced by electrospinning, a suitable technique for
regulating drug release due to a homogeneous drug distributed inside
the polymer nanofiber. Recently, the fabrication of biopolymers to gen-
erate electrospun polymer nanofibers using electrospinning has be-
come an exciting approach to deliver several substances in the drug
delivery systems due to its increased surface-to-mass ratio, ease of oper-
ation, and cost-effectiveness [14]. Moreover, drug-loaded electrospun
polymer nanofibers can overcome several key challenges, such as low
solubility of drugs, loading efficiency, low bioavailability of drugs, and
control drug release behavior [15]. Therefore, an implantable drug de-
livery system (IDDS) made of electrospun polymer nanofibers was de-
signed to provide prolonged vitamin D3 delivery to treat vitamin D
deficiency. Using nanofiber in this study hence provides a new way
to develop an implantable drug delivery system. Among the different
biocompatible polymers used to obtain electrospun nanofibers,
cellulose acetate (CA) and polycaprolactone (PCL) have been chosen
as carrier polymers. Cellulose acetate provides essential advantages
such as renewability, affordability, and ease of mass production. It
also has strong solubility in organic solvents, making it an ideal
material for electrospinning. Cellulose acetate is biocompatible, semi-
biodegradable, non-irritating, and non-toxic [16]. In recent years,
researchers have mainly concentrated on developing a CA nanofiber
mat through a process commonly known as electrospinning and used
it in the topical delivery system to carry different drugs [17]. PCL is
more applicable for long-term than short-term drug delivery systems
due to its slow biodegradation rate. PCL's biocompatibility has also
been approved by the FDA [18]. The PCL polymer solution could be fab-
ricated to create an electrospun PCL nanofiber membrane using
electrospinning [19]. PCL nanofibers membrane can also be modified
to the capsule by thermally sintering to improve its long-termed me-
chanical properties and control drug releases due to reduced
electrospun pore size and porosity [20,21].
Khoshnevisan, Kamyar, et al. produced a propolis-impregnated CA/
PCL nanofibrous mat using electrospinning and applied topically for
wound healing due to propolis has antibacterial and antioxidant effects
[22]. Several studies have been designated co-electrospinning PCL/CA
polymers with various (w/w) ratios. The PCL/CA composite nanofibers
have a smooth and uniform surface with excellent mechanical strength,
hydrophilicity, and enhanced biocompatibility [23–25]. In short, PCL
was blended with CA and successfully fabricated to produce PCL/CA
composite nanofiber by electrospinning. The blended PCL/CA was
used to enhance physicochemical and biological properties to use in
biomedical applications.
The aim of this study introduces a new IDDS to deliver vitamin D3
that can be implanted in subcutaneous tissue. The main objectives
are 1) to control drug release and improve mechanical properties
of the IDDS, the implantable drug delivery system's core consists of
a single rod of CA nanofiber loading with vitamin D3 enclosed in a
thin-wall sintered PCL capsule. The sintered PCL capsule is a rate-
limiting polymer membrane produced from the PCL nanofibrous
membrane by thermal sintering (at 58 °C) [20]. In this way, we stud-
ied the effect of various drug concentrations (6%, 12%, and 20% w/w)
on the surface morphology and fiber diameter and studied the PCL
layer's impact on the in-vitro drug release and tensile testing. 2) In
order, long-termed good biocompatibility and slow biodegradability,
CA and PCL polymers biomaterial have been chosen to formulate the
IDDS. For this purpose, in-vitro biodegradation of the IDDS was stud-
ied. In-vitro cell cytotoxicity and cell viability are also measured
against human skin fibroblast cells using Trypan Blue exclusion test
83
International Journal of Biological Macromolecules 181 (2021) 82–98
and MTs assay. To the best of our knowledge, no work has been re-
ported on electrospun nanofiber use to develop a rod of implantable
drug delivery system. This work would be the first to use electrospun
nanofiber in the formulation of IDDS.
2. Experimental
2.1. Materials
Vitamin D3 (25(OH) vitamin D3, Mw 384.64 g/mL, C1357) was
used as a drug model, Cellulose acetate (CA, Mn = 30,000, 180,955)
and poly ɛ-caprolactone (PCL, Mn = 80,000) were used as the
electrospinning polymer. Organic solvents including acetone (99.5%),
N, N-dimethylacetamide (99%), N, N-dimethylformamide (99.9%),
ethanol (99.5%), and dichloromethane (99%) were purchased from
Sigma Aldrich. Methanol and acetonitrile, both HPLC grade, were pur-
chased from Merck. Phosphate buffer saline (PBS, pH 7.4 ± 0.2,
PB0354), used to prepare the release medium, was purchased from
Vivantis Technologies Sdn. Bhd., Malaysia. Dulbecco's Modified Eagle
Medium (DMEM, High Glucose (4.5 g/L), with stable glutamine and
sodium pyruvate, DMEM-HPSTA), and fetal bovine serum (FBS-11A)
were purchased from Capricorn Scientific. Phosphate buffer saline
(PBS, PH 7.4, 806552) was purchased from Merck. Trypsin-EDTA
solution (0.25%, T4049), Trypan Blue solution (0.4%, T8154), and glutar-
aldehyde solution (Grade I, 25% in H2O, G5882-50mL) were purchased
from Sigma Aldrich. MTs Cell Proliferation, assay Kit (MBS841516),
and 25-HVD3 (25-(OH) Vitamin D3) ELISA Kit (96T, MBS2510792)
were purchased from MyBioSource. Antibiotic-antimycotic (100×,
15240062) was purchased from Thermo Fisher Scientific. Cell lines of
the human skin fibroblasts (1184) at passage 11 were received from
the Tissue Engineering Laboratory, Biosciences Department - Faculty
of Science, UTM.
2.2. Preparation of polymer solutions
CA solution and CA blended vitamin D3 (VD) solutions for
electrospinning have been made in a ternary solvent system
consisting of acetone/DMAc/ethanol (3:2:1, v/v). The CA polymer so-
lution was made up to 17% by weight. Vitamin D3 blended CA solu-
tions in 6, 12, and 20% w/w (based on the weight of CA polymer)
have also been prepared by adding 0.13 g, 0.278 g, and 0.51 g of
pure vitamin D3 powder to the 12 mL of 17% CA solution, separately.
10% of PCL polymer solution was prepared by dissolved 1 g of PCL
pellets in mixed solvents of DCM/DMF (4:1) [26]. Each solution
was then stirred continuously on a magnetic stirrer until a homoge-
neous and transparent solution was obtained. A Brookfield DV-II+
pro programmable viscometer and a BC3020 conductivity meter
were used to measure the intrinsic viscosity (ŋ), and conductivity
at different shear rates (s−1) of the prepared spinning solutions. All
measurements have done in triplicates, and the result was presented
as mean ± standard division.
2.3. Electrospinning fabrication
The electrospinning setup consisted of a syringe pump (NE-300)
used to feed the polymer solution. Gamma High Voltage Research
RR30-2P/DDPM is used to charge the polymer solution held in a 10-
mL disposable syringe with high voltage DC power. A flat plate collector
was used to collect the generated nanofiber webs that are wrapped with
aluminum foil. A five mL syringe, filled with the spinning polymer solu-
tion, was placed at the top of the constant flow pump. The stainless steel
needle (gauge-23) of the syringe was connected to the Gamma High
Voltage's positive electrode. The produced electrospun fiber was col-
lected on a plate covered with Al-foil. The experimental parameters
were set as represented in Table 1. Electrospinning was done at
20–23 °C and 40–60% relative humidity. After electrospinning, all
M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al. International Journal of Biological Macromolecules 181 (2021) 82–98

Table 1
Experimental process parameters of fiber fabrication.

Electrospinning Polymer concentration (w/v) Drug% w/w based on polymer Voltage Distance Flow rate (mL/h) Abbreviation of e-spun
solutions (kV) (cm) fibrous mat

Pure CA 17% 0 10.5 12 0.7 CANF

Blended Vit.D3 and CA 6 9.5–10.5 0.5–0.7 CAVD6
17% 12 12 CAVD12
20 CAVD20
PCL 10% 0 11 13 1 PCLNF

CA: cellulose acetate; PCL: polycaprolactone, W: weight, V: volume, Vit.D3: vitamin D3.

electrospun fiber membranes were put in a vacuum oven at 40 °C for
24 h to remove the residual solvent.
2.4. Design of an implantable drug delivery system
An implantable drug delivery system was created from CA nanofiber
loading with vitamin D3 (CAVD) and PCL nanofiber membrane. The
implant's core consists of a single CAVD enclosed in thin-wall a sintered
PCL capsule, as illustrated in Fig. 1.The sintered PCL capsule is a rate-
control polymer membrane formed from the PCL nanofiber membrane
by thermal sintering (at 58 °C) [20]. This study aimed to produce differ-
ent vitamin D3-loaded CA nanofiber (CAVD6, CAVD12, and CAVD20)
based on the amounts of vitamin D3 (Table 1). Vitamin D3-loaded CA
nanofibrous mate was then manually rolling to a rod-shaped that is
4 cm long and around 2.5 mm diameter and enclosed in a thin-walled
PCL membrane (Fig. 1). Finally, the electrospun PCL membrane was
modified to a capsule using thermal sintering at 58 °C. The sample's di-
ameter was measured using a digital micrometer (0–25 mm/0–1 “Opti-
cal external micrometer IP65 OXD3316110 K”) screw gauge. The
samples were inserted into zip-lock plastic bags placed into a standard
recirculating water bath (WCB-10 L, Model DSB-500D, temperature sta-
bility ±0.2 °C) for 3 h at 58 °C in the sintering process. An SEM cross-
section image was used to observe the top surface morphology of the
rod CAVD/PCL. The PCL thickness layer was measured using the Image
J software from the cross-section image, and the diametric shrinkage
capsule was determined using the following Eq. (1), the data shown in
Table 2:
 
t 0 −t 1
%shrinkage ¼ 100⁎ ð1Þ
t0
where t0 is the original thickness of the PCL layer, and t1 is the PCL thick-
ness after sintering.
2.5. Morphology characteristics
The nanofibers were sputter-coated with platinum for 50 s at the
current strength of 30 mA using a Quorum Q150R Thin-Film Coater to
prevent charging. Their morphologies were detected using field-
emission scanning electron microscopy (FESEM; Hitachi SU8020). The
average diameter and nanofibers diameter distribution of 100 nanofi-
bers were measured using IMAGE J software.
2.6. Thermal properties
The thermal properties of CA, vitamin D3-loaded CA nanofiber
(CAVD12 and CAVD20), PCLNF, and sintered PCL mats were investi-
gated using differential calorimeter scanning (DSC 25-TA instrument,

Fig. 1. Schematic of formulation CAVD/PCL with cross-sectional SEM image.

84
M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al.
Table 2
Detail information on the rod-shaped implantable drug delivery system.
Measurements
Implants core
Diameter of core (mm)
Diameter CAVD/PCL before sintering (mm)
Diameter CAVD/PCL after sintering (mm)
PCL's thickness (μm) after sintereda
% shrinkage
The data are expressed as mean ± SD, n = 3.
a
Image J software used to measure PCL's thickness at cross-section layers.
USA). The crystallinity degree of PCL (Xm) was calculated from a DSC
analysis base on the following Eq. (2).
 
ΔH m
X m of PCL ¼ ⁎100 ð2Þ
139:5 J=g
where ΔHm is the melting enthalpy of PCL fibers, and it was obtained
from the DSC curve. The 139.5 J/g is the melting enthalpy of 100% crys-
talline PCL (for the PCL molecular weight of 80,000 g/mol) [27].
2.7. Mechanical testing
The tensile strength, breaking elongation, and elastic modulus of the
prepared samples was carried out using a universal testing machine
(Zwick/Roell Z020, Zwick, Germany) at room temperature, which a
load cell of 500 N, gauge length 5 cm at a crosshead speed of 5 mm/
min. For mechanical testing of samples under wet conditions, the sam-
ples were immersed in PBS (pH 7.4) for 24 h. The experiment was re-
peated five times for each of these measurements, and the data were
presented as mean ± standard deviation.
2.8. Water contact angle
The surface wettability of nanofiber mats was measured using a ses-
sile drop method-based video contact angle system (VCA Optima, AST
Products, Billerica, MA). Approximately 5 μl of distilled water was
dropped on the membrane's surface, and the contact angle was mea-
sured over time by recording multiple images on the system. Five read-
ings for each type of fibrous mat were measured, and its average was
reported.
2.9. Degree of swelling and weight of loss
The weight loss and swelling behavior of CANF, CAVD12/PCL, and
CAVD20/PCL were performed by total immersed in phosphate buffer so-
lution (pH 7.4) at 37 °C for 30-days. The weight loss percentage and de-
gree of swelling were calculated using the following equations.
W 0 −W i
%weight loss ¼ ⁎100
W0
W s −W 0
%degree of swelling ¼ ⁎100
W0
where wi is the weight of the dried sample after the ith days that was
immersed in the phosphate buffer, ws is the weight of the swelling sam-
ple after wiping with tissue paper, and w0 is the initial weight of the
sample in the dry state.
2.10. Stability of vitamin D3 after loaded in CA fibrous mats
Due to the application of a high electrical potential to blend CA/Vita-
min D3 solutions during electrospinning, Vitamin D3 stabilization has
International Journal of Biological Macromolecules 181 (2021) 82–98
Implantable drug delivery system
CAVD6/PCL CAVD12/PCL CAVD20/PCL
CAVD6 CAVD12 CAVD20
2.35 ± 0.04 2.3 ± 0.11 2.29 ± 0.05
3.44 ± 0.03 3.3 ± 0.10 3.45 ± 0.04
2.55 ± 0.02 2.5 ± 0.10 2.5 ± 0.05
200 ± 16.20 200 ± 18.42 210 ± 28.43
81.70 80.00 81.90
been evaluated using a UV–Visible spectrophotometer (Thermo Scien-
tific GENESYS 10S, Germany) and high-performance liquid chromatog-
raphy (HPLC) Series 1100/1200 (Agilent Technologies, USA) [28]. The
chromatographic Vitamin D3 determination was performed on a
reversed-phase Luna® 5 μm C18, 100 Å, LC Column 150 × 4.6 mm
(CN: 00F-4252-E0, Phenomenex, USA) at 40 °C, the acetonitrile–
methanol (95:5, v/v) was used as a mobile phase at a flow rate of
1.2 mL/min [29]. The injection volume was set at 10 μL, and detection
was carried out at 265 nm.
For chromatographic analysis, reference was prepared by dissolving
2 mg of standard vitamin D3 in 1 mL of methanol, then diluted to the de-
sired concentration. Besides, vitamin D3 was extracted from CA nanofi-
bers by incubating an amount of Vitamin D3-loaded CA fibrous
membrane in methanol overnight with continuous stirring then filtered
through a polytetrafluoroethylene (PTFE-20/25) filter (average pore
size = 0.2 μm) before injection. Methanol was used in the chromato-
graphic analysis to dissolve and extract vitamin D3 from the CA
nanofibrous mat without dissolving CA polymer. Moreover, methanol
is a mobile phase content and could avoid a solvent peak.
For UV-spectra analysis, reference vitamin D3 solution (10 mg/mL)
and an amount of vitamin D3-loaded CA nanofiber was dissolved in ac-
etone then analyzed against the solvent in the range of wavelength
190–400 nm. Cellulose acetate and vitamin D3 have been completely
dissolved in acetone. A baseline can eliminate the interfere of CA spectra
and acetone solvent. Moreover, this method cost-effective to measure
higher concentrations of vitamin D3 (mg).
2.11. In-vitro drug release study
2.11.1. Actual 25-vitamin D3 content
2.11.1.1. Vitamin D3 encapsulation efficiency. To determine the amount of
vitamin D3 loaded in the CA nanofiber mats (wt. of drug in the fiber) was
started by drying drug-loaded CA nanofiber mats in a drying oven
(LFDO-101) at 40 °C. After that, the sample was weighed and dissolved
in 10 mL of acetone; the absorbance of vitamin D3 in the prepared solu-
tion was measured against CA blank solution by UV–Visible spectropho-
tometer at 326 nm. The amount of vitamin D3 was obtained from the
ð3Þ calibration curve of vitamin D3 (y = 3029× + 0.1775, R2 = 0.99881).
The encapsulation efficiency (EE) was calculated by the following
Eq. (5):
ð4Þ
wt:of drug in the sample
EE ð%Þ ¼ ⁎100 ð5Þ
theoretical weight of drug in the sample
2.11.2. Preparation of releasing medium
Due to vitamin D3 has limit solubility in an aqueous solution, release
medium prepared from 50 mL of PBS (20×) and 210 mL of ethanol
(95%) then diluted in 1 L of de-ionized water (PBS, 1×, pH ~ 7.4 + ethanol
(20%)). Ethanol was utilized as a co-solvent to increase the solubility of
vitamin D3.
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M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al.
2.11.3. Drug release study
In-vitro drug release of the CAVD/PCL was performed by total im-
mersion. A known weight of the CAVD/PCL was incubated in the recep-
tor chamber of the Franz Diffusion Cells (Premerger's V3B Manual
Diffusion System) that filled with 6 mL of releasing medium. The sam-
ples were entirely immersed in the receptor chambers, and the temper-
ature of the releasing medium was maintained at 37 ± 1 °C
(physiological body temperature) with a stirring speed of 500 ±
10 rpm. The compartments were also covered with Al-foil to protect
against light exposure. For each sample, the experiment was carried
out in triplicate. At predetermined time intervals, 1 mL of soaking solu-
tion was collected to determine the amount of vitamin D3 by an
enzyme-linked immunosorbent assay (ELISA) kit for vitamin D3 due to
the low concentration of vitamin D in the released medium. The re-
maining medium was removed and replaced with another 6 mL of
fresh media to maintain the sink condition [30]. According to the man-
ufacturer's instructions, the amount of vitamin D3 was determined in
the collected sample solutions at 450 nm using the 25(OH) vitamin D
ELISA kit against the predetermined calibration curve for vitamin D3
[31]. The drug released was calculated by the following Eq. (6):
wt:of released drug ðngÞ
Drug release ¼
Initial weight of the drug incorporated in the sample ðmgÞ
ð6Þ
The experimental data were also fitting to mathematical kinetic
models to investigate the mechanism of vitamin D3 release. For this,
four kinetic models were used, including the zero-order model
Mt

[33,34].
(M ∞
¼ K 0 tÞ, first-order model log M t ¼ log M∞ þ 2:303
Kt
, Higuchi
Mt Mt
model M ∞
¼ K H t 0:5 , and Korsmeyer-Peppas model M ∞
¼ K R t n used.
Where Mt and M∞ are total cumulative quantity of drug release at
time t and infinite time, respectively, K0, K1, KH, and KR are the
respective release constant. Moreover, n presents the exponent of diffu-
sion, indicating the Fickian diffusion (n ≤ 0.45) or non-Fickian
(0.45 < n < 1) release mechanism.
2.12. In-vitro cytotoxicity
Cell lines of the human skin fibroblast (HSF, 1184) were used for in-
vitro cytotoxicity study. Briefly, HSF cells were thawed in T-25 flasks
then subculture in T-75 flasks using DMEM containing 10% FBS and 1%
antibiotic-antimycotic. The cells were incubating at 37 °C in a humidi-
fied atmosphere containing 5% CO2. Culture media was replaced after
every two days. Before cell-culture, the fibrous mats were placed in a
24-wall plate and sterilized by UV light in a biosafety cabinet for 1 h
on each side. When the cell confluence reached 80%, they were de-
tached from the flask surface using Trypsin/EDTA (0.25% Trypsin in
0.04). A cell suspension at the density of 5 × 103 cells/well was seeding
on the sterilized sintered PCL, PCL, and CA nanofibers membrane for
MTs assay and Trypan Blue dye exclusion test, separately.
2.12.1. MTs assay
MTs cell proliferation assay was used to assess cell viability and pro-
liferation rates on PCL and CA nanofiber membranes. This method re-
duces the MTs tetrazolium compound by viable cells to produce a
purple formazan product soluble in cell culture media. In brief, 3 and
7 days after seeding 5 × 103 cells/well on the membranes in the 24-
well plates, DMEM was removed and replaced by 1000 μL new DMEM
with 50 μL/well MTs reagent in each well and incubated for 2 h at
37 °C under standard culture conditions. The plates were covered with
Al-foil due to MTs are sensitive to light. After 2 h of incubation, the
medium-containing MTs were gently taken out from the incubator
and transferred 200 μL of the medium to a 96-well plate with five repli-
cates per sample. Then read the absorbance directly at OD = 490 nm
using a plate reader. Cells cultured on wells without samples were
86
International Journal of Biological Macromolecules 181 (2021) 82–98
used as a control, while well contained only DMEM was set as blank.
This experiment was performed in triplicate.
2.12.2. Dye exclusion method (Trypan Blue)
The Trypan Blue dye exclusion test evaluates cell Live/Dead viability
by cell counting at 3, 5, and 7-days. HSF cells at a density of 5 × 103 cells/
well were cultured with sterilized PCL and CA membrane. Besides, the
cells cultured on the plate without any electrospun membrane were
used as the control. After each time point, the cell detached, and suspen-
sion cells were collected to count viable and dead cells [32]. The total
number of viable cells NV and total number of dead cells Nd in four
major squares (corner) of a haemocytometer were counted and used
in the following equation (Eq. (7)) to calculate the percentage of viabil-
ity cell. Triplicate cell counting was done for each sample.
NV
%Cell viability ¼  100 ð7Þ
NV þ Nd
2.12.3. Cell adhesion study
Cell adhesion, proliferation, and morphological characterization on
the modified PCL, PCL and CA nanofibrous membranes were observed
after 3, 5, and 7 days using FESEM (Hitachi SU8020). After each time
point, the membranes were moved to another well and rinsed with
PBS two times. Subsequently, fixed with 2.5% glutaraldehyde in 0.1 M
PBS overnight before to dehydration in a graded series of ethanol
(30%, 50%, 70%, 90%, 95% and 100%, respectively) for every 15 min.
Then the samples were dried by air drier in a fume hood. The dried sam-
ples were sputter-coated with platinum and observed by FESEM
2.13. Statistical analysis
The experimental data are presented as a mean ± SD. The compari-
son between the two groups was performed using a pair-sample t-test,
and multiple comparisons were made using one-way ANOVA followed
by Tukey's test to evaluate the substantial difference in P-values <0.01
and <0.05 between the data. Both statistical analyses were conducted
using the Origin Lab and the SPSS-V25.
3. Results and discussion
3.1. Nanofiber morphology
SEM images of CA nanofiber, PCL nanofiber, CAVD6, CAVD12, and
CAVD20 nanofibrous membranes, with their average diameters and fre-
quency distributions, are shown in Fig. 2A–D, respectively. The smooth
and beadless CA nanofiber with a uniform surface structure was ob-
served. The average diameters (n = 100) for CA was 325 ± 101 nm.
The diameter of nanofibers is a vital factor in the regulation of drug re-
lease. In this study, mean CA diameters were smaller than that of CA di-
ameter reported in previous works [16,35–37]. So, 17% CA polymer
solution was selected and used to load vitamin D3 based on its unifor-
mity and the fiber diameter results shown in Fig. 2.
The average diameters (n = 100) for vitamin D3-loaded CA nanofi-
bers with percentages of 6, 12, and 20 (w/w) were 324 ± 118 nm,
401 ± 131 nm, and 428 ± 129 nm, respectively. It was increased from
324 ± 118 to 428 ± 129 nm as the percentage of vitamin D3 increased.
Ullah et al. reported a similar finding. They found that average CA fiber
diameter increased from 324 ± 94 nm to 420 ± 178 and 448 ± 180 nm
by increased amounts of blumea oil from 15% to 20%, respectively [38].
The electrospun fiber diameter was affected by many processing param-
eters, including applied voltage, flow rate, polymer concentration, vis-
cosity and conductivity of the polymer solutions, nature of the solvent,
and tip to collector distance [39,40].
The increased CA fiber diameter of the CAVD6, CAVD12, and CAVD20
in the present study is related to increased viscosity and decreased
M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al. International Journal of Biological Macromolecules 181 (2021) 82–98

Fig. 2. SEM images and diameter distributions of (A) CANF, (B) CAVD6, (C) CAVD12, and (D) CAVD20 at the magnification value of 10,000× and length scale of 5 μm.

conductivity of the blended CA/Vitamin D3 solution by increasing the

amount of vitamin D3 as seen in Table 3, which was in agreement
with the previous article [35,41].
Table 3
Tawwab, Marwa Y. Abdel, et al. [42] stated that higher viscosity
Shear viscosity and electrical conductivity of neat CA and vitamin D3-containing CA
resistance resulted from higher concentrations of vitamin D3 in solutions.
the blended solution, which fails to maintain its flow at the tip of the
Taylor cone. Consequently, a large size of nanofibers was produced. Sample Conductivity Viscosity (cP) at a different shear rate
μS
Radisavljevic et al. reported an increase in the average fiber diameter 0.4 s−1 0.5 s−1 0.6 s−1 0.7 s−1 0.8 s−1
associated with increased viscosity and reduced conductivity of the Neat CA 7.59 ± 0.7 213 ± 2 410 ± 1 506 ± 1 529 ± 1 541 ± 1
blend solution. They also argued that increasing the solution's conduc- CAVD12 4.88 ± 0.7 398 ± 2 530 ± 3 595 ± 2 600 ± 5 650 ± 2
tivity would increase the droplet surface charge and cause the fiber di- CAVD20 4.53 ± 0.5 504 ± 3 608 ± 2 650 ± 4 642 ± 2 717 ± 1
ameter to decrease [27]. In other words, the vitamin D3-loaded CA The data are expressed as mean ± SD, n = 3.

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M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al. International Journal of Biological Macromolecules 181 (2021) 82–98

Fig. 3. SEM images and diameter distributions of (A) PCL and (B) sintered PCL membrane at 58 °C at the magnification value of 15,000× and length scale of 3 μm.

nanofibers were smooth and did not affect their morphology, as shown SEM image of PCL showed that PCL nanofiber was beadles, smooth,
in Fig. 2B–D. While neither vitamin D3 crystal particles nor other types and the fibers were randomly oriented with a uniform surface structure,
of drug aggregates were found on CA fiber's surfaces, there is strong ev- as shown in Fig. 3A. The average PCL's diameter (n = 100) was 333 ±
idence that vitamin D3 is well incorporated into the CA fiber membrane. 79.9 nm. The average PCL diameters were also lower in the current

Fig. 4. An image to show the size and shape of (A) CAVD6/PCL, (B) CAVD12/PCL, (C) CAVD20/PCL with their cross-sectional SEM image, and (D) schematic representation of reservoir drug
delivery system.

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M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al.
study than in previous studies [27,43]. The electrospun PCL fiber also
displays cylindrical, isolated fiber morphologies, free from fiber-fiber
connections with a highly porous microstructure with significant pore
interconnectivity. PCL morphology was dramatically changed after ther-
mal sintered at 58 °C nanofibers. It was more fiber-fiber interactions,
bonding, and less porosity Fig. 3B. This result goes beyond previous re-
ports, indicating a significant decrease in pore size by increasing
sintering temperature, and it was optimally achieved at 58 °C and
59 °C as reported in the previous studies [20,21]. According to this find-
ing, vitamin D3 release can be tailored by enclosed CAVD within PCL
nanofiber membrane and modified its nanostructure morphology to
less porosity by the thermal sintering.
3.2. Implants design
A rod implantable drug delivery system with 4 cm long and around
2.5 mm diameter has been developed to deliver vitamin D3 that can be
implanted in subcutaneous tissue, and the applicator equipment has al-
ready been designed for implants of this size [44]. The CAVD6/PCL,
CAVD12/PCL, and CAVD20/PCL were formulated in the current research
(Fig. 4A–C and Table 4) that were loaded with 6.1 ± 0.66, 12.08 ± 0.8,
and 19.57 ± 0.28 mg of vitamin D3, respectively. These implants were
developed to deliver vitamin D3 and used to treat vitamin D deficiency
by implanting it in the subcutaneous tissue.
The core of the CAVD/PCL implants was produced from vitamin D3-
loaded CA nanofiber. The drug was homogeneously dispersed inside the
implant's core, covered with the permeable and slow biodegradable PCL
capsule (rate-limiting polymer membrane around the core). As illus-
trated in Fig. 4D, this type of device can be described as a reservoir-
type implant [45,46]. To date, the reservoir-based system is one of the
most common controlled drug delivery systems. It is most useful for
long-termed drug delivery because the drug release rate from a reser-
voir implant is controlled by drug diffusion across a rate-controlling
membrane and the drug partition coefficients between the implant
core and surrounding media.
3.3. Thermal analysis of the nanofibrous membranes
The DSC heating-cooling-heating thermograms of CA nanofiber
(CANF), vitamin D3-loaded CA nanofibers (CAVD12 and CAVD20), and
pure vitamin D3 are shown in Fig. 5. The DSC heating-cooling-heating
thermograms of PCL nanofiber (PCLNF) and sintered PCL membrane
are seen in Fig. 6.
The DSC thermograms of the pure vitamin D3 showed two endother-
mic and one exothermic peak. As stated by Tsai, Shu-Yao et al. [47] and
Branton, A., & Jana, S. [48], the sharp endothermic peak at 86.7 °C is the
melting point of crystal vitamin D3 while the exothermic peak and sec-
ond endothermic peak at 271.18 °C and 219.4 °C, respectively, may be
due to the decomposition of vitamin D3 and slow degradation of inter-
mediates produced during the thermal degradation, as displayed in
Fig. 5A. In comparison, the melting temperature of vitamin D3 in the
CAVD20 showed a broad endothermic peak at 58.51 °C in the first
heat cycle (Fig. 5B) and a sharp endothermic peak at 52.31 °C in the sec-
ond heat cycle (Fig. 5D). The broad endothermic peak in the first heating
thermograms of the CAVD20 ascribed to overlaps of the desorption
water outlet of the CAVD20 nanofibrous mats and melting temperature
peak of vitamin D3. A sharp peak in the second heating cycle at the same
Table 4
The specifications of each modal CAVD/PCL.
Modal Wt. (mg) of CAVD Wt. (mg) with PCL layer
CAVD6/PCL 102.6 ± 11.10 124.4 ± 4.20
CAVD12/PCL 101.7 ± 8.80 130.1 ± 5.60
CAVD20/PCL 100.2 ± 10.50 137 ± 4.50
Wt.: weight; VD3: vitamin D3, CAVD was aforementioned. The data are expressed as mean ± SD, n = 3.
International Journal of Biological Macromolecules 181 (2021) 82–98
temperature is associated with the vitamin D3 melting point only due to
the moisture being evaporated, and the sharp peak appeared. The
CAVD20 fibrous membrane also exhibited an endothermic peak at
214 °C associated with the CA melting point (Fig. 5B).
The CANF and CAVD12 fibers showed two endothermic peaks in the
first heat cycle. The first peak corresponds to the desorption water mois-
ture in both of them [16], was noticed at 75.41 °C and 69.8 °C, respec-
tively, and omitted in the second heat cycle (Fig. 5D). The second
endothermic peak of the CA and CAVD12 nanofiber membrane was de-
tected at 231 °C and 206 °C that is corresponding to the CA's melting
temperature. This result is close to the data reported in the literature
[49].
The CA melting temperature in the CAVD12 and CAVD20
nanofibrous mats was slightly decreased to 206 °C and 214 °C, by load-
ing 12% and 20% of vitamin D3, respectively, as reported in Table 5. Ad-
ditionally, by increasing the amount of drug loading into CA, an
endothermic melting peak with a lower enthalpy was observed,
which can be attributed to a decrease in the crystalline structure of CA
by increasing the vitamin D3 loading into fibers due to the uniform dis-
persion of the drug in the CA matrix. This finding is directly consistent
with the previous study that reported decreased fusion enthalpy by in-
creasing (%) drug loading [50]. In conclusion, the thermal analysis of the
CA and CA loaded with vitamin D3 discovered three points. The first
point, CA nanofibers endothermic melting temperature by loading
12%, and 20% of vitamin D3 was slightly decreased. Second, by increas-
ing the drug concentrated loading, the CA fusion enthalpy decreased,
as shown in Table 5.
Third, the endothermic melting temperature of vitamin D3 has not
appeared in the DSC thermogram of CAVD12, and it was verified by
cooling and the second heat cycle. It may be due to the low concentra-
tion of vitamin D present in the fibrous CAVD12 membrane. In contrast,
the endothermic melting temperature peak of vitamin D3 in the
CAVD20 was observed in the first and second heat cycles, and the exo-
thermic crystallization peak of vitamin D3 in CAVD20 was also observed
in the cooling curve of the DSC (Fig. 5C), maybe this amount of vitamin
D3 is enough to detect by DSC instrument. Furthermore, the glass tran-
sition temperature (Tg) of the CA polymer was still noticeable in the sec-
ond heat cycle. It means that the content of amorphous material is high,
and CA nanofiber could preserve its amorphous properties after the first
heating because it had sufficient time to cool in the cooling cycle
(Fig. 5C).
DSC thermograms of the PCL nanofiber and sintered PCL mem-
brane are represented in Fig. 6A, and B. PCLNF exhibited glass transi-
tion (Tg ) at −61.9 °C, melted at 60.04 °C and 54.79 °C for first and
second heat cycles, respectively, and crystallized during the cooling
run at 28.26 °C (Fig. 6A), which is following the literature [27]. In
the first PCL heating cycle, a broad melting peak temperature was ob-
served, but in the second heating cycle, the ΔHm and degree of crys-
tallinity (χm) was dropped significantly (P < 0.01), it was 63.32 J/g to
47.47 J/g for ΔHm and from 45.39% to 34.029% for χm, respectively.
The melting peak temperature was also shifted from 60.04 °C to
54.79 °C. This significant decrease in ΔHm and χm means quick disen-
tanglement of PCL nanofiber once the melting has started. It can be
clarified that the melted PCL nanofiber does not regenerate its nano-
structure due to did not have enough time to cool down during the
cooling process, and its nanostructure was transformed into another
morphology [20,21].
CAVD/PCL's diameter CAVD/PCL's length (cm) Wt. (mg) of VD3 loaded
2.55 ± 0.03 4 ± 0.15 6.1 ± 0.66
2.5 ± 0.102 4 + 0.12 12.08 ± 0.80
2.5 ± 0.04 4 ± 0.14 19.57 ± 0.28
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M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al. International Journal of Biological Macromolecules 181 (2021) 82–98

Fig. 5. DSC curve (A) heating-cooling-heating scans of the vitamin D3, (B) first heat cycle, (C) cooling cycle, and (D) second heat cycle of the CANF, and vitamin D3-loaded CANF.

Fig. 6. DSC curve of the heating-cooling-heating scans of (A) PCLNF and (B) sintered PCL.

Fig. 6B reveals that Tg, Tm, and Tc for sintered PCL membrane was
Table 5
−60.14 °C, 64.36 °C, and 27.5 °C, respectively. It was almost identical
DSC analysis data for pure CANF and vitamin D3-loaded CANF.
to the PCL nanofiber. However, the sintered PCL showed twin melting
Samples Heating Midpoint Tg Tm ΔHm Tv ΔHv peaks in the first heat cycle, which could not be detected in the second
Cycle (°C) (°C) (J/g) (°C) (J/g)
heat. The exhibition of twin melting endothermic peaks can explain that
CANF 1st cycle 192.91 231.17 12.08 75.41 110.13 sintered PCL membrane, including two different crystal morphologies
2nd cycle 190.53 231.70 7.04 (Fig. 3B) and converted into a single morphology after the first heating.
CAVD12 1st cycle 188.70 206.80 8.69 69.79 140.71
DSC results also exhibited that the ΔHm and χm of the sintered PCL
2nd cycle 184.25 206.60 5.52
CAVD20 1st cycle 174.17 210.38 8.86 58.51 108.00 membranes were higher than those of neat PCL nanofiber in which
2nd cycle 175.67 210.70 3.95 ΔHm was increased significantly (P < 0.05) from 63.32 J/g to 80.19 J/g,
Midpoint Tg: midpoint glass transition temperature, Tv: water evaporation of temperature, and χm was also statistically increased from 45.39% to 57.49%. This find-
ΔHv: enthalpy of vaporization. ing indicates that PCL nanofiber could lead to loss of nanofiber

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M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al.
entanglement and potential recrystallization by thermal sintering near
its melting point, as stated by Nelson, M. Tyler, et al. [21].
3.4. Mechanical properties
The necessity of desirable mechanical properties in terms of tensile
strength and flexibility is crucial to withstand the handling and insert
into target tissues during implantation. Therefore, mechanical proper-
ties including modulus (Et), elongation at break (εb %), and tensile
strength (σm) of rod-shaped CA nanofiber, CAVD nanofiber, and
CAVD/PCL under dry and wet condition were measured and compared.
The Et, σm, and εb of these samples are summarised in Table 6, obtained
from stress-strain curves, while the comparison between these samples
for modulus and tensile strength is illustrated in Fig. 7. The findings re-
vealed the lowest mechanical properties with a modulus of 56.1 MPa
and tensile strength of 7.48 MPa for CANF. While the modulus substan-
tially increased (P = 0.003) to 131.7 MPa for vitamin D3 loaded CA
(CAVD), although the tensile strength increased to 8.16 MPa but not sig-
nificant. The elongation at break of CAVD nanofibers (14.16%) decreased
compared to the pure CANF (17.29%.) The decreasing elongation of
CAVD is attributable to the vitamin D3 nanoparticle filler in the polymer
matrix serves as a tension aggregation point, allowing the polymer to
fail at lower elongations [51]. This interpretation is in agreement with
the previous articles [52,53].
In order to increase mechanical properties, CAVD was enclosed
within the PCL nanofiber membrane and modified using thermally
sintered at 58 °C (Section 2.4). A study reported that PCL nanofibers
have better tensile strength than CA nanofibers [54], another study
showed that PCL's mechanical properties could be improved by thermal
sintering near the melting temperature [21] due to its recrystallization.
The mechanical properties of CAVD after enclosed in PCL were im-
proved, as shown in Table 6 and Fig. 7. The modulus and tensile strength
of CAVD/PCL were 161 ± 14 and 13.07 ± 2.5 MPa, respectively, under
dry conditions. While for CAVD, the Et and σM value was 132 ±
52 MPa and 8.16 ± 2.36 MPa, respectively. The modulus difference be-
tween them was 29.26 ± 18.77 MPa, but not significantly increased. The
tensile strength was significantly increased in CAVD/PCL (dry) com-
pared to CAVD, with a mean difference of 4.9.
Moreover, the difference between CAVD/PCL (dry) and CANF for
modulus and tensile strength was 105.03 MPa and 5.58 MPa, respec-
tively, which was significantly (t-test, P < 0.01) increased (Fig. 7). This
improvement in CAVD/PCL's mechanical properties might be related
to increased fiber-fiber interactions, bonding, and reducing porosity in
the PCL membrane and increasing degree of crystallinity of PCL [21]
that is explained in Section 3.3. A study reported that PCL nanofiber
membrane lost almost all surface porosity and microstructure by ther-
mal sintering at 58 and 59 °C [20].
Additionally, CAVD/PCL's mechanical properties in wet conditions
were also tested due to considering the risk of withdrawing at an emer-
gency. The result showed the core CANF fractured at a maximum of the
tensile strength (19.38 ± 2.66%) while the PCL outer layer necked, and
the modulus and tensile strength of the CAVD/PCL (wet) decreased
compared to CAVD/PCL (dry), but not statistically significant. The mod-
ulus of CAVD/PCL in the dry condition was 161 ± 14 MPa and reduced
to 125.2 ± 35.4 MPa when wetted, which was decreased by 22.28%.
Table 6
Mechanical properties of CANF, CAVD, CAVD/PCL under dry and wet state.
Sample ID Diameter d0 (mm)
Neat CANF 2.39 ± 0.13
CAVD 2.37 ± 0.17
CAVD/PCL (dry) 2.38 ± 0.24
CAVD/PCL (wet) 2.41 ± 0.16
The data are expressed as mean ± SD, n = 5.
International Journal of Biological Macromolecules 181 (2021) 82–98
The tensile strength also declined from 13.065 to 10.19 MPa. The tensile
strength of CAVD/PCL (wet) still is higher than the CAVD and CANF.
The reduction mechanical properties of CAVD/PCL (wet) may be due
to fibers' swelling. Studies reported that fiber's diameter and wrapped
angles were increased by swollen fiber in the wet state [55]. The friction
coefficient has a more significant effect on the fiber's displacement and
entanglements' loss than the wrapped angles. Due to swollen fiber in
the wet condition, the friction coefficient significantly reduced leads to
decreased tensile strength and modulus [56].
3.5. Water contact angle
The surface wettability of fibrous membranes is vital for drug re-
lease, cell attachment, and proliferation [57]. The mean contact angle
of the CA nanofiber membrane was 131.22°, as shown in Table 7. It
has a hydrophobic nature (>90). This result is in agreement with a
study [58]. Incorporating vitamin D into CA nanofiber, the water contact
angle decreased with an increase in vitamin D from 6% to 20%. The water
contact angle of CAVD6, CAVD12, CAVD20 were 114.57°, 114.06°, and
110.68° at 0 s, respectively. It may be related to the presence of –OH
groups in the vitamin D3 (25-hydroxyvitamin D₃) while their surface
still has a hydrophobic nature (>90). Ullah, Azeem, et al. claimed that
a decreasing water contact angle and improved hydrophilicity could
help adhere to a cell and migrate by the presence of water-loving func-
tional groups (primarily OH-CH groups) of Manuka honey incorporated
in CA nanofibrous mat [59].
The water contact angles of CANF and vitamin D3-loaded CA nanofi-
ber membranes at 60 s were 126.73°, 100.04°, 99.6°, and 77.96°, respec-
tively, indicating the increasing surface wettability during the time was
observed in all membranes.
Additionally, the water contact angles of both electrospun PCL nano-
fiber and sintered PCL have been measured, and the data are shown in
Table 7 as well. The PCLNF water contact angles were 127.59° and
125° at 0 and 120 s, respectively. It means that the electrospun PCL
membrane is hydrophobic and in agreement with the literature [60].
Thermal sintering treatment can significantly (P < 0.01) improve the
PCL membrane's hydrophilicity and wettability as water contact angles
reduced from 127.59° before treatment to 72.9° after treatment.
3.6. The weight loss and degree of swelling
The degree of swelling and weight loss of CANF, CAVD12/PCL, and
CAVD20/PCL were measured in a phosphate buffer solution (pH 7.4, at
37 °C) with constant shaking, and results were shown graphically
(Fig. 8).
Fig. 8A displays the swelling behavior of CANF, CAVD12/PCL, and
CAVD20/PCL for the period of 30-days. The water absorption ability in
all samples increases up to 1 day after that, a plateau trend was ob-
served. Among all samples, CANF has the highest water uptake. The
water up taking decreased significantly (P-value < 0.01) from 337% of
CANF to 150% and 91.8% of CAVD12/PCL and CAVD20/PCL, respectively.
Tungprapa et al. reported that loading CA fiber with hydrophobic drug
exhibited lower swelling values relative to neat e-spun CA fiber mats
[35]. CANF provides higher water uptake properties than other samples
related to water engulfed in the porous structure. It is in agreement with
Et (MPa) σM (MPa) εB (%)
56.1 ± 21.78 7.48 ± 1.07 17.29 ± 2.01
132 ± 52 8.16 ± 2.36 14.13 ± 2.17
161 ± 14 13.07 ± 2.52 20.04 ± 1.33
125.2 ± 35.4 10.19 ± 2.88
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M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al. International Journal of Biological Macromolecules 181 (2021) 82–98

Fig. 7. Statistical significant differences between the means of the CANF, CAVD, CAVD/PCL (dry), and CAVD/PCL (wet) for modulus and tensile strength, *P < 0.01.

the literature [15,61]. The size of fibers may be relevant to these effects.
According to the previous section, fiber diameter increased with an in-
creasing amount of vitamin D3 in the CA fibrous mat. As a result, the
smaller fiber diameters of pure CANF had a larger surface area per vol-
ume, and greater porosity contributes to a higher amount of water re-
tention [62]. By increasing the amount of vitamin D3, the intake of
water decreased dramatically. It may be related to the hydrophobic na-
ture of vitamin D3 [34]. A study reported a declining pattern in the
swelling of nanofiber mats with an increase in the amount of Zataria
multiflora (ZM) is an essential oil due to its hydrophobic nature [63].
It has supported the above interpretation to decrease water intake in
CAVD12/PCL and CAVD20/PCL.
Furthermore, the lower water absorption of CAVD12/PCL and
CAVD20/PCL can be connected to PCL's outer layer is more stable to
the PBS solution. The degree of swelling and rate of degradation is the
main factors for drug release, in which increased swelling and degrada-
tion will lead to accelerated release of drugs. Therefore, CAVD/PCL is an
appropriate model for delivering vitamin D3 due to its slow degrading
and low water absorption [64].
Fig. 8B shows the weight loss of the neat CANF, CAVD12/PCL, and
CAVD20/PCL. The CANF has the highest percentage of weight loss
among all samples over 30-days and an average of 4.48%. In comparison,
the rate of weight loss decreased dramatically in CAVD12/PCL and
CAVD20/PCL (P < 0.05). The mean difference between them was
3.21% and 3.59%, respectively. Besides, the weight loss of CAVD20/PCL
(0.9%) was lower than that of CAVD12/PCL (1.27%). The higher weight
loss of CAVD12/PCL compared to CAVD20/PCL was most likely due to
the lower PCL layer thickness of CAVD12/PCL (200 μm) than that of
CAVD20/PCL (210 μm) (Table 2), resulting in more water accumulated
and weight loss. Samples with higher water levels retention, which
meant that they were more swollen and more surrounded by solvent,
had a higher weight loss degree, as reported by a study [62]. The find-
ings indicate that the enclosed rod CAVD within the PCL membrane is
followed by sintering, which is a helpful technique to reduce the degra-
dation rate, and the weight loss can also be reduced by increasing the
thickness of the PCL layer.
3.7. Drug stability

Drug stability during loading is a critical factor in the successful for-

mulation of a drug delivery system. Vitamin D3 is considered to be un-
Table 7 stable to stress factors such as light, heat, and chemicals. Therefore,
Effect of vitamin D3 on the water contact angle of CANF and sintering on the PCLNF. the stability of vitamin D3 after loaded in CA nanofiber was investigated
S/N Samples Mean ± SD water contact angle in degree (°)
in the current study using HPLC-PAD [65,66] and UV–Visible spectro-
photometers (190-400 nm).
0s 60 s 120 s
The results obtained from the current analysis indicate similar chro-
1 CANF 131.28 ± 5.67 126.73 ± 4.88 matograms (Fig. 9A) with a single high peak at 8.077, 8.053, and
2 CAVD6 114.57 ± 9.33 100.04 ± 11.62
8.055 min of retention times for vitamin D3, CAVD20, and CAVD12, re-
3 CAVD12 114.06 ± 11.42 99.6 ± 12.74
4 CAVD20 110.68 ± 9.00 77.96 ± 13.50 spectively. The single peak in the chromatogram of the CAVD12 and
5 PCLNF 127.59 ± 1.80⁎ 125.00 ± 1.78⁎ CAVD20 (Fig. 9Ab and Ac) is evidence of non-degradation of vitamin
6 Sintered PCL 72.90 ± 3.65 65.91 ± 3.39 D3 compared to standard vitamin D3 (Fig. 9Aa). Previous studies have
The data are expressed as mean ± SD, n = 5. shown that vitamin D3 degradation can occur due to isomerization
⁎ P < 0.01, significant declined. under various stress conditions. Thermal degradation of vitamin D3

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M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al.
Fig. 8. (A) Swelling behavior CANF, CAVD12/PCL, and CAVD20/PCL; (B) comparison of weight loss between CAND, CAVD12/PCL and CAVD20/PCL in the phosphate buffer at 37 °C, *P < 0.05,
**P < 0.01.
resulted in the formation of pre-vitamin D3 product, while a trans con-
figuration of vitamin D3 has formed under cis/trans isomerization by io-
dine and Isotachysterol produced under low pH isomerization from
vitamin D3.
In general, if vitamin D3 degraded under each condition, an
additional peak appeared next to the peak of vitamin D3 in the chro-
matogram [29,65,66].
93
International Journal of Biological Macromolecules 181 (2021) 82–98
In terms of confirming vitamin D3 stability, the UV-spectra of pure
vitamin D3, CANF, CAVD12, and CAVD20 are measured by UV–Visible
spectrophotometers. CANF has a single peak at 210 nm, while vitamin
D3, CAVD12, and CAVD20 exhibited two peaks at 210 and 326 nm
(Fig. 9B). The presence of an additional peak in CAVD12 and CAVD20
corresponding to vitamin D3. The spectral similarity between pure vita-
min D3 and vitamin D3-loaded CA nanofiber showed stability of vitamin
M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al.
Fig. 9. (A) HPLC-PAD chromatograms of (a) vitamin D3 powder dissolved in methanol (1 μg/ml), (b) vitamin D3 extracted from (b) CAVD20, (c) CAVD12 by methanol; (B) UV spectra of
CANF, vitamin D3 powder (2 mg/mL), CAVD12, and CAVD20 dissolved in acetone.
D in each sample that is verified the results of HPLC herein. Depended
on the obtained data can be concluded that encapsulation of vitamin
D in CA nanofiber using electrospinning is a harmless method.
3.8. In-vitro drug release study
3.8.1. Drug loading efficiency
The actual quantity of vitamin D3 in the CAVD6, CAD12, and CAVD20
was 99.15%, 98.86%, and 96.7% from the UV- Vis spectrophotometric
analysis. The high encapsulation potency of vitamin D3 was found in
all samples due to the high solubility of vitamin D3 in the polymer solu-
tion. Vitamin D3 is soluble in acetone, although we added ethanol to the
solvent system to increase its solubility [29,67].
3.8.2. In-vitro drug release study
Burst drug release is related to drugs on the surface of the
electrospun nanofibrous membrane that is a critical challenge. The
rod CAVD was enclosed within the PCL capsule in order to eliminate
the burst drug release and well-controlled drug release from the
IDDS. The PCL capsule functions as a rate-limiting polymer mem-
brane formed from the PCL membrane using the thermal sintering
method [20].
Fig. 10A shows the vitamin D3 release profile of CAVD6/PCL,
CAVD12/PCL, and CAVD20/PCL. It can be seen that drug release had a bi-
phasic release. The first phase shows a linear relationship between the
cumulative drug release (ng/mL) and time (5-days). After that, a steady
rise in the drug release followed, reaching a plateau in the second phase,
which can be found approximately after 15 days. The linear relation-
ships between cumulative drug release (ng/mL) and time (days) were
observed for all of the CAVD/PCL in the early five days, that is fitting
with the zero-order model by determining the highest R2 value
(>0.96), as shown in Fig. 10B.
Drug release rate depends on many factors, including swelling, degra-
dation rate, and drug solubility in the medium [62]. The slower release of
vitamin D3 from CAVD6/PCL, CAVD12/PCL, and CAVD20/PCL can be due to
the PCL layer that provided a barrier to regulating the penetration and
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International Journal of Biological Macromolecules 181 (2021) 82–98
diffusion of media and drug from the environment to the core region
and reverses. The water absorption and in-vitro degradation study herein
confirmed this interpretation and showed a significant decline in CAVD/
PCL's swelling and degradation trend compared to CANF (Fig. 8).
Upon the media penetration, only a portion of the drug particles can
be dissolved due to the drug's limited solubility in released media,
resulting in a saturated drug concentration maintained in the inner re-
gion. The drug is dissolved and released and is replaced quickly by par-
tial dissolution of the remaining drug particles. The drug concentration
in the inner region remains stable as long as the out layer is stable. The
drug release follows a zero-order model as expected from a reservoir
system [68]. These results were broadly in line with a previous study
[9]. The drug release behavior is also broadly in line with the drug deliv-
ery systems mediated by controlled swelling [44].
These findings indicate that vitamin D3-loaded CA nanofibers enclosed
within the thin layer of sintered PCL membrane could be an optimal solu-
tion for drug release applications over a long period. Because in the case of
zero-order kinetics, the release of the drug is the only function with time,
and the mechanism takes place at a constant rate regardless of the drug's
concentration. Literature shows that this form of drug release is suitable
for a long-termed drug delivery system [69].
The release kinetics was further studied by fitting the drug released
data to the four mathematical kinetic models described above, as seen in
Fig. 10C–F. The related correlation coefficients (R2), drug release con-
stant (K), and release exponent (n) of each model are shown in
Table 8. These models were previously used to investigate the kinetics
of drug release [70].
Among all the mathematical fitting models, the Korsmeyer-Peppas
model is the best-fitting model with the higher correlation value
(R2 > 0.84) for the drug release profile of all kinds CAVD/PCL, as shown
in Fig. 10F. The straight-line regression of the Korsmeyer-Peppas curve
was used to achieve a release index (n). The n value is an exponent that
describes the release of the kinetics process [71]. It was smaller than
0.31 in all CAVD/PCL. This finding indicated that the drug released from
CAVD/PCL was mainly the Fickian diffusion mechanism under these con-
ditions by swollen the whole sample and pores filled with water [62,72].
M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al. International Journal of Biological Macromolecules 181 (2021) 82–98

Fig. 10. Cumulative vitamin D3 release profile of (A) CAVD20/PCL, CAVD12/PCL, and CAVD6/PCL, (B) its linear regression of fitting experimental data to the zero-order model at five days,
and (C–F) different mathematical fitting curves of drug release.

Besides, the results showed that the CAVD/PCL drug release profile, re- 3.9. Cell culture
gression period between 0 and 5 days, liner manner according to the
zero-order equation (R2 = 0.962). 5 × 103 cells/well of HSF seeded in 24-well plates based on the opti-
mizing cell-seeding density is represented in supplementary materials.
Table 8 In-vitro cytotoxicity and cell viability assay were conducted using the
Results of fitting the experimental data with four kinetic models. MTs cell proliferation assay (colorimetric) and the Trypan Blue Dye Ex-
clusion Method. The level of viability and cell proliferation rates are
Implants Zero-order First-order Higuchi Korsmeyer-Peppas
good indicators of cell health.
2 2 2
R K0 R K1 R Kh R2 Kk-p n

CAVD6/PCL 0.73 2.415 0.114 0.72 0.76 1.53 0.84 6.27 0.31 3.9.1. MTs assay and Trypan Blue dye exclusion method
CAVD12/PCL 2.717 1.61 0.31 MTs assay indirectly evaluates the number of viable cells based on
CAVD20/PCL 3.022 1.7 0.31
reducing the MTs tetrazolium compound by viable active cells,

95
M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al.
reflecting the normal function of mitochondria, while Trypan Blue is
based on the principle that living cells possess intact cell membranes
that are not stained.
In-vitro cytotoxicity results showed CANF, PCLNF, and sintered PCL
membranes no cytotoxicity over seven days of culture (Fig. 11A and
B). The CA and PCL nanofiber membrane and modified PCL membrane
were non-toxic to human skin fibroblast cells. Cell viability and prolifer-
ation were well on the membranes, and no significant differences
(P > 0.05) were observed. Besides, according to the optical density of
MTs assay, the number of cells increases continuously over seven days
of culture (Fig. 11A), the rate of cell proliferation was approximately
2.5 times on day seven compared to that of 3 days, indicating that all
membranes are non-toxic.
Doostan, Maryam, et al. reported that the CA mat cell viability did
not differ statistically from that of the control group after 48 and 72 h in-
cubation. They also concluded the non-cytotoxic CA fibrous mat to
human fibroblasts [61].
The number of viable and dead cells in four replicates was counted
by the dye exclusion method, and the mean ± SD cell viability percent-
age of HSF cells at intervals of 3, 5, and 7 days for CA, PCL, and sintered
PCL mats, as shown in Fig. 11B. There were no dead cells in control
throughout 3 to 5-day culture time observed, while cell viability in the
CA, PCL, and sintered PCL was observed in a range between 95 and
98%. In 7-days, the cell viability in the CA, PCL, and sintered PCL mats in-
creased to a range of 97%–98%. It concluded that the number of HSF cells
on each membrane increased over time at a different rate.
The cell viability above 80% is considered non-toxic under ISO-
10993-5 [38]. MTs assay and dye exclusion method showed a higher
cell viability of more than 97%. These experiments are scientific evi-
dence of the non-toxicity of PCLNF, CANF, and PCL sintered membrane
to HSF cells. This finding is well linked to previous research in which
PCL is an FDA-approved biopolymer for biomedical applications
[73,74]. Besides, in-vitro cytotoxicity assay findings indicate that CA
nanofibers are biocompatible and non-toxic to fibroblast cells with
higher growth and proliferation rates. This result is in agreement with
the results of many studies [15,34,38].
3.9.2. Cell adhesion studies using SEM
Adhesion, proliferation, and morphological appearance of HSF
cells on sintered PCL and CA nanofibrous membranes at the third,
fifth, and seventh cultured days are showed in Fig. 12. The HSF cells
were attached to the CA and PCL membrane's surface at different
rates. The cell proliferation was also increasing continuously with in-
creasing culture time, indicating that both membranes are non-toxic.
The cells spreading became more noticeable on the sintered PCL
membranes than on the CANF membrane. The hydrophilic
Fig. 11. (A) Cell viability and proliferation study by MTs assay (data are representative of three independent experiments and are plotted as mean ± SD (n = 3)), (B) cell viability
percentage of the HSF cell study by cell counting using Trypan Blue dye exclusion method (data represented as the mean ± SD of n = 4).
96
International Journal of Biological Macromolecules 181 (2021) 82–98
properties of sintered PCL (Table 7) are consistent with the high
cell distribution on its surface than that of CA. The hydrophobic sur-
faces of CANF lead to lower cell adhesion than that of hydrophilic
surfaces of sintered PCL, and cell proliferation is significantly influ-
enced by the hydrophilicity of the membrane [32,34,60]. Due to
sintered PCL's low porosity and tiny pore size, cells were spread
without infiltrating the surface membrane. While, on the surface of
CANF, flattened cells were observed and spread between CA fibers.
Cell elongation with supporting filopodia was also observed on the
surface of both membranes. The filopodia is a particular structure re-
lated to fibroblasts' cell motility (Fig. 12d–f for PCL and Fig. 12b
for CA).
To sum up, using sintered PCL membrane as an out-layer to the
rod CAVD/PCL helps improve biocompatibility. This system is
designed for long-term drug delivery applications due to PCL being
slowly degraded.
4. Conclusion
Cellulose acetate nanofibrous membrane loaded with different
amounts of vitamin D3 and PCL nanofibrous membrane was success-
fully fabricated using electrospinning. The FESEM images of CA, CA
loaded with vitamin D3, and PCL nanofibers showed narrow distribu-
tion, uniform structures, and smooth morphology. HPLC and UV
spectra indicate incorporating vitamin D3 into CA nanofiber using
electrospinning is a harmless process. A new implantable drug deliv-
ery system (CAVD/PCL) was successfully created from a single rod of
vitamin D 3-loaded CA nanofiber enclosed in a rate-limiting PCL
membrane.
Drug release of the implantable drug delivery system could be closely
regulated by modified PCL nanofibrous membrane using thermal
sintering. The drug release rate tends to be gradual release in CAVD6/
PCL, CAVD12/PCL, and CAVD20/PCL. The kinetic drug release shows
zero-order in the initial phase. This finding confirmed that the outer PCL
membrane plays a crucial role in long-termed control drug release.
The mechanical properties of the CAVD/PCL have also been affected
by the modified PCL membrane. The finding showed the mechanical
properties of CAVD/PCL significantly improved compared to CA
and CAVD.
The in-vitro cytotoxicity studies showed high cell viability and
proliferation of HSF cells with CA nanofiber and modified PCL mem-
brane. The study also revealed high cell distribution and adherence
on the modified PCL membrane surface due to the PCL surface's hy-
drophilic feature. Based on these results of the in-vitro cytotoxicity
studies, CA and PCL nanofibers are not toxic to implants in the sub-
dermal human body.
M.A. Wsoo, S.I.A. Razak, S.P.M. Bohari et al.
Fig. 12. SEM images of HSF adhered and proliferated on the surfaces of CANF and sintered PCL membranes at different times cultured, (a) flattened cells spread on the fibers, (b, d, e,
f) elongated cell with supporting filopodia, (c) flattened cells spread between fibers.
CRediT authorship contribution statement
Mohammed Ahmad Wsoo: Conceptualization; Methodology; Writ-
ing – Original draft; Investigation; Visualization; Data curation. Shafinaz
Shahir: Validation; Co-supervisor. Siti Pauliena Mohd Bohari: Valida-
tion; Co-supervisor. Rabie: Methodology. Mohammed Rafiq Abdul
Kadir: Formal analysis. Nadirul Hasraf Mat Nayan: Formal analysis.
Saiful Izwan Abd Razak: Supervisor; Validation; Writing - review &
editing. All authors have read and agreed to the published version of
the manuscript.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
This research has received financial support from Universiti
Teknologi Malaysia, Research Grant (04G53 and 4C354). All authors
are grateful to Universiti Teknologi Malaysia for their assistance and fi-
nancial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2021.03.108.
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